10 research outputs found
In vivo identification of brain structures functionally involved in spatial learning and strategy switch
Spatial learning is a complex behavior which includes, among others, encoding of space, sensory and motivational processes, arousal and locomotor performance. Today, our view on spatial navigation is largely hippocampus-centrist. Less is known about the involvement of brain structures up- and downstream, or out of this circuit. Here, I provide the first in vivo assessment of the neural matrix underlying spatial learning, using functional manganese-enhanced MRI (MEMRI) and voxel-wise whole brain analysis. Mice underwent place-learning (PL) vs. response-learning (RL) in the water cross maze (WCM) and its readout was correlated to the Mn2+ contrasts. Thus, I identified structures involved in spatial learning largely overlooked in the past, due to methods focused on region of interest (ROI) analyses. These structures include several sensory-related structures and differ between place-learners and response-learners, with the former (PL) comprising mostly structures involved in different properties of visual processing, such as horizontal gaze (e.g. nucleus prepositus) and saccade (e.g. fastigial nucleus), or provide vision-input and eye movement information from parahippocampal (e.g. presubiculum, perirhinal, postrhinal and ectorhinal areas) and other regions (e.g. orbital area, superior colliculus and vestibular ocular-reflex from the vestibular nucleus) likely to head-direction, grid- and place-cells; and the latter (RL) presenting structures related to more basic rodent sensory computations, like odor (e.g main and accessory olfactory bulb, cortical amygdala, piriform, endopiriform and postpiriform areas) and acoustic stimuli representation (e.g. auditory area, nucleus of the lateral lemniscus and superior olivary complex), or sensory-motor properties, such as body representation (e.g. somatosensory area – upper limbs) and head-direction signal. Add-on experiments pointed to preferential Mn2+ accumulation towards projection terminals, suggesting that our mapping was mostly formed by projection sites of the originally activated structures. This is corroborated by in-depth analysis of MEMRI data after WCM learning showing mostly downstream targets of the hippocampus. These differ between fornical afferences from vCA1 and direct innervation from dCA1/iCA1 (for PL), and structures along the longitudinal association bundle originating in vCA1 (for RL).
To elucidate the pattern of Mn2+ accumulation seen on the scans, I performed c-fos expression analyses following learning in the WCM. This helped me identify the structures initially activated during spatial learning and its underlying connectivity to establish the matrix.
Finally, to test the causal involvement of selected structures from our previous findings I inhibited them (through DREADDs) while mice performed the WCM task. I also focused on the causal involvement of the vHPC-mPFC circuit on strategy switch during WCM learning.
I believe that this study might shed light into new brain structures involved in spatial learning and strategy switch and complement the current knowledge on these circuits’ connectivity. Moreover, I elucidated some functional mechanisms of MEMRI, clarifying the interpretation of data obtained with this method and its possible future applications
In vivo identification of brain structures functionally involved in spatial learning and strategy switch
Spatial learning is a complex behavior which includes, among others, encoding of space, sensory and motivational processes, arousal and locomotor performance. Today, our view on spatial navigation is largely hippocampus-centrist. Less is known about the involvement of brain structures up- and downstream, or out of this circuit. Here, I provide the first in vivo assessment of the neural matrix underlying spatial learning, using functional manganese-enhanced MRI (MEMRI) and voxel-wise whole brain analysis. Mice underwent place-learning (PL) vs. response-learning (RL) in the water cross maze (WCM) and its readout was correlated to the Mn2+ contrasts. Thus, I identified structures involved in spatial learning largely overlooked in the past, due to methods focused on region of interest (ROI) analyses. These structures include several sensory-related structures and differ between place-learners and response-learners, with the former (PL) comprising mostly structures involved in different properties of visual processing, such as horizontal gaze (e.g. nucleus prepositus) and saccade (e.g. fastigial nucleus), or provide vision-input and eye movement information from parahippocampal (e.g. presubiculum, perirhinal, postrhinal and ectorhinal areas) and other regions (e.g. orbital area, superior colliculus and vestibular ocular-reflex from the vestibular nucleus) likely to head-direction, grid- and place-cells; and the latter (RL) presenting structures related to more basic rodent sensory computations, like odor (e.g main and accessory olfactory bulb, cortical amygdala, piriform, endopiriform and postpiriform areas) and acoustic stimuli representation (e.g. auditory area, nucleus of the lateral lemniscus and superior olivary complex), or sensory-motor properties, such as body representation (e.g. somatosensory area ? upper limbs) and head-direction signal. Add-on experiments pointed to preferential Mn2+ accumulation towards projection terminals, suggesting that our mapping was mostly formed by projection sites of the originally activated structures. This is corroborated by in-depth analysis of MEMRI data after WCM learning showing mostly downstream targets of the hippocampus. These differ between fornical afferences from vCA1 and direct innervation from dCA1/iCA1 (for PL), and structures along the longitudinal association bundle originating in vCA1 (for RL). To elucidate the pattern of Mn2+ accumulation seen on the scans, I performed c-fos expression analyses following learning in the WCM. This helped me identify the structures initially activated during spatial learning and its underlying connectivity to establish the matrix. Finally, to test the causal involvement of selected structures from our previous findings I inhibited them (through DREADDs) while mice performed the WCM task. I also focused on the causal involvement of the vHPC-mPFC circuit on strategy switch during WCM learning. I believe that this study might shed light into new brain structures involved in spatial learning and strategy switch and complement the current knowledge on these circuits? connectivity. Moreover, I elucidated some functional mechanisms of MEMRI, clarifying the interpretation of data obtained with this method and its possible future applications
Whole-brain R1 predicts manganese exposure and biological effects in welders
Manganese (Mn) is a neurotoxicant that, due to its paramagnetic property, also functions as a magnetic resonance imaging (MRI) T1 contrast agent. Previous studies in Mn toxicity have shown that Mn accumulates in the brain, which may lead to parkinsonian symptoms. In this article, we trained support vector machines (SVM) using whole-brain R1 (R1 = 1/T1) maps from 57 welders and 32 controls to classify subjects based on their air Mn concentration ([Mn]Air), Mn brain accumulation (ExMnBrain), gross motor dysfunction (UPDRS), thalamic GABA concentration (GABAThal), and total years welding. R1 was highly predictive of [Mn]Air above a threshold of 0.20 mg/m3 with an accuracy of 88.8% and recall of 88.9%. R1 was also predictive of subjects with GABAThal having less than or equal to 2.6 mM with an accuracy of 82% and recall of 78.9%. Finally, we used an SVM to predict age as a method of verifying that the results could be attributed to Mn exposure. We found that R1 was predictive of age below 48 years of age with accuracies ranging between 75 and 82% with recall between 94.7% and 76.9% but was not predictive above 48 years of age. Together, this suggests that lower levels of exposure (< 0.20 mg/m3 and < 18 years of welding on the job) do not produce discernable signatures, whereas higher air exposures and subjects with more total years welding produce signatures in the brain that are readily identifiable using SVM
Dissecting the neuronal basis of threat responding in mice
Environmental threats demand adaptive defensive responses of an organism that ensure its survival. Extreme stressors, however, can unbalance stress homeostasis and lead to long-term changes that impair appropriate defensive behaviors and emotional responses. In my thesis, I assessed (1) the interaction of two stress-related neuromodulatory systems, (2) the effects of a traumatic incident on brain volume and hyperarousal, and (3) sonic vocalization as a defensive behavior in mice, and discussed the topics in three independent studies.In the first study, I evaluated the interaction of two regulatory systems with respect to fear, anxiety, and trauma-related behaviors. Although the endocannabinoid and the corticotropin-releasing factor (CRF) systems are well described in modulating stressrelatedresponses, the direct interaction of both systems remained poorly understood. The generation of a new conditional knockout mouse line that selectively lacked the expression of the cannabinoid type 1 (CB1) receptor in CRF-positive neurons presented no differences in various tests of fear and anxiety-related behaviors under basal conditions or after a traumatic event. Also stress hormone levels were unaffected. However, male knockout animals exhibited a significantly increased acoustic startle response thus suggesting a specific involvement of CB1-CRF interactions in controlling arousal.In the second study, I assessed the consequences of a traumatic experience on behavior and grey matter volume in mice. Whole-brain deformation-based morphometry (DBM) by means of magnetic resonance imaging (MRI) after incubation of a traumatic incident showed changes in the dorsal hippocampus and the reticular nucleus. Using the severity of hyperarousal as regressor for cross-sectional volumetric differences between traumatized mice and controls revealed a negative correlation with the dorsal hippocampus. Further, longitudinal analysis including volumetric measurements before and after the traumatic incident showed that volume reductions in the globus pallidus reflect trauma-related changes in hyperarousal severity.In the third study, I characterized sonic vocalization as a defensive behavior in mice. Mice bred for high anxiety-related behavior (HAB) were found to have a high disposition to emit audible squeaks when taken by the tail which was not the case for any of the other five mouse lines tested. The calls emitted had a fundamental frequency of 3.8 kHz and were shown to be sensitive to anxiolytic but not panicolytic compounds. Manganese-enhanced MRI (MEMRI) scans pointed towards an increased tonic activity, among others, in the periaqueductal grey (PAG). Inhibition of the dorsal PAG by muscimol not only completely abolished sonic vocalization, but also reduced anxiety-like behavior. This suggests that sonic vocalization of mice is related to anxiety and controlled by the PAG. To explore the ecological relevance of defensive vocalization, I performed playback experiments with conspecifics and putative predators. Squeaks turned out to be aversive to HAB mice but became appetitive to both mice and rats when a stimulus mouse was present during playback.Collectively, the results of this thesis provide novel insights into fear and anxiety-related behaviors and shine light onto their mechanistic basis and ecological relevance
Caracterização das funções neurotróficas da proteína precursora de amilóide de Alzheimer
Doutoramento em BiomedicinaThe Amyloid Precursor Protein (APP) is a type 1 membrane glycoprotein,
mainly known as the precursor of the amyloid β-peptide, a central player in
Alzheimer’s disease. Nevertheless, APP has been established as a
neuromodulator of developing and mature nervous system. Alterations in the
level or activity of APP and APP fragments seem to play a critical role in
several neurodegenerative and neurodevelopment disorders. APP is a complex
molecule due to the intricate relationships between its intracellular trafficking,
posttranslational modifications, proteolytic cleavages, and multiple protein
interactors. Various studies currently address the physiological roles of APP
and its fragments, but there are contradictory results and missing pieces that
need further work. The main objective of this thesis was to contribute to the
characterization of the role of APP in neuronal differentiation. Particularly, we
focused on mechanisms mediated by APP, its fragment sAPP, and APP
phosphorylation at serine 655.
First, we characterized the APP protein in Retinoic Acid (RA)-induced SHSY5Y
cell differentiation. The comprehensive analysis of this model exposed a
biphasic temporal response: a first early phase (D0-D4), where a sAPP/APP
peak assists the emergence of new processes and their elongation into
neurites; and a second phase (D4-D8) when increased holoAPP protein levels
are necessary to sustain neuritic elongation and stabilization.
In line with our main aim, we subsequently characterized the relationship
between APP and the neurotrophic EGF-EGFR-ERK signaling pathway. We
showed, for the first time, that APP interacts with proEGF, and confirmed the
interaction with EGFR. Furthermore, we showed that combined APP and EGF
have a synergistic effect on neuronal-like differentiation, related to enhanced
ERK1/2 activation, and observed that APP modulates EGFR expression levels
and trafficking. Both ERK1/2 activation and EGFR seem to be modulated by
the APP S655 phosphorylation state, and phosphorylation at this residue
favours dendritogenesis in mice cortical neurons.
Finally, we focused on discovering APP protein interactors dependent on S655
phosphorylation and with a role in neuronal differentiation. SH-SY5Y
differentiated cells, overexpressing APPWt or S655 phosphomutants, were
used to immunoprecipitate the specific APP proteins and their respective
interacting partners, later identified by mass spectrometry. The
dephosphoS655 APP interactome was enriched in functions associated with
cytoskeleton organization, and these cells were particularly associated with
actin remodeling. The phosphoS655 APP interactome included proteins
involved in the regulation of survival and differentiation, and in various signaling
pathways, correlating well with an enhanced neurite outgrowth displayed by
these cells.
We hope that the knowledge here gathered can contribute to a better
comprehension of APP-driven neurotrophic roles and underlying mechanisms.A Proteína Precursora de Amilóide (APP) é uma proteína membranar mais
conhecida por ser precursora do péptido Amilóide β, tendo por isso um papel
central na doença de Alzheimer. Não obstante, a APP tem sido reconhecida
como neuromodulador do sistema nervoso central. Alterações nos níveis ou na
atividade da APP e seus fragmentos estão implicadas em diferentes doenças
neurológicas. As relações entre o seu transporte intracelular, modificações
pós-traducionais, corte proteolítico, e proteínas com as quais interage são
complexas e multifacetadas. Talvez por isso, estudos focados no papel
fisiológico da APP apresentem resultados contraditórios e muitas questões em
aberto. O objetivo deste trabalho consistiu na caracterização do papel
fisiológico da APP na diferenciação neuronal. Particularmente, focámo-nos nos
mecanismos mediados pela APP e fragmento sAPP, e a fosforilação da APP
no resíduo serina 655.
Inicialmente, caracterizámos a proteína APP ao longo da diferenciação de
células SH-SY5Y com ácido retinóico (RA). A análise sistemática deste modelo
permitiu delimitar uma resposta bifásica: na primeira fase (D0-D4), um pico de
sAPP/APP acompanha o aparecimento de novos processos e o crescimento a
neurites; na segunda fase (D4-D8) o aumento nos níveis da APP suporta o
crescimento e manutenção das neurites.
Caracterizámos posteriormente a relação entre a APP e a via de sinalização
EGF-EGFR-ERK na diferenciação neuronal. Demonstrámos, pela primeira vez,
que a APP interage com o proEGF, e confirmámos a sua ligação ao EGFR.
Adicionalmente, observámos que a APP e o EGF têm um efeito sinérgico na
diferenciação tipo-neuronal e aumento da ativação da ERK1/2, e que a APP
afeta os níveis e transporte do EGFR. Estes mecanismos são modulados pela
fosforilação da APP na S655, que favorece a dendritogénese em neurónios
corticais de ratinho.
Por último, focámo-nos na identificação de proteínas interatoras da APP
dependentes da fosforilação em S655 e com função na diferenciação neuronal.
Usando células SH-SY5Y diferenciadas e a sobrexpressar a APPWt ou
fosfomutantes da S655, imunoprecipitámos as diferentes APPs e seus
interatores, posteriormente identificados por espectrometria de massa. O
interatoma da APP desfosforilada é enriquecido em funções associadas à
organização do citoesqueleto, levando a uma maior reorganização da actina. O
interatoma da APP fosforilada incluí proteínas envolvidas na regulação de
sobrevivência e diferenciação, e em várias vias de sinalização, o que se
correlaciona com o favorecimento de neurites nestas células.
Com este trabalho esperamos ter contribuído para uma melhor compreensão
do papel neurotrófico da APP e dos mecanismos subjacentes a este