Literatura académica sobre el tema "GCaMP6f"

<b><i>Introduction:</i></b> Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. <b><i>Methods:</i></b> We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. <b><i>Results:</i></b> Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. <b><i>Conclusion:</i></b> Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.

OBJECTIVECortical spreading depolarization (CSD) has been linked to poor clinical outcomes in the setting of traumatic brain injury, malignant stroke, and subarachnoid hemorrhage. There is evidence that electrocautery during neurosurgical procedures can also evoke CSD waves in the brain. It is unknown whether blood contacting the cortical surface during surgical bleeding affects the frequency of spontaneous or surgery-induced CSDs. Using a mouse neurosurgical model, the authors tested the hypothesis that electrocautery can induce CSD waves and that surgical field blood (SFB) is associated with more CSDs. The authors also investigated whether CSD can be reliably observed by monitoring the fluorescence of GCaMP6f expressed in neurons.METHODSCSD waves were monitored by using confocal microscopy to detect fluorescence increases at the cortical surface in mice expressing GCaMP6f in CamKII-positive neurons. The cortical surface was electrocauterized through an adjacent burr hole. SFB was simulated by applying a drop of tail vein blood to the brain through the same burr hole.RESULTSCSD waves were readily detected in GCaMP6f-expressing mice. Monitoring GCaMP6f fluorescence provided far better sensitivity and spatial resolution than detecting CSD events by observing changes in the intrinsic optical signal (IOS). Forty-nine percent of the CSD waves identified by GCaMP6f had no corresponding IOS signal. Electrocautery evoked CSD waves. On average, 0.67 ± 0.08 CSD events were generated per electrocautery episode, and multiple CSD waves could be induced in the same mouse by repeated cauterization (average, 7.9 ± 1.3 events; maximum number in 1 animal, 13 events). In the presence of SFB, significantly more spontaneous CSDs were generated (1.35 ± 0.37 vs 0.13 ± 0.16 events per hour, p = 0.002). Ketamine effectively decreased the frequency of spontaneous CSD waves (1.35 ± 0.37 to 0.36 ± 0.15 CSD waves per hour, p = 0.016) and electrocautery-stimulated CSD waves (0.80 ± 0.05 to 0.18 ± 0.08 CSD waves per electrocautery, p = 0.00002).CONCLUSIONSCSD waves are detected with far greater sensitivity and fidelity by monitoring GCaMP6f signals in neurons than by monitoring IOSs. Electrocautery reliably evokes CSD waves, and the frequency of spontaneous CSD waves is increased when blood is applied to the cortical surface. These experimental conditions recapitulate common scenarios in the neurosurgical operating room. Ketamine, a clinically available pharmaceutical agent, can block stimulated and spontaneous CSDs. More research is required to understand the clinical importance of intraoperative CSD.

Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a combination of recently developed optical tools to monitor neural population at the meso-scale level and to mould the cortical patterns of targeted neuronal population. Our goal was to investigate the propagation of neuronal activity over the mouse cortex that is triggered by optogenetic stimulation in the contralateral hemisphere. Towards this aim, we developed a wide-field fluorescence microscope that is characterized by a double illumination path allowing for the optogenetic stimulation of the transfected area in the left hemisphere and the simultaneous recording of cortical activity in the right hemisphere. The microscope was further implemented with a custom shutter in order to split the LED illumination path, resulting in a half-obscured field of view. By avoiding the spectral crosstalk between GCaMP6f and channelrhodopsin 2 (ChR2), this system offered the possibility of simultaneous “pumping and probing” of inter-hemispheric functional connectivity on Thy1-GCaMP6f mice.

Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities.

It is hypothesized that perinatal cerebellar injury leads to long-term functional deficits due to circuit dysmaturation. Using a novel integration of GCaMP6f fiber photometry with automated measurement of cerebellar behavior using the ErasmusLadder, we causally link cerebellar injury to altered Purkinje cell responses during maladaptive behavior. Chemogenetic inhibition of neonatal Purkinje cells is sufficient to phenocopy the effects of perinatal cerebellar injury. Our results uncover a direct link between perinatal cerebellar injury and activity-dependent maturation of cerebellar cortex.

Abstract Introduction Chronic sleep disruption (CSD) in young adult mice leads to phenotypes consistent with early (pre-plaque) Alzheimer’s Disease (AD), including increased Aβ and hippocampal neuron loss. Mechanisms underlying this injury are not known. Both acute sleep loss and AD activate cofilin, a regulator of actin dynamics. Activated cofilin (AC) in AD mouse models can impart neural injury, increase Aβ, and cofilin translocation to the mitochondria delays cytosolic Ca2+ clearance. We are critically testing the role of AC in chronic short sleep (CSS) and sleep fragmentation (SF) neural injury. Methods Synapse loss was studied using STED confocal microscopy and Imaris in CSS (n=9) and rested (n=10) mice. Synapses were identified as overlaps of pre- and postsynaptic densities. Percent area of cofilin was measured with FIJI. To further understand if and how wake-induced cofilin activation induces sleep-loss synapse and neural injury, we implanted AAV9CAMKII-GCaMP6f and then GRIN lenses, and later studied CAMKII calcium transients in CA1 of WT controls (n=4) and SF mice (n=4) by measuring GCaMP6f calcium transients. We developed a Shiny R application to analyze the frequency of Ca2+ spikes, ΔF/F0, and the rising and clearance patterns of spikes. To directly test cofilin’s role in delayed calcium clearance, we studied the calcium transients in hAPP mice (n=2) after injection of AAV-CAMKII-CofilinS3A to express AC and GCaMP6f. All data were analyzed with two-way ANOVA or unpaired t-tests. Results Results reveal significant synapse loss in CA1 of CSS mice (CSS=48.8±10.3; Rested=83.4±8.4), t(16)=2.63, p&lt;0.02, and increased cofilin activation (AC=19.8±3.41; Rested=8.76±1.95), t(16)=8.43, p&lt;0.0001. SF mice reveal an increase in NREM sleep firing rates, F(1,1)=22.0, p&lt;0.001. In contrast, hAPP-AC mice show significantly increased ΔF/F0, F(1,1)=356, p&lt;0.0001, prolonged calcium influx, F(1,1)=18.6, p&lt;0.02, and prolonged calcium clearance duration, F(1,1)=23.9, p&lt;0.01, but not increased firing frequencies. Conclusion CSS induces CA1 synapse loss and cofilin activation in WT mice. Increased CAMKII calcium ΔF/F0 occurs through different pathways in SF and AC, suggesting additional factors in CSD neural injury. Support (If Any) NIH AG054104; AG064231

Abstract INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ flux imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p &lt; 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p &lt; 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bear pertinence to the pathogenesis of neuro-cognitive decline and tumor associated epilepsy.

Plasmodium falciparum e Toxoplasma gondii são protozoários parasitas pertencentes ao filo Apicomplexa. Apirases são enzimas metabolizadoras de nucleotídeos extracelulares. Nesta tese mostramos pela primeira vez a presença de um membro desta família de enzimas em P. falciparum, o qual foi capaz de degradar ATP extracelular. Análises por RT-qPCR revelaram a expressão da apirase durante todo o ciclo intraeritrocítico. A adição de inibidores desta classe de enzimas foi capaz de prejudicar o desenvolvimento dos parasitas e a invasão de novas hemácias pelos merozoitos, sugerindo assim um papel da apirase nestes processos. A via de sinalização por Ca2+ é universal e vital para todas as células. Para melhor entender a fisiologia celular de P. falciparum construímos uma nova linhagem de parasitas transgênicos, PfGCaMP3, que nos tornam capazes de monitorar a dinâmica de Ca2+ sem o uso de protocolos invasivos de marcação. De modo semelhante utilizamos uma nova linhagem de T. gondii expressando de forma estável o indicador de Ca2+ GCaMP3 para estudar o papel deste íon na saída da célula. T. gondii possui o Ca2+ necessário para promover este processo, entretanto Ca2+ extracelular age como um fator intensificador neste passo essencial do ciclo lítico.
Plasmodium falciparum and Toxoplasma gondii are protozoan parasites that belong to phylum Apicomplexa. Apirases are metabolizing enzymes of extracellular nucleotides. In this work we show for the first time the presence of an apyrase in P. falciparum, which was able to degrade extracellular ATP. RTqPCR analysis revealed the expression of apyrase throughout the intraerythrocytic cycle. Addition of apyrase inhibitors was able to impair the development of the parasites and the invasion of new erythrocytes by merozoites, thus suggesting a role of apyrase in these processes. Calcium signaling is universal and vital to all cells. To better understand the cellular physiology of P. falciparum we construct a new strain of transgenic parasites, PfGCaMP3, which enable us to monitor the Ca2+ dynamics without using invasive protocols. Similarly we use a new strain of T. gondii that stably express the Ca2+ indicator GCaMP3 to study the role Ca2+ in parasite egress. T. gondii has the Ca2+ required to promote this process, however extracellular Ca2+ acts as an enhancer factor in this crucial step of the lytic cycle.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire
Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes

Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

In my PhD thesis I have studied the changes in functional and structural plasticity induced by a photothrombotic stroke in mouse primary motor cortex. In order to dissect the multiple aspects consequent to the damage we exploit fluorescent imaging techniques that allow to investigate the functional and structural rearrangement of the cortex at different scale, from the entire hemisphere, with wide-field calcium imaging, up to the single synapse with two-photon microscopy. To promote a functional recovery of the mouse forelimb we applied different rehabilitative strategies in order to both foster the stabilization of regions of the cortex linked to the stroke core, and stimulate the remodelling of peri-infarct areas. We took advantage of a robotic platform (M-Platform), developed by our collaborator in Pisa, to perform the rehabilitation of mouse forelimb through a repetitive motor training. Together with this approach we applied different strategies to mould cortical activity. We temporary inhibited the healthy primary motor cortex, with an intracortical injection of Botulin Neuro Toxin E, in order to counterbalance the iper-excitability of the healthy hemisphere and to promote the structural and functional remodelling of the peri-infarct cortex. This combined rehabilitative protocol promotes the recovery of cortical maps of activation during motor training and the rewiring of interhemispheric connectivity, both from functional and structural level. Then we applied an optogenetic approach as a pro-plasticizing treatment by stimulating with light the region of the cortex surrounding the damage. By coupling this treatment with an intense motor training on the M-Platform we observed a generalized recovery of forelimb functionality in terms of manual dexterity and cortical profiles of activation. In this study, we have shown that different rehabilitative protocols that combines repetitive motor training and neuronal modulation of specific cortical regions induce a synergic effect on neuronal plasticity that promotes the recovery of structural features of healthy neuronal networks.

Neurotransmitter release is modulated by multiple regulatory mechanisms that control several stages of synaptic vesicle (SV) exocytosis. At the final stage, SV fusion with the presynaptic membrane requires calcium influx through voltage-gated calcium channels, and regulatory mechanisms that alter the surface expression or conductance of calcium channels have large effects on neurotransmitter release. To determine how these mechanisms contribute to synapse-specific modulations of neurotransmitter release and synaptic strength, we require a means to monitor presynaptic calcium transients at individual synapses. Genetically encoded calcium indicators (GECIs), engineered proteins that change their fluorescence emission properties upon calcium binding, generally lack the sensitivity to measure such transients in response to isolated stimuli. Therefore, we modified the GECI, GCaMP3, by altering its sensitivity for calcium. Our results suggest the modified GCaMP-based presynaptically targeted GECIs are excellent tools to quantify presynaptic calcium transients at individual synapses in response to isolated action potentials.

The ability of animals to sense, interpret, and respond appropriately to social stimuli in their environment is essential for identifying and distinguishing between members of their own species. In mammals, social interactions both within and across species play a key role in determining if an animal will live to pass on its genes to the next generation or else be removed from the gene pool. The result of this selection pressure can be observed in specialized neural circuits that respond to social stimuli and orchestrate appropriate behavioral responses. This highly conserved network of brain structures is often referred to as the Social Behavior Network (SBN). The medial amygdala (MeA) is a central node in the SBN and has been shown to be involved in transforming information from olfactory sensory systems into social and defensive behavioral responses. Previous research has shown that individual neurons in the MeA of anesthetized mice respond selectively to different chemosensory social cues, a characteristic not observed in its upstream relay, the accessory olfactory bulb (AOB). However, the cause of this stimulus selectivity in the MeA is not yet understood. Here, I hypothesize that a subpopulation of neurons in the MeA that express the enzyme aromatase are involved in the sensory representation of social stimuli in awake, behaving animals. To test this hypothesis, I designed and built a novel behavioral apparatus that allows for discrete presentations of social stimuli in a highly controllable and reproducible environment. I then injected the adeno-associated virus (AAV) AAV-Syn-Flex-GCAMP6s into the MeA of Aromatase:Cre transgenic mice and implanted a fiber optic cannula slightly above the injection site. The combination of this transgenic mouse line and conditional AAV caused GCaMP6s expression to be exclusive to aromatase-expressing neurons. By coupling my novel behavioral apparatus to a fiber photometry system, I successfully recorded the moment-to-moment activity of aromatase neurons in the MeA of awake, behaving animals as they investigated various social stimuli. Aromatase neurons in the MeA of adult male mice respond strongly to conspecific social stimuli, including live adult mice, mouse pups, and mouse urine samples. Sniffing and investigative behaviors correlated strongly with increased GCaMP6s signal in aromatase neurons, reflecting increases in their neural activity. Interestingly, after repeated investigations of the same stimuli the activity of aromatase neurons gradually diminished. Presenting a novel stimulus following repeated investigations of a familiar stimulus reinstated some, but not all of the initial GCaMP6s signal. This points to the potential role that aromatase neurons may play in the habituation to social stimuli that are consistently present in their environment. Investigations of predator stimuli did not evoke significant responses from aromatase neurons, nor did investigations of non-social stimuli. These results demonstrate that aromatase expressing neurons in the MeA of awake, behaving animals encode the sensory representation of conspecific social stimuli, and their responses are highly selective to the type of stimulus presented.

In this PhD thesis, we performed functional imaging of zebrafish larvae with a multi-modal and multi-scale approach. In particular, we focused our research on measuring neuronal activity in both physiological and pathological conditions. We adopted a pharmacological model of epilepsy, by administering pentylentetrazole at different concentrations and inducing seizures of different entity in zebrafish larvae expressing in all CNS neurons the Ca2+ reporter GCaMP6s. Owing to the relation between neuronal activity (i.e. action potentials) and intracellular Ca2+ concentration, we were able to measure neuronal activity by recording the changes in fluorescence of GCaMP indicator. Using a custom-made widefield fluorescence microscope, we measured activity during the onset and propagation of seizures, investigating the dynamics between different brain regions along with tail locomotor activity. We implemented commonly used zebrafish high-throughput drug screening assays, measuring only behavioural parameters (i.e. velocity of swimming, total travelled length), with a direct measure of the overall brain activity, thus laying the foundation for novel drug screening methods capable of improved efficacy. In order to improve the spatio-temporal resolution of brain activity recordings, being able to perform optical sectioning of the transparent zebrafish brain, we performed Bessel beam illumination light-sheet fluorescence microscopy measurements. Indeed, with respect to conventional Gaussian illumination, Bessel beams, owing to their nondiffractive and self-healing properties, allow for a substantial reduction of haemodynamic artefacts jeopardizing functional recordings in conventional measurements. We applied a custom analysis pipeline to produce 3D maps of neuronal activity, with single cell resolution. Finally, in order to perform real-time whole-brain measurements with singleneuron resolution, we devised a novel two-photon light-sheet microscope able to image the entire larval brain at 1 Hz. Applying a pixel-wise custom analysis we were able to identify functional circuitries involved both in physiological and pathological neuronal communication.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
A sinalização de cálcio e um mecanismo ubíquo, responsável pela regulação de vários processos fisiológicos celulares, mas também pela comunicação entre células. Destes estão incluídos transcrição, regulação do ciclo celular, diferenciação, motilidade/migração celular, apoptose, contracção/relaxamento muscular, fertilização e excitabilidade neuronal. De forma a fazer uso de tal sinal, as células necessitam de estar equipadas com diversas ferramentas sofisticadas para regular precisamente estes sinais de cálcio. A concentração de cálcio intracelular ronda os 100 nM sendo que os níveis extracelulares são de 2 mM. Este nível intracelular pode aumentar ate mais do que 1 μM apos as células serem estimuladas, por exemplo, por activação hormonal (como por exemplo por adenosina trifosfato – ATP). Este aumento de [Ca2+]i e responsável por uma versátil sinalização de cálcio, portanto e importante compreender o mecanismo pelo qual as células procedem a sua regulação. Os canais de cloreto ativados pelo cálcio (do ingles Calcium Activated Chloride Channels – CaCCs) são canais aniónicos selectivos que são ativados pelo aumento de concentrações citosólicas de cálcio. Os CaCCs participam em múltiplas funções fisiológicas, entre as quais: secreção de electrólitos/fluidos, excitabilidade do musculo liso, repolarização e duração de potencial de acção nos neurónios, estabilização de potencial de membrana nos fotorreceptores e fertilização em oócitos. Em 2008 a proteína TMEM16A (ANO1, Anoctamina 1) foi caracterizada como sendo um CaCC e pertence a uma família de proteínas transmembranares constituída por 10 membros (TMEM16A-K ou ANO1-10), que apresentam semelhança estrutural: dez segmentos transmembranares, um poro e cujos terminais amina e carboxilo se encontram localizados na face citoplasmática da membrana plasmática ou vesícula. A função das anoctaminas e ainda incerta, mas existem evidencias de que estas proteínas estão envolvidas na sinalização intracelular de Ca2+. Recentemente foi demonstrado que, em intestino de murganhos knockout para a ANO1, a secreção de Cl- dependente de Ca2+ e significativamente reduzida. Também foram sugeridos três mecanismos pelos quais a ANO1 facilita a sinalização de Ca2+: ou funcionando como um counter ion channel – onde, ao transportar Cl-, mantém a neutralidade eléctrica da célula; ou ativando os canais K+ dependentes de Ca2+, facilitando a secreção de Cl- dependente do Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); ou possibilitando a activação apical do canal CFTR através da inibição de fosfatases dependentes de Ca2+ e/ou ativando a Proteína Cinase C (PKC). Para alem disso, noutro estudo foi também demonstrado que a ANO1 interage, nas jangadas lipídicas da membrana plasmática, com o receptor de inositol trifosfato (IP3R) do reticulo endoplasmático, resultando num tethering entre estes dois componentes celulares, o que revelou novas evidencias de uma sinalização compartimentalizada de Ca2+ nestes domínios membranares. Curiosamente a proteína de levedura IST2, que e caracterizada como sendo um homologo das anoctaminas, e uma proteína importante para o tethering que detém a membrana plasmática em conjunto com o reticulo endoplasmático, mantendo juncões entre estes dois compartimentos. A IST2 e localizada na membrana do reticulo e demonstra um alto nível de homologia com a ANO10. Foram também apresentadas fortes evidencias de que, em osteoblastos, a ANO6 permite a entrada de Ca2+ e regula a sua sinalização indiretamente ao potenciar a atividade dos canais antiporte de Na+/Ca2+ (NCX). Estas evidências prévias de regulação da sinalização de Ca2+ dependente da ANO1 e da ANO6 permitiram uma melhor compreensão deste mecanismo de sinalização, porem, e necessário um conhecimento mais detalhado sobre esta regulação. O trabalho elaborado teve como objetivo estudar num sistema celular recombinante e em dois modelos animais se também outras proteínas da família TMEM16 alteram a concentração intracelular de Ca2+ ([Ca2+]i) ou próxima da membrana plasmática ([Ca2+]P). O âmbito do trabalho visava a regulação de células epiteliais, onde a ANO1, -4, -5, -6, -7, -8, -9 e -10 são expressas, sendo que ANO2 e a ANO3 não foram estudadas porque são maioritariamente expressas em neurónios. Deste modo, a ANO1, -4, -5, -6, -7, -8, -9 ou -10 foram sobreexpressas em células HeLa e as alterações da [Ca2+]i induzidas por ATP foram medidas, utilizando o indicador de Ca2+ fluorescente Fura-2 AM. Este indicador da informação sobre alterações da [Ca2+]i apos um estimulo, onde e possível obter um pico inicial, que representa a libertação de Ca2+ armazenado no reticulo endoplasmático e um posterior plateau que representa o influxo de Ca2+ celular. Foi assim possível estudar as consequências da sobre expressão destas ANOs na regulação destes processos de sinalização de cálcio. Para cada um destes processos de sinalização foi possível distinguir as ANOs em dois grupos. A sobre expressão da ANO5 ou da ANO6 aumenta a libertação de Ca2+ armazenado no reticulo endoplasmático, enquanto a da ANO4 diminui esta libertação. O influxo celular de Ca2+ e inibido pela sobre expressão da ANO4, -6, -8 ou -9 e activado pela sobre expressão da ANO5. A sobre expressão da ANO1, da ANO7 e da ANO10 não suscitou nenhuma alteração significativa em termos de alterações de [Ca2+]i. Quando uma variante da proteína GFP sensível ao cálcio expressa na membrana plasmática (PM-GCaMP2) foi co-expressa com as diferentes ANOs mencionadas anteriormente, foi possível medir alterações especificas da [Ca2+]P. Este novo indicador revela um sinal transiente que representa a libertação de Ca2+ armazenado no reticulo endoplasmático quando induzida por ATP. Foi possível dividir os elementos da família TMEM16 estudados em dois grupos: a sobre expressão da ANO1, -5 ou -10 leva a um aumento da [Ca2+]P, enquanto que a sobre expressão ANO4, -8, ou -9 a diminui. Não foi possível porem, observar alterações da [Ca2+]P dependentes da sobre expressão da ANO6 e da ANO7. A utilização de inibidores específicos para anoctaminas, como Acido Niflumico (NFA) e CaCC-AO1, demonstrou que a atividade destas proteínas e necessária para esta regulação. Estudos da ANO6 e da ANO10 endógenas nestas células demonstraram que estas proteínas são necessárias para as alterações de Ca2+ induzidas por ATP, anteriormente observadas. Foram realizados estudos adicionais de localização celular e co localização destas proteínas com recetores de IP3 e a bomba SERCA de forma a perceber como diferenças na localização subcelular levam a diferentes alterações da [Ca2+]P. Quando sobre expressas, a ANO1 e a ANO6 localizam-se na membrana plasmática das celulase HeLa, enquanto a ANO4, -5, -7, -8, -9 e -10 foram observadas nas membranas de outros organelos celulares. Graças aos estudos de imunocitoquímica e utilizando um polieno antibiótico, Filipina, que se associa ao colesterol, levando a destabilização das jangadas lipídicas, foi observado que a ANO1 e a ANO4 são expressas em diferentes localizações celulares. Quando estes domínios transmembranares foram destabilizados, observou-se um aumento da sinalização de cálcio, tanto quando a ANO1 ou a ANO4 foram sobre expressas, tendo-se obtido um sinal não especifico, e concluiu-se que o indicador PM-GCaMP2 e expresso nas jangadas lipídicas da membrana plasmática. A sobre expressão da ANO1 demonstrou uma co-localização desta proteína com o recetor de IP3 e resultou numa distribuição deste ate a membrana plasmática, corroborando as evidencias de que esta anoctamina e responsável por um tethering do reticulo plasmático a membrana, aumentando a [Ca2+]P em compartimentos de jangadas lipídicas. Observou-se também que a ANO4 colocaliza-se com a bomba SERCA, noutros compartimentos celulares, diminuindo a [Ca2+]P. Finalmente foi realizado o estudo em dois modelos animais que não expressam a ANO10 em túbulos proximais do rim ou em criptas do jejuno intestinal, de forma a perceber como esta proteína endogenamente expressa regula a sinalização de Ca2+. As alterações de [Ca2+]i induzidas por ATP foram medidas com o indicador Fura-2 AM e comparadas com murganhos wild-type (wt). Observou-se que a ANO10 potencia a sinalização de Ca2+ nestes dois tipos de células epiteliais, sendo que os efeitos na sinalização de Ca2+ anteriormente observados foram corroborados. Concluindo, neste trabalho são discutidas evidencias inovadoras de uma nova função das anoctaminas na sinalização de cálcio. E sugerido que estas proteínas estão envolvidas na regulação de uma sinalização de Ca2+ compartimentalizada em jangadas lipídicas, resultante do tethering reticulo endoplasmático–membrana plasmática, ou simplesmente envolvidas na regulação de sinalizacao de Ca2+ como counter ion channels, elucidando assim, as amplas funções celulares das anoctaminas.
Ca2+ activated TMEM16 Cl- channels (CaCCs; Anoctamins, ANOs) are anionselective channels activated by an increase in cytosolic Ca2+ concentration. The function of these CaCCs is still unclear, although there are evidences that ANOs could modulate intracellular Ca2+ signaling. Recently it was demonstrated that in mouse intestine lacking expression of ANO1, Ca2+ dependent Cl- secretion was significantly reduced. The study also suggested that ANO1 may facilitate Ca2+ signaling as a counter ion channel, or as a ER tethering protein or possibly activates the apical CFTR indirectly. ANO6 has been shown to regulate Ca2+ signaling indirectly by supporting the activity of the Na+/Ca2+ exchangers (NCX). Moreover, it has been reported that ANO1 interacts with IP3R, in lipid rafts, tethering the ER, giving new evidences of compartmentalized Ca2+ signaling in this membrane compartments. A recombinant cell system and two animal models were further examined in order to study whether also other proteins of the TMEM16 family change intracellular Ca2+ concentration close to the plasma membrane ([Ca2+]P). Therefore, ANO1, -4, -5, -6, -7, -8, -9 and -10 were overexpressed in HeLa cells and ATP-induced changes of [Ca2+]P were measured, using a plasma membrane targeted calcium sensitive GFP protein. It was observed that overexpression of ANO1, -5 or -10 enhanced, while ANO4, -8, or -9 decreased changes of [Ca2+]P and that the activity of these proteins is relevant for this regulation. Further studies of cellular localization and colocalization with IP3 receptors and SERCA pump suggested a localization of ANO1 and ANO4 in different lipid rafts, which could explain the different effect on changes of [Ca2+]P. For ANO10 the effects on Ca2+ signaling were confirmed in murine renal proximal tubules and intestinal epithelial knockout cells. This study describes innovative evidences for a new role of anoctamins in intracellular Ca2+ signaling. It is suggested that anoctamins may regulate a compartmentalized Ca2+ signaling in different lipid rafts or simply act as counter ion channels, which may give a hint to the wide cellular functions of anoctamins. Broader Key words: Anoctamin, Ca2+ signaling, PM-GCaMP2, Endoplasmic Reticulum, Calcium activated Chloride Channels (CaCCs).

We present a new implementation of adaptive optics for light-sheet microscopy, with a direct extended-scene wavefront sensing measurement for fast aberration correction. We report AO-enhanced images of GCaMP in freshly dissected drosophila brains.