Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Plasma membrane calcium ATPase 4 (PMCA4) is a calcium extrusion pump which may also modulate Ca2+-triggered signal transduction pathways. Previous studies postulate that PMCA4 modulates signalling via an interaction with neuronal nitric oxide synthase (nNOS) in localised plasmalemmal microdomains. The effect of PMCA4 on vascular contractility is unclear. This project has utilised PMCA4 ablated mice (PMCA4 KO (-/-)) and a novel specific PMCA4 inhibitor (termed AP2) to study the role of PMCA4 in mouse resistance artery contractility.Immunohistochemistry, Western blotting and polymerase chain reaction (PCR) confirmed the absence of PMCA4 in the brain, vasculature and ear snips obtained from PMCA4 KO (-/-) mice whereas it was present in those from wild type (WT (+/+)) mice. Pressure myography was employed to assesss contractile function of isolated, pressurised (to 60 mmHg) mesenteric resistance arteries from 3 months old male PMCA4 KO (-/-) and WT (+/+) mice, in response to high K+ physiological salt solution (KPSS) (40mM & 100mM) and noradrenaline (NA) (Log[NA] -9.0 to -5.0M). Passive lumen diameter and left and right wall thicknesses of arteries from PMAC4 KO (-/-) and WT (+/+) mice were taken at transmural pressures of 5-140 mmHg. Effects of acute PMCA4 inhibition with AP2 (10µM and 1µM), nitric oxide synthase (NOS) inhibition with LNNA (100µM) and specific nNOS inhibition with Vinyl-L-Nio (10µM) were also investigated. Effects of PMCA4 ablation and AP2 (10µM) on global intracellular Ca2+ changes ([Ca2+]i) in pressurised mesenteric arteries were assessed after loading arteries with the Ca2+-sensitive indicator indo-1. PMCA4 ablation had no effect on the magnitude of arterial constrictions or on the changes of [Ca2+]i in response to KPSS (40mM & 100mM) or to noradrenaline. The passive intra-lumen diameter, wall thickness, wall to lumen diameter and cross sectional area of mesenteric arteries across the intravascular pressure range studied were also not modulated by PMCA4 ablation. A leftwards shift in the stress to strain relationship and significant increase in beta elastic modulus (β) were revealed in arteries from PMCA4 KO (-/-) mice compared to those from WT (+/+) mice, suggesting that PMCA4 ablation reduces mesenteric arterial distensibility. Acute PMCA4 inhibition with AP2, significantly reduced arterial constrictions and the increase in [Ca2+]i in response to noradrenaline in arteries from WT (+/+) mice, but had no effect on arterial constrictions elicited by arteries from PMCA4 KO (-/-) mice. Inhibitory effects of AP2 were not present in arteries after NOS inhibition by LNNA and also after nNOS inhibition with Vinly-L-Nio. Hence, PMCA4 inhibition with AP2 reduces vascular constriction by a nNOS-dependent mechanism.In conclusion, the main findings of the study were that ablation and acute inhibition of PMCA4 with AP2 have different effects on mouse mesenteric resistance arterial contractility. This study provides more insight into PMCA4 as a significant modulator of signalling within the vasculature via effects on nNOS.

Indiana University-Purdue University Indianapolis (IUPUI) / Stimulation of N-­methyl-­D-­aspartic acid receptors (NMDARs) and the resulting activation of neuronal nitric oxide synthase (nNOS) are critical for fear memory formation. A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear memory, but they also affect many other CNS functions. Following NMDAR stimulation, efficient activation of nNOS requires linking nNOS to a scaffolding protein, the postsynaptic density protein 95 (PSD95). We hypothesized that PSD95-­nNOS interaction in critical limbic regions (such as amygdala and hippocampus) during fear conditioning is important in regulating fear memory formation, and disruption of this protein-­protein binding may cause impairments in conditioned fear memory. Utilizing co-­immunoprecipitation, electrophysiology and behavioral paradigms, we first showed that fear conditioning results in significant increases in PSD95-­nNOS binding within the basolateral amygdala (BLA) and the ventral hippocampus (vHP) in a time-­dependent manner, but not in the medial prefrontal cortex (mPFC). Secondly, by using ZL006, a small molecule disruptor of PSD95-­ nNOS interaction, it was found that systemic and intra-­BLA disruption of PSD95-­ nNOS interaction by ZL006 impaired the consolidation of cue-­induced fear. In contrast, disruption of PSD95-­nNOS interaction within the vHP did not affect the consolidation of cue-­induced fear, but significantly impaired the consolidation of context-­induced fear. At the cellular level, disruption of PSD95-­nNOS interaction with ZL006 was found to impair long-­term potentiation (LTP) in the BLA neurons. Finally, unlike NMDAR antagonist MK-­801, ZL006 is devoid of adverse effects on many other CNS functions, such as motor function, social activity, cognitive functions in tasks of object recognition memory and spatial memory. These findings collectively demonstrated that PSD95-­nNOS interaction within the conditioned fear network appears to be a key molecular step in regulating synaptic plasticity and the consolidation of conditioned fear. Disruption of PSD95-­nNOS interaction holds promise as a novel treatment strategy for fear-­ motivated disorders, such as post-­traumatic stress disorder and phobias.

Indiana University-Purdue University Indianapolis (IUPUI) / Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories.

Recentemente, o óxido nítrico (NO) tem sido relacionado com a depressão. A administração de inibidores da NO sintase (NOS) induz efeitos do tipo antidepressivo em modelos animais e há um aumento da expressão da NOS em estruturas do sistema límbico em indivíduos depressivos e em animais expostos a estresse. Além disso, sabe-se que o estresse causa um aumento da ativação de neurônios localizados em estruturas do sistema límbico e que o tratamento com antidepressivos bem como com inibidor da NOS, diminui essa marcação. Contudo, ainda não se sabe como o sistema nitrérgico dessas estruturas está relacionado com os comportamentos relacionados à depressão. Assim nosso objetivo é testar a hipótese de que o desenvolvimento do desamparo (comportamento relacionado à depressão) em ratos seria causado por um aumento de atividade de neurônios que contém nNOS em estruturas envolvidas com a resposta emocional ao estresse, e que os diferentes tratamentos induzem efeitos do tipo antidepressivo no modelo apresentando através de um efeito final comum de diminuir essa ativação e, portanto, diminuir os níveis de NO. Para isso, ratos foram submetidos ao modelo do desamparo aprendido e tratados com drogas antidepressivas. Após o teste, foi feita a imunohistoquímica com marcação para Fos (Fos-IR; marcador de atividade neuronal) e nNOS (nNOS-IR). O tratamento repetido com desipramina (DES, na dose de 25, mas não na de 12,5 mg/Kg), fluoxetina (FXT, na dose de 15, mas não na de 30 mg/Kg) e imipramina (IMI, 15mg/Kg) induziu efeito do tipo-antidepressivo no teste do desamparo aprendido (LH). O tratamento agudo apenas com imipramina, mas não com FXT ou IMI, induziu o mesmo tipo de efeito. O tratamento com DES, FXT ou IMI também aumentou o número de cruzamentos entre choques no LH, porém não induziu aumento de atividade locomotora no teste do campo aberto. O tratamento repetido com DES diminuiu a Fos-IR na amígdala basolateral (BlAm), amígdala lateral (LAm), córtex pré-frontal medial (mPFC), região CA1 e CA3 do hipocampo dorsal (dHPC) e região CA3 do hipocampo ventral (vHPC). O tratamento agudo com DES induziu um aumento de Fos-IR na amígdala central (CeAm), amígdala medial (MeAm) e CA1 e CA3 do dHPC. O tratamento repetido com FXT diminuiu Fos-IR na BlAm e LAm, enquanto o tratamento agudo aumentou Fos-IR na CeAm. O tratamento repetido com IMI aumentou nNOS-IR na MeAm e a dupla marcação no núcleo leito da estria terminal (BST); e diminuiu o Fos-IR na região CA1 do dHPC e na região parvocelular do núcleo paraventricular do hipotálamo (pPVN). Por fim, foram encontradas relações positivas entre o número de células Fos-IR e o número de falhas em fugir ou escapar dos choques no LH na BlAm, LAm, CA1 e CA3 do dHPC e CA3 do vHPC; i.e., quanto mais células ativadas nessas estruturas, maior o número de choques que os animais receberam sem consegui fugir. Os resultados aqui apresentados são, em parte, corroborados pela literatura, mostrando a participação das estruturas analisadas no comportamento do desamparo aprendido e no efeito das drogas antidepressivas. Nesse contexto, acredita-se que o BST funcionaria como um núcleo de processamento da informação vinda do mPFC, HPC e amígdala, enviando projeções para o PVN e regulando o funcionamento do eixo HPA. O trabalho abre caminho para a identificação de subpopulações específicas de neurônios que expressam a nNOS, buscando compreender o papel destas na modulação das respostas comportamentais numa situação de estresse, na busca pela formulação de um cenário cada vez mais completo da participação do sistema nitrérgico dentro do complexo neurocircuito que regula as emoções / Recently, nitric oxide (NO) has been related with the neurobiology of depression. The NO synthase (NOS) inhibition induces antidepressant-like effects in animal models and there is an increase in the NOS expression in limbic structures of depressed patients or in stress exposed animals. Besides, it is well known that stressful events causes an increase in limbic structures neuronal activation and that antidepressant treatment as well as NOS inhibition attenuates this effect. However, it is still unknown how the limbic nitrergic system is related with depression-related behaviors. Then, the aim of this work is to test the hypothesis that the helplessness behavior development (a depression-related behavior) in rats would be induced by an increased activity of nNOS-containing neurons in structures related with the neurobiology of stress responses. Furthermore, the antidepressant-like effect induced by antidepressants treatment in this model would share a final effect, decreasing the activation of such neurons, and decreasing the levels of NO in these structures. For this aim, male rats were submitted to the learned helplessness model and treated with antidepressants. After the test, immunohistochemistry assay were performed, with double labeling for c-Fos (Fos-IR; neuronal activity marker) and nNOS (nNOS-IR). The repeated treatment with desipramine (DES, 25 mg/kg but not 12,5mg/kg), fluoxetine (FXT, 15 mg/Kg, but not 30 mg/Kg) and imipramina (IMI, 15 mg/KG) induced antidepressant-like effects in the learned helplessness test (LH). The acute treatment with IMI, but not with DES or FXT, induced the same effect. The repeated treatment with DES, FXT or IMI also increased the number of intertrial crossings in the LH, but not the locomotor activity score on the open field score. The repeated treatment with DES decreased the number of Fos-IR into the basolateral amygdala (BlAm), lateral amygdala (Lam), medial prefrontal cortex (mPFC), CA1 and CA3 regions of the dorsal hippocampus (dHPC), and CA3 region of the ventral hippocampus (vHPC). The acute treatment with DES increased the Fos-IR into the central amygdale (CeAm), medial amygdala (MeAm), and CA1 and CA3 regions of the dHPC. The repeated treatment with FXT decreases the number of Fos-IR into the BlAm and Lam, while the acute treatment increases the Fos-IR into the CeAm. The repeated treatment with IMI increased the nNOS-IR into the MeAm and the double- labeled cells into the bed nucleus of stria terminalis (BST); and decreased the Fos-IR into the CA1 region of the dHPC and into the parvocellular region of the paraventricular nucleus of the hypothalamus. Finally, positive correlations between the number of Fos-IR and the number of failures in escaping or avoiding the foot shocks on the LH were found into the BlAm, Lam, CA1 and CA3 of the dHPC, and CA3 of the vHPC, i.e., with more activated cells into these structures mentioned, more foot shocks the rats received. These results are (partially) corroborated with previous scientific papers, showing the analyzed structures participation in the learned helplessness behavior as well as in the antidepressant effect of antidepressant administration. Within this context, the BST would work as a relay center, processing the information coming from the mPFC, HPC and amygdaloid nuclei, and sending the output to the PVN, modulating the HPA axis. This work open some questions about the identification of specific nNOS-containing neuronal subpopulations, aiming to clarify their role in the stress response, and searching for the formulation of a more complete scenario of the nitrergic system participation in this complex emotion-regulating neurocircuit

Recentemente, o óxido nítrico (NO) tem sido relacionado com a depressão. A administração de inibidores da NO sintase (NOS) induz efeitos do tipo antidepressivo em modelos animais e há um aumento da expressão da NOS em estruturas do sistema límbico em indivíduos depressivos e em animais expostos a estresse. Além disso, sabe-se que o estresse causa um aumento da ativação de neurônios localizados em estruturas do sistema límbico e que o tratamento com antidepressivos bem como com inibidor da NOS, diminui essa marcação. Contudo, ainda não se sabe como o sistema nitrérgico dessas estruturas está relacionado com os comportamentos relacionados à depressão. Assim nosso objetivo é testar a hipótese de que o desenvolvimento do desamparo (comportamento relacionado à depressão) em ratos seria causado por um aumento de atividade de neurônios que contém nNOS em estruturas envolvidas com a resposta emocional ao estresse, e que os diferentes tratamentos induzem efeitos do tipo antidepressivo no modelo apresentando através de um efeito final comum de diminuir essa ativação e, portanto, diminuir os níveis de NO. Para isso, ratos foram submetidos ao modelo do desamparo aprendido e tratados com drogas antidepressivas. Após o teste, foi feita a imunohistoquímica com marcação para Fos (Fos-IR; marcador de atividade neuronal) e nNOS (nNOS-IR). O tratamento repetido com desipramina (DES, na dose de 25, mas não na de 12,5 mg/Kg), fluoxetina (FXT, na dose de 15, mas não na de 30 mg/Kg) e imipramina (IMI, 15mg/Kg) induziu efeito do tipo-antidepressivo no teste do desamparo aprendido (LH). O tratamento agudo apenas com imipramina, mas não com FXT ou IMI, induziu o mesmo tipo de efeito. O tratamento com DES, FXT ou IMI também aumentou o número de cruzamentos entre choques no LH, porém não induziu aumento de atividade locomotora no teste do campo aberto. O tratamento repetido com DES diminuiu a Fos-IR na amígdala basolateral (BlAm), amígdala lateral (LAm), córtex pré-frontal medial (mPFC), região CA1 e CA3 do hipocampo dorsal (dHPC) e região CA3 do hipocampo ventral (vHPC). O tratamento agudo com DES induziu um aumento de Fos-IR na amígdala central (CeAm), amígdala medial (MeAm) e CA1 e CA3 do dHPC. O tratamento repetido com FXT diminuiu Fos-IR na BlAm e LAm, enquanto o tratamento agudo aumentou Fos-IR na CeAm. O tratamento repetido com IMI aumentou nNOS-IR na MeAm e a dupla marcação no núcleo leito da estria terminal (BST); e diminuiu o Fos-IR na região CA1 do dHPC e na região parvocelular do núcleo paraventricular do hipotálamo (pPVN). Por fim, foram encontradas relações positivas entre o número de células Fos-IR e o número de falhas em fugir ou escapar dos choques no LH na BlAm, LAm, CA1 e CA3 do dHPC e CA3 do vHPC; i.e., quanto mais células ativadas nessas estruturas, maior o número de choques que os animais receberam sem consegui fugir. Os resultados aqui apresentados são, em parte, corroborados pela literatura, mostrando a participação das estruturas analisadas no comportamento do desamparo aprendido e no efeito das drogas antidepressivas. Nesse contexto, acredita-se que o BST funcionaria como um núcleo de processamento da informação vinda do mPFC, HPC e amígdala, enviando projeções para o PVN e regulando o funcionamento do eixo HPA. O trabalho abre caminho para a identificação de subpopulações específicas de neurônios que expressam a nNOS, buscando compreender o papel destas na modulação das respostas comportamentais numa situação de estresse, na busca pela formulação de um cenário cada vez mais completo da participação do sistema nitrérgico dentro do complexo neurocircuito que regula as emoções / Recently, nitric oxide (NO) has been related with the neurobiology of depression. The NO synthase (NOS) inhibition induces antidepressant-like effects in animal models and there is an increase in the NOS expression in limbic structures of depressed patients or in stress exposed animals. Besides, it is well known that stressful events causes an increase in limbic structures neuronal activation and that antidepressant treatment as well as NOS inhibition attenuates this effect. However, it is still unknown how the limbic nitrergic system is related with depression-related behaviors. Then, the aim of this work is to test the hypothesis that the helplessness behavior development (a depression-related behavior) in rats would be induced by an increased activity of nNOS-containing neurons in structures related with the neurobiology of stress responses. Furthermore, the antidepressant-like effect induced by antidepressants treatment in this model would share a final effect, decreasing the activation of such neurons, and decreasing the levels of NO in these structures. For this aim, male rats were submitted to the learned helplessness model and treated with antidepressants. After the test, immunohistochemistry assay were performed, with double labeling for c-Fos (Fos-IR; neuronal activity marker) and nNOS (nNOS-IR). The repeated treatment with desipramine (DES, 25 mg/kg but not 12,5mg/kg), fluoxetine (FXT, 15 mg/Kg, but not 30 mg/Kg) and imipramina (IMI, 15 mg/KG) induced antidepressant-like effects in the learned helplessness test (LH). The acute treatment with IMI, but not with DES or FXT, induced the same effect. The repeated treatment with DES, FXT or IMI also increased the number of intertrial crossings in the LH, but not the locomotor activity score on the open field score. The repeated treatment with DES decreased the number of Fos-IR into the basolateral amygdala (BlAm), lateral amygdala (Lam), medial prefrontal cortex (mPFC), CA1 and CA3 regions of the dorsal hippocampus (dHPC), and CA3 region of the ventral hippocampus (vHPC). The acute treatment with DES increased the Fos-IR into the central amygdale (CeAm), medial amygdala (MeAm), and CA1 and CA3 regions of the dHPC. The repeated treatment with FXT decreases the number of Fos-IR into the BlAm and Lam, while the acute treatment increases the Fos-IR into the CeAm. The repeated treatment with IMI increased the nNOS-IR into the MeAm and the double- labeled cells into the bed nucleus of stria terminalis (BST); and decreased the Fos-IR into the CA1 region of the dHPC and into the parvocellular region of the paraventricular nucleus of the hypothalamus. Finally, positive correlations between the number of Fos-IR and the number of failures in escaping or avoiding the foot shocks on the LH were found into the BlAm, Lam, CA1 and CA3 of the dHPC, and CA3 of the vHPC, i.e., with more activated cells into these structures mentioned, more foot shocks the rats received. These results are (partially) corroborated with previous scientific papers, showing the analyzed structures participation in the learned helplessness behavior as well as in the antidepressant effect of antidepressant administration. Within this context, the BST would work as a relay center, processing the information coming from the mPFC, HPC and amygdaloid nuclei, and sending the output to the PVN, modulating the HPA axis. This work open some questions about the identification of specific nNOS-containing neuronal subpopulations, aiming to clarify their role in the stress response, and searching for the formulation of a more complete scenario of the nitrergic system participation in this complex emotion-regulating neurocircuit

Respostas adaptativas ao estresse podem ser acompanhadas por alterações nos comportamentos emocionais, em particular relacionados com medo e ansiedade, bem como alterações na sensibilidade dolorosa. Ainda, tem sido investigado o papel do óxido nítrico em áreas encefálicas relacionadas ao comportamento defensivo. Embora várias evidências têm demonstrado que o enriquecimento ambiental promove melhora nos processos de memória, no aprendizado e em respostas nociceptivas, a relação entre o estresse crônico e as vantagens da utilização do enriquecimento ambiental ainda são pouco investigadas. O presente estudo teve como objetivo investigar se o enriquecimento ambiental promove alteração do comportamento emocional, da sensibilidade nociceptiva, bem como na imunorreatividade à nNOS no núcleo central da amígdala, na formação hipocampal e na região dorsolateral da substância cinzenta periaquedutal, em ratos submetidos ao estresse por isolamento social ou estresse crônico variado e mantidos em ambiente enriquecido ou sem enriquecimento. Ratos machos Wistar (~70g) foram divididos aleatoriamente em dois grandes grupos experimentais: Ambiente Padrão (Padrão) ou Ambiente Enriquecido (EE), mantidos por 38 dias. Cada grupo foi subdividido dependendo do tipo de estresse crônico: Controle (sem estresse), Isolamento Social (por 38 dias) e Estresse Crônico Variado (do dia 28 ao dia 37). Ao fim do tempo experimental (dia 38) os ratos foram avaliados quanto ao comportamento emocional pelos testes de labirinto em cruz elevado (LCE) e claro/escuro (TCE) e sensibilidade nociceptiva pelo teste da placa quente (a qual foi realizada em duas etapas, sendo a primeira medida no dia 0 e outra no dia 38). A eutanásia dos ratos ocorreu no dia 39, para coleta do encéfalo para análise da imunorreatividade à óxido nítrico sintase neuronal (nNOS). Levando-se em consideração o comportamento emocional e a sensibilidade nociceptiva, os diferentes tipos de estresse crônico diminuíram a porcentagem de tempo, a frequência de entrada e a exploração da extremidade dos braços abertos e na frequência de mergulho de cabeça no teste do LCE, apesar de não alterar a sensibilidade nociceptiva. Por outro lado, o enriquecimento ambiental aumentou a porcentagem de tempo, a frequência de entrada e a exploração da extremidade dos braços abertos no teste do LCE, apesar de não alterar a sensibilidade nociceptiva. Foi observado aumento da imunorreatividade à nNOS na formação hipocampal em diferentes tipos de estresse crônico. Em particular, na região de CA3 houve interação significante entre os fatores estresse por isolamento social e ambiente de manutenção. Deste modo, os resultados obtidos neste trabalho sugerem que a formação hipocampal desempenha importante função no efeito ansiogênico exercido pelos diferentes tipos de estressores crônicos (aqui representados pelo isolamento social e pelo estresse crônico variado) provavelmente pela ativação do sistema nitrérgico e sugere-se que o enriquecimento possa prevenir o comportamento do tipo ansioso. / Adaptive responses to stress may be accompanied by changes in emotional behaviors, in particular related to fear and anxiety, as well as changes in pain sensitivity. Furthermore, the role of nitric oxide in brain areas related to defensive behavior has been investigated. Although several evidences have shown that environmental enrichment improves memory processes, learning and nociceptive responses, the relationship between chronic stress and the advantages of using environmental enrichment is still poorly investigated. The present study aimed to investigate whether environmental enrichment promotes alteration of the emotional behavior, nociceptive sensitivity, as well as immunoreactivity to neuronal nitric oxide synthase (nNOS) in the central nucleus of the amygdala, hippocampal formation and dorsolateral periaqueductal gray matter in rats submitted to social isolation stress or chronic unpredictable stress and reared in enriched environment or standard environment. Male Wistar rats (~ 70g) were randomly divided into two major experimental groups: Standard Environment (Standard) or Enriched Environment (EE), maintained for 38 days. Each group was subdivided according to the type of chronic stress: Control (without stress), Social Isolation (for 38 days) and Chronic Unpredictable Stress (from day 28 to day 37). At the end of the experimental time (day 38), the rats were evaluated for emotional behavior by elevated plus maze (EPM) and light/dark box (LDBT) tests and nociceptive sensitivity by the hot plate test (which was performed in two steps , the first being measured on day 0 and the other on day 38). Euthanasia of rats occurred on day 39, to collect the brain for nNOS immunoreactivity analysis. Taking into account emotional behavior and nociceptive sensitivity, the different types of chronic stress decreased the percentage of time, the frequency of entry of the open arms, end-arm exploration and the head dipping frequency in the EPM, despite of not altering the nociceptive sensitivity. On the other hand, environmental enrichment increased the percentage of time, the frequency of entry of the open arms and the end arm-exploration in the EPM test, although it did not alter the nociceptive sensitivity. Increased immunoreactivity to nNOS in hippocampal formation was observed in different types of chronic stress. In particular, in the CA3 region there was a significant interaction between stress factors due to social isolation and maintenance environment. Thus, the results obtained in this study suggest that hippocampal formation plays an important role in the anxiogenic effect exerted by the different types of chronic stressors (represented here by social isolation and by chronic chronic stress) probably due to the activation of the nitrergic system and it is suggested that environmental enrichment can prevent of anxiety-like behavior.

Respostas adaptativas ao estresse podem ser acompanhadas por alterações nos comportamentos emocionais, em particular relacionados com medo e ansiedade, bem como alterações na sensibilidade dolorosa. Ainda, tem sido investigado o papel do óxido nítrico em áreas encefálicas relacionadas ao comportamento defensivo. Embora várias evidências têm demonstrado que o enriquecimento ambiental promove melhora nos processos de memória, no aprendizado e em respostas nociceptivas, a relação entre o estresse crônico e as vantagens da utilização do enriquecimento ambiental ainda são pouco investigadas. O presente estudo teve como objetivo investigar se o enriquecimento ambiental promove alteração do comportamento emocional, da sensibilidade nociceptiva, bem como na imunorreatividade à nNOS no núcleo central da amígdala, na formação hipocampal e na região dorsolateral da substância cinzenta periaquedutal, em ratos submetidos ao estresse por isolamento social ou estresse crônico variado e mantidos em ambiente enriquecido ou sem enriquecimento. Ratos machos Wistar (~70g) foram divididos aleatoriamente em dois grandes grupos experimentais: Ambiente Padrão (Padrão) ou Ambiente Enriquecido (EE), mantidos por 38 dias. Cada grupo foi subdividido dependendo do tipo de estresse crônico: Controle (sem estresse), Isolamento Social (por 38 dias) e Estresse Crônico Variado (do dia 28 ao dia 37). Ao fim do tempo experimental (dia 38) os ratos foram avaliados quanto ao comportamento emocional pelos testes de labirinto em cruz elevado (LCE) e claro/escuro (TCE) e sensibilidade nociceptiva pelo teste da placa quente (a qual foi realizada em duas etapas, sendo a primeira medida no dia 0 e outra no dia 38). A eutanásia dos ratos ocorreu no dia 39, para coleta do encéfalo para análise da imunorreatividade à óxido nítrico sintase neuronal (nNOS). Levando-se em consideração o comportamento emocional e a sensibilidade nociceptiva, os diferentes tipos de estresse crônico diminuíram a porcentagem de tempo, a frequência de entrada e a exploração da extremidade dos braços abertos e na frequência de mergulho de cabeça no teste do LCE, apesar de não alterar a sensibilidade nociceptiva. Por outro lado, o enriquecimento ambiental aumentou a porcentagem de tempo, a frequência de entrada e a exploração da extremidade dos braços abertos no teste do LCE, apesar de não alterar a sensibilidade nociceptiva. Foi observado aumento da imunorreatividade à nNOS na formação hipocampal em diferentes tipos de estresse crônico. Em particular, na região de CA3 houve interação significante entre os fatores estresse por isolamento social e ambiente de manutenção. Deste modo, os resultados obtidos neste trabalho sugerem que a formação hipocampal desempenha importante função no efeito ansiogênico exercido pelos diferentes tipos de estressores crônicos (aqui representados pelo isolamento social e pelo estresse crônico variado) provavelmente pela ativação do sistema nitrérgico e sugere-se que o enriquecimento possa prevenir o comportamento do tipo ansioso. / Adaptive responses to stress may be accompanied by changes in emotional behaviors, in particular related to fear and anxiety, as well as changes in pain sensitivity. Furthermore, the role of nitric oxide in brain areas related to defensive behavior has been investigated. Although several evidences have shown that environmental enrichment improves memory processes, learning and nociceptive responses, the relationship between chronic stress and the advantages of using environmental enrichment is still poorly investigated. The present study aimed to investigate whether environmental enrichment promotes alteration of the emotional behavior, nociceptive sensitivity, as well as immunoreactivity to neuronal nitric oxide synthase (nNOS) in the central nucleus of the amygdala, hippocampal formation and dorsolateral periaqueductal gray matter in rats submitted to social isolation stress or chronic unpredictable stress and reared in enriched environment or standard environment. Male Wistar rats (~ 70g) were randomly divided into two major experimental groups: Standard Environment (Standard) or Enriched Environment (EE), maintained for 38 days. Each group was subdivided according to the type of chronic stress: Control (without stress), Social Isolation (for 38 days) and Chronic Unpredictable Stress (from day 28 to day 37). At the end of the experimental time (day 38), the rats were evaluated for emotional behavior by elevated plus maze (EPM) and light/dark box (LDBT) tests and nociceptive sensitivity by the hot plate test (which was performed in two steps , the first being measured on day 0 and the other on day 38). Euthanasia of rats occurred on day 39, to collect the brain for nNOS immunoreactivity analysis. Taking into account emotional behavior and nociceptive sensitivity, the different types of chronic stress decreased the percentage of time, the frequency of entry of the open arms, end-arm exploration and the head dipping frequency in the EPM, despite of not altering the nociceptive sensitivity. On the other hand, environmental enrichment increased the percentage of time, the frequency of entry of the open arms and the end arm-exploration in the EPM test, although it did not alter the nociceptive sensitivity. Increased immunoreactivity to nNOS in hippocampal formation was observed in different types of chronic stress. In particular, in the CA3 region there was a significant interaction between stress factors due to social isolation and maintenance environment. Thus, the results obtained in this study suggest that hippocampal formation plays an important role in the anxiogenic effect exerted by the different types of chronic stressors (represented here by social isolation and by chronic chronic stress) probably due to the activation of the nitrergic system and it is suggested that environmental enrichment can prevent of anxiety-like behavior.

Despite very extensive studies on molecular mechanisms of learning and memory formation it is little known about individual variation in the learning skills within a random animal population and about the differences in the brain biochemistry behind this variation. In the present study, we have focused on the expression and distribution of nitric oxide synthase (NOS), one of the molecules implemented in activity-dependent neuroplasticity, in the rat hippocampus, the structure critical for episodic memory in humans and animals. The aim of the present study was to investigate the differences in expression of three different NOS isoforms: neural (n), epithelial (e), and inducible (i), in four hippocampal subregions (CA1, CA3, DG, and hilus) between Wistar rats classified on the basis of their performance in partially baited 12-arm radial maze as &ldquo / good&rdquo / and &ldquo / poor&rdquo / learners. The NOS isoforms were visualized on coronal hippocampal sections using fluorescent immunohistochemistry technique and n- and eNOS images were processed using ImageJ software, while iNOS immunoreactivity (IR) was assessed by counting immunoreactive cells. In this study, overall hippocampal levels of nNOS were significantly higher than those of eNOS and iNOS. The level of n and eNOS was higher in CA1 compared to DG/hilus areas, but lower than that in CA3 region. The expression of iNOS was the highest in CA1 and the lowest in hilus region. nNOS IR was significantly higher in &ldquo / poor&rdquo / than in &ldquo / good&rdquo / learners but only in CA1 region. No significant between-group differences were found in eNOS expression. iNOS expression was higher in &ldquo / poor&rdquo / learners but it did not reach the required significance level.

The activity of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were regulated by kinase through phosphorylation. The cyclin-dependent kinase 5 (Cdk5) by associating with its neuron-specific activator p35 has been demonstrated to be essential for neurodegenerative neuronal death. This study focuses on the functional regulation of nNOS and eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. We found that nNOS associated with Cdk5 by immunoprecipitation (IP) and in vitro phosphorylated by Cdk5 by autoradiograph. Nitrite (NO2-) production was significantly reduced in Cdk5 over-expressing N18 cells, suggested that Cdk5 down-regulated nNOS enzymatic activity. In addition, Cdk5 phosphorylated eNOS both in vitro and in vivo on Ser 113, and the Cdk5 inhibitor roscovitine suppressed the phosphorylation of eNOS. Interaction of wild-type eNOS and S113A mutant eNOS with Cdk5 was observed in co-immunoprecipitation experiments. Co-expression of S113A eNOS and Cdk5/p35 resulted in 2-fold enhancement nitrite (NO2-) generation than co-expression of eNOS and Cdk5/p35 in SH-SY5Y cells. These data indicate that Cdk5 phosphorylated nNOS and eNOS, as well as down regulated nNOS and eNOS activity. Our results supposed that Cdk5 associated with and regulated the activity of nNOS and eNOS through phosphorylation.