Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado-Joseph disease

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.Fundação para a Ciência e Tecnologia through the projects [FEDER/FCT, POCI/SAU-MMO/60412/2004], [PTDC/SAU-GMG/64076/2006]. This work was supported by Fundação para a Ciência e Tecnologi

Nucleus accumbens microcircuit underlying D2-MSN-Driven increase in motivation

Accepted manuscriptThe nucleus accumbens (NAc) plays a central role in reinforcement and motivation. Around 95% of the NAc neurons are medium spiny neurons (MSNs), divided into those expressing dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R). Optogenetic activation of D2-MSNs increased motivation, whereas inhibition of these neurons produced the opposite effect. Yet, it is still unclear how activation of D2-MSNs affects other local neurons/interneurons or input terminals and how this contributes for motivation enhancement. To answer this question, in this work we combined optogenetic modulation of D2-MSNs with in loco pharmacological delivery of specific neurotransmitter antagonists in rats. First, we showed that optogenetic activation of D2-MSNs increases motivation in a progressive ratio (PR) task. We demonstrated that this behavioral effect relies on cholinergic-dependent modulation of dopaminergic signalling of ventral tegmental area (VTA) terminals, which requires D1R and D2R signalling in the NAc. D2-MSN optogenetic activation decreased ventral pallidum (VP) activity, reducing the inhibitory tone to VTA, leading to increased dopaminergic activity. Importantly, optogenetic activation of D2-MSN terminals in the VP was sufficient to recapitulate the motivation enhancement. In summary, our data suggests that optogenetic stimulation of NAc D2-MSNs indirectly modulates VTA dopaminergic activity, contributing for increased motivation. Moreover, both types of dopamine receptors signalling in the NAc are required in order to produce the positive behavioral effects.This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Part of the work was supported by the Janssen Neuroscience Prize (1st edition) and by a BIAL Grant (30/2016). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038 and of project POCI-01-0145-FEDER-016428info:eu-repo/semantics/publishedVersio

Preclinical assessment of mesenchymal-stem-cell-based therapies in spinocerebellar ataxia type 3

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.This research was funded by the National Ataxia Foundation (NAF) and by Portuguese national funds, through the Foundation for Science and Technology (FCT)—projects UIDB/50026/2020, UIDP/50026/2020, POCI-01-0145-FEDER-029206, and through the Santa Casa Neuroscience Awards (Santa Casa da Misericórdia Lisboa)—project MC-04/17. Additionally, this project was funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). S.C.S. received an individual fellowship within the project TUBITAK/0007/2014. The FCT funded individual fellowships to J.S C., A.N.-C., B.M.- P., F.G.T., R.L., S.M., N.A.S., C.S.-C., and S.D.-S. (SFRH/BD/140624/2018, SFRH/BPD/118779/2016, SFRH/BD/120124/2016, SFRH/BPD/118408/2016, PD/BDE/127836/2016, CEECIND/01902/2017, CEECIND/04794/2017, CEECIND/03887/2017, and CEECIND/00685/2020)

Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction

Emerging studies implicate Tau as an essential mediator of neuronal atrophy and cognitive impairment in Alzheimer's disease (AD), yet the factors that precipitate Tau dysfunction in AD are poorly understood. Chronic environmental stress and elevated glucocorticoids (GC), the major stress hormones, are associated with increased risk of AD and have been shown to trigger intracellular Tau accumulation and downstream Tau-dependent neuronal dysfunction. However, the mechanisms through which stress and GC disrupt Tau clearance and degradation in neurons remain unclear. Here, we demonstrate that Tau undergoes degradation via endolysosomal sorting in a pathway requiring the small GTPase Rab35 and the endosomal sorting complex required for transport (ESCRT) machinery. Furthermore, we find that GC impair Tau degradation by decreasing Rab35 levels, and that AAV-mediated expression of Rab35 in the hippocampus rescues GC-induced Tau accumulation and related neurostructural deficits. These studies indicate that the Rab35/ESCRT pathway is essential for Tau clearance and part of the mechanism through which GC precipitate brain pathology.work was supported by NIH grants R01NS080967and R21MH 104803 to C.L.W., Portuguese Foundation for Science & Technology (FCT) PhD fellowships to J. Vaz-Silva and T. Meira (PD/BD/105938/2014; PD/BD/113700/2015, respectively), and the following grants to I.S.: FCT Investigator grant IF/01799/2013, the Portuguese North Regional Operational Program (ON.2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER), the Project Estratégico co-funded by FCT (PEst-C/SAU/LA 0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01 -0124-FEDER-037298) as well as the project NORTE- 01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio

Constitutive deficiency of the neurogenic hippocampal modulator AP2γ promotes anxiety-like behavior and cumulative memory deficits in mice from juvenile to adult periods

The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.H2020 -“la Caixa” Foundation(101003187

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Papel dos neurónios do núcleo accumbens que expressam recetores de dopamina D1 e D2 na recompensa e motivação

Tese de Doutoramento em Ciências da SaúdeThe reward circuit, mainly comprised by dopaminergic fibres arising from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is one of the most widely studied brain circuits due to its role in mediating reward perception and reinforcement. Dopamine signals from the VTA are essentially decoded by two major populations of GABAergic medium spiny neurons (MSNs) in the NAc, which are canonically segregated into those expressing dopamine receptor D1 (D1R) (D1-MSNs), representing the direct pathway, and those expressing the dopamine receptor D2 (D2R) (D2-MSNs), comprising the indirect pathway. For many years a functional dichotomy between the direct and the indirect pathways has been assumed in motor control, and lately the same was proposed for valenced stimuli: whereas D1-MSNs mediate reward and reinforcement, D2-MSNs have been associated with aversion and punishment. Interestingly, previous work from our team has shown that in utero exposure to high levels of the synthetic glucocorticoid (GC) dexamethasone (iuGC), induces a decrease in dopaminergic innervation of the NAc together with epigenetic and expression changes of D2R (but not other dopamine receptors). At a behavioural level, iuGC animals presented anhedonia and increased drug-seeking behaviour, suggestive of an imbalance of the reward circuit. In this thesis we show that iuCG exposure negatively affects motivational drive towards natural rewards and induces prominent D2R changes in different brain regions. Importantly, normalization of dopamine levels by systemic administration of levodopa rescues this phenotype, and this seems dependent on D2R but not D1R signalling. To get further insight on the role of D1- and D2-MSNs in behaviour, we used optogenetic tools to selectively activate (and inhibit) these neuronal populations during reward-related behaviours. Contrary to the dominant perspective in the field, our results suggest that the classic view of D1-D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2-MSNs may play a more prominent pro-reward role than originally anticipated. We demonstrate that both D1- and D2-MSNs are recruited during tasks requiring different degrees of motivation, and that optogenetic activation of either neuronal population enhances motivation in mice. Because data regarding D2-MSNs was paradoxical, we used a different methodological approach in rats, and further show that activating NAc D2-MSNs increases motivation and is able to induce behavioural conditioning, i.e, is reinforcing. Conversely, optogenetic inhibition of D2-MSNs decreases motivation and induces aversion. These results were extended to the iuGC model, in which we show that brief D2-MSNs activation rescues their motivational deficits. In vivo electrophysiological recordings in downstream target regions confirmed the selectivity of the pathway activation and further supported the behavioural data. We next performed hybrid experiments in which optogenetic activation of D2-MSNs was coupled with pharmacological manipulation, and showed that the increase in motivation required acetylcholine-induced dopamine release from VTA dopaminergic terminals into the NAc, and that both D1R and D2R are essential for this behavioural effect. Lastly, we show for the first time that NAc D2-MSNs can bi-directionally modulate valenced behaviours depending on their neuronal activation pattern: brief stimulation of these neurons is reinforcing whereas prolonged D2-MSNs stimulation is aversive. In conclusion, with this work we prove for the first time that the classic view of D1- and D2- MSNs functional antagonism is not true for all dimensions of reward behaviours and that D2-MSNs play a more pro-motivation/reward role than initially anticipated.O sistema de recompensa, constituído principalmente por fibras dopaminérgicas originárias da área tegmental ventral (VTA) para o núcleo accumbens (NAc), é um dos circuitos do cérebro mais estudados devido ao seu papel na percepção de recompensas e no reforço. Os sinais dopaminérgicos do VTA são principalmente descodificados por duas populações GABAérgicas de neurónios espinhosos médios (MSNs) no NAc, que são tipicamente segregados nos que expressam o recetor de dopamina D1 (D1R) (D1-MSNs), que constituem a via direta, e aqueles que expressam o receptor de dopamina D2 (D2R) (D2-MSNs), representando a via indireta. Ao longo de vários anos foi proposta uma dicotomia funcional entre a via direta e a via indireta no que respeita ao controlo motor, e, recentemente, o mesmo foi sugerido para estímulos com valência: enquanto que os D1-MSNs medeiam recompensa e reforço positivo, os D2-MSNs têm sido associados a aversão e punição ou reforço negativo. Trabalho realizado anteriormente pelo nosso grupo demonstrou que a exposição in utero a elevados níveis do glicocorticóide (GC) sintético dexametasona (iuGC), induzia uma diminuição da inervação dopaminérgica do NAc, bem como alterações epigenéticas e de expressão do D2R (mas não de outros recetores de dopamina). A nível comportamental, os animais iuGC apresentavam anedonia e aumento de drug-seeking behavior, o que sugeria um desequilíbrio do sistema de recompensa. Nesta tese mostrámos que a exposição a iuCG afeta negativamente a motivação para obter recompensas naturais, e induz alterações proeminentes nos D2R em diferentes regiões do cérebro. A normalização dos níveis de dopamina através da administração sistémica de levodopa, foi capaz de resgatar este fenótipo, dependendo da sinalização via D2R (mas não D1R). De forma a compreender melhor qual o papel dos D1- e D2-MSNs no comportamento, usamos a técnica de optogenética para seletivamente ativar (ou inibir) populações neuronais durante tarefas de recompensa. Os nossos resultados sugerem que a visão clássica de antagonismo funcional entre D1-D2, não é válida para todas as dimensões do comportamento de recompensa, e que os D2-MSNs podem desempenhar um papel pró-recompensa mais saliente do que inicialmente previsto. Demonstramos que tanto os D1- como os D2-MSNs são recrutados durante tarefas que requerem diferentes graus de motivação, e que a ativação optogenética de qualquer uma destas populações neuronais aumenta a motivação em murganhos. Uma vez que os dados sobre os D2- MSNs eram paradoxais, usámos uma abordagem metodológica diferente em ratos. Confirmamos que a ativação dos D2-MSNs no NAc aumenta a motivação e, além disso, mostramos que induz condicionamento, ou seja, é recompensador. Por outro lado, a inibição optogenética dos D2-MSNs diminuiu a motivação e induziu aversão. Mostramos ainda que ativação breve dos D2-MSNs durante a tarefa reverte os défices motivacionais dos animais iuGC. Gravações electrofisiológicas in vivo em regiões alvo a jusante do NAc confirmaram a seletividade da ativação da via. De seguida, realizámos experiências híbridas em que a ativação optogenética dos D2-MSNs foi emparelhada com manipulação farmacológica. O aumento na motivação induzida pela activação dos D2-MSNs depende da libertação de dopamina mediada por acetilcolina no NAc pelos terminais dopaminérgicos do VTA. Tanto os D1R como os D2R são essenciais para este efeito comportamental. Por fim, mostrámos que os D2-MSNs do NAc podem modular bidireccionalmente comportamentos de valência, dependendo do seu padrão de ativação neuronal: a estimulação breve destes neurónios é recompensadora, enquanto que uma estimulação mais prolongada é aversiva. Em conclusão, com este trabalho provámos pela primeira vez que a visão clássica de antagonismo funcional dos D1- e D2-MSNs não é verdade para todas as dimensões de comportamentos de recompensa e que os D2-MSNs desempenham um papel mais prómotivacional do que o antecipado.The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho. Financial support was provided by a PhD grant (SFRH/BD/51992/2012) from the FCT - Foundation for Science and Technology -, by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145-FEDER-007038; and by the project NORTE- 01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Pavlovian and instrumental behaviour in a model of prenatal exposure to glucocorticoids : an integrative approach

Dissertação de mestrado em Ciências da SaúdeExposure to early life stress (ELS) or elevated levels of glucocorticoids (GC) has an enduring effect in the central nervous system (CNS), which can lead to maladaptive behaviour in adult life. Several studies suggest that ELS increases the vulnerability to develop psychiatric disorders, such as schizophrenia, depression or addiction. Synthetic GCs such as dexamethasone (DEX) are frequently prescribed in clinics, to ensure foetal lung maturation in preterm risk pregnancies. Despite its beneficial effects in the lungs, it has been shown that GCs can lead to long-lasting effects on brain function. Interestingly, neuronal dopamine (DA) populations seem to be particularly vulnerable to elevated levels of GCs. For example, adult rats exposed in utero to GC (iuGC) showed significant alterations in DAergic reward mesolimbic circuit, namely in the ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala, alterations that occurred in parallel with increased drug-seeking behaviour. To further assess the integrity of the reward circuitry in iuGC animals, we decided to analyse their performance in the Pavlovian-to-instrumental (PIT) test, a paradigm that involves the ability of environmental stimuli to activate the memory of sensory-specific features of the outcome/reward used in instrumental conditioning. Interestingly, iuGC animals were impaired in both non-selective and selective protocols of PIT. Since PIT performance strongly depends on prefrontal cortex (PFC), we decided to perform a morphometric analysis of this brain region. iuGC treatment induced significant changes in the volume, cell numbers and neuronal morphology of the medial PFC and less so on the orbitofrontal cortex. Molecular analysis of these brain regions further revealed alterations on the expression levels of dopamine receptors (Drd) 1 and 2 and in molecules important for neuronal remodelling. Altogether, our results suggest that iuGC exposure alters the reward circuitry, impairing PIT performance. This reduction in the incentive salience attributed to cues predicting natural rewards can ultimately contribute for an increased vulnerability for non-natural rewards, and eventually explain the observed increased sensitivity and preference to drugs of abuse.A exposição a stress pré-natal (SPN) ou a níveis elevados de glucocorticóides (GC), tem um impacto duradouro no sistema nervoso central (SNC), o que pode levar a alterações comportamentais na vida adulta. Vários estudos sugerem que o SPN aumenta a vulnerabilidade para o desenvolvimento de doenças psiquiátricas, tais como a esquizofrenia, depressão ou adição. GCs sintéticos como a dexametasona (DEX) são frequentemente prescritos na prática clínica, para assegurar maturação pulmonar fetal em situações de gravidez com risco de parto pré-termo. Apesar dos efeitos benéficos nos pulmões, vários estudos apontam para um efeito deletério no SNC. Curiosamente, as populações neuronais dopaminérgicas parecem ser particularmente vulneráveis a elevados níveis de GCs. Por exemplo, ratos adultos expostos in utero a GCs (iuGC) exibem alterações significativas no circuito dopaminérgico de recompensa – a via mesolimbica, nomeadamente na área tegmental ventral, no núcleo accumbens e amígdala; alterações essas associadas a um aumento de vulnerabilidade para drogas de abuso. A fim de avaliar a integridade do sistema de recompensa, analisamos a performance dos animais iuGC no teste Pavlovian-to-instrumental (PIT) transfer, que é um paradigma que envolve a capacidade de estímulos ambientais ativarem a memória de características sensoriais de recompensa no condicionamento instrumental. Uma vez que o PIT depende em grande parte do córtex pré-frontal (PFC), decidimos analisar esta região a nível estereológico e morfológico. Verificamos que a exposição a iuGC induz alterações significativas no volume, número de células e morfologia do PFC medial, com menor impacto no córtex orbitofrontal. Análise molecular destas regiões revelou ainda alterações na expressão dos receptores de dopamina 1 e 2, e de moléculas importantes para a remodelação neuronal. De uma forma global, os nossos resultados sugerem que exposição a iuGC altera o sistema de recompensa, inibindo a performance do PIT. Esta redução na motivação atribuída a pistas que predizem recompensas naturais pode, em último caso, contribuir para uma vulnerabilidade acrescida para recompensas não-naturais (drogas), e eventualmente explicar o aumento da sensibilização e preferência para drogas de abuso observada nestes animais