Neuroimagen molecular preclínica en estimulación cerebral profunda

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Farmacología, leída el 28/01/2021. Tesis formato europeo (compendio de artículos)Deep brain stimulation (DBS) is a very powerful neurostimulation therapy for the palliative treatment of, mainly, resistant cases of motor disorders. The fruitful results obtained in the neurological scenario led to explore the possibility of extending its application to different psychiatric pathologies. Thus, DBS emerges as a potential alternative to the traditional neurosurgical interventions, which consist of local or major ablative procedures, performed in specific brain regions involved in the symptomatology with the aim to palliate it. In this sense, although it is indeed an invasive technique, it is actually a focal therapy, with potential reversibility of the effects induced by the electric stimulation, and offering the possibility of adjusting the stimulation parameters according to the patient’s needs. Nevertheless, despite the huge efforts and extensive research history in the DBS field, the specific mechanism of action of this therapy remains unknown. Several theories have tried to explain the effects obtained under specific pathologies and stimulation circumstances. However, regardless of DBS undoubted efficacy, a wide number of unanswered questions remain open regarding the specific physiological effects and long-term consequences. Therefore, the application of DBS in the majority of the proposed neuropsychiatric pathologies to potentially benefit from this treatment remains at a research level. Objectives: The main aim of this thesis is to evaluate the effects of DBS, by means of in vivo functional neuroimaging techniques, in healthy rats and in two models of murine obesity. In particular, the main contribution of this thesis is to describe the neuromodulation consequences of DBS on brain metabolism using [18F]FDG-PET at different stages: 1st) after the electrodes insertion, 2nd) during the electrical stimulation, 3rd) after a chronic and intermittent DBS protocol, and 4th) after a chronic and continuous DBS protocol...La estimulación cerebral profunda (DBS) es una potente terapia de neuroestimualción dedicada al tratamiento paliativo de, en su mayoría, casos avanzados de patologías motoras. Los fructíferos resultados obtenidos dentro del ámbito de la neurología llevaron a explorar la posibilidad de extender la aplicación de la DBS a diferentes patologías psiquiátricas. Así, la DBS emerge como una alternativa a las tradicionales intervenciones neuroquirúrgicas, las cuales consisten en procedimientos de ablación, local o extensa, realizados en regiones cerebrales específicas involucradas en la sintomatología a paliar. En este sentido, aunque la DBS es de hecho una técnica invasiva, se trata de una terapia local, con potencial reversibilidad de los efectos inducidos por la estimulación eléctrica, y que proporciona la posibilidad de ajustar in vivo los parámetros de estimulación de acuerdo a las necesidades del paciente. Sin embargo, a pesar de los enormes esfuerzos y la extensa historia de investigación en el campo de la DBS, los mecanismos de acción específicos de esta terapia todavía se desconocen. Varias teorías han intentado explicar los efectos obtenidos bajo circunstancias patológicas y de estimulación concretas. No obstante, a pesar de la indudable eficacia de la DBS, un amplio número de preguntas sin responder permanecen abiertas en relación a los efectos fisiológicos específicos de la DBS, así como a las consecuencias a largo plazo de la misma. Por este motivo, la aplicación de la DBS en la mayoría de las patologías neuropsiquiátricas que han sido propuestas de poder potencialmente beneficiarse de su tratamiento, permanecen en una etapa de investigación. Objetivos: La principal motivación de esta tesis consiste en evaluar los efectos de la DBS, por medio de técnicas de imagen funcional in vivo, en ratas sanas y en dos modelos murinos de obesidad. En concreto, la principal contribución de este trabajo radica en describir las consecuencias de la neuromodulación inducida por la DBS sobre el metabolismo cerebral, utilizando [18F]FDG-PET en diferentes niveles: 1º) después de la inserción de los electrodos; 2º) durante la estimulación eléctrica; 3º) después de un protocolo crónico e intermitente de DBS; y 4º) tras un protocolo crónico y continuo de DBS...Fac. de MedicinaTRUEunpu

Biological impact of Quantum Dots in rodents

I Quantum Dots (QDs) sono nanoparticelle fluorescenti largamente impiegate nell’imaging in vivo, ex vivo ed in vitro, grazie alle loro straordinarie proprietà ottiche. In particolare, i QTracker® 800 (Invitrogen) sono una tipologia commerciale di QDs con emissione nel Near-Infra-Red (800nm), disegnati appositamente per l’esecuzione di studi in vivo di diagnosi nell’uomo o drug delivery in piccoli animali da laboratorio. Uno dei principali problemi relativi a queste nanoparticelle è la presenza di metalli pesanti, fondamentali per le proprietà ottiche, che, nel caso dei QTracker®, sono CdSeTe. Gli studi della possibile tossicità dei QDs che si trovano in letteratura sono stati effettuati principalmente in vitro e potrebbero non corrispondere in modo soddisfacente alla risposta scatenata in vivo dalle nanoparticelle. Lo scopo di questa tesi è quindi lo studio di biodistribuzione e potenziali effetti dannosi dei QDs in sistemi viventi, con particolare attenzione al sistema nervoso centrale, prima di poter utilizzare tali nanocomposti in campo clinico. In seguito al trattamento di topi Balb-c con QDs (con iniezione intravenosa di 10μl/g, 40pM) è stato eseguito uno studio di biodistribuzione, tramite visualizzazione in vivo con la tecnica di Optical Imaging, seguita dalla conferma con microscopia elettronica e microscopia confocale e quantificazione dei metalli pesanti mediante ICP-MS a 24h, 1, 2 e 3 settimane dopo l’iniezione. Si è constatato che i QDs sono in grado di penetrare attraverso i vasi nei tessuti animali e di accumularsi, in particolare, nel reticolo endoteliale di fegato e milza. Inoltre i QDs sono in grado di attraversare la barriera ematoencefalica e di accumularsi intorno ai vasi sanguigni e nel parenchima in diverse aree cerebrali, soprattutto nella corteccia. L’ICP-MS ha rivelato un accumulo di metalli pesanti nel cervello immediatamente dopo l’iniezione, e significativamente elevato per tutte le 3 settimane dello studio. Nel cervello, ad una settimana dal trattamento, si trovano aggregati di alcune decine di nanoparticelle, distribuiti in neuroni, cellule epiteliali e cellule della glia, sia nel citosol che nel nucleo, talora anche a livello di sinapsi, sulle guaine mieliniche che ricoprono gli assoni neuronali e nei mitocondri. Abbiamo quindi valutato se la presenza sporadica delle nanoparticelle possa causare anomalie comportamentali, ad esempio anomalie nell’attività locomotoria, nell’attività cognitiva ecc. Dati recenti indicano che tutte le cellule cerebrali dialogano fra loro, e che l'attività dei neuroni è profondamente influenzata dall'intera orchestra cellulare. L’ipotesi di base è che se i nanocomposti, presenti a livello del parenchima, innescano una risposta infiammatoria prolungata, questo può riverberarsi in una modificazione dell’eccitabilità del neurone e conseguentemente in una modificazione del comportamento dell’animale. La presenza di tali nanoparticelle a livello del sistema nervoso centrale ha indotto un’alterazione della memoria di riconoscimento a 3 settimane dopo il trattamento, valutata con il test del Novel Object Recognition (senza alterazioni delle capacità locomotorie). La presenza di tali nanoparticelle induce una risposta infiammatoria che rimane elevata per le 3 settimane, caratterizzata da un aumento di microglia e astrociti a livello della corteccia entorinale e dell’ippocampo (giro dentato, aree CA1, CA2, CA3), che corrispondono alle aree associate alla codifica dei processi mnesici. La Long Term Potentiation, un modello di plasticità sinaptica d’altra parte non ha evidenziato nessuna alterazione delle funzioni ippocampali ad 1 e 3 settimane dal trattamento. Abbiamo valutato poi se la presenza nel parenchima cerebrale di NPs possa portare ad alterazioni del ciclo sonno/veglia e dell’attività dell’animale nell’home cage. I dati ottenuti indicano che occorre una valutazione accurata degli effetti dei nanocomposti sulla salute umana, ed occorrono molti studi approfonditi sia in vitro che in vivo prima del loro utilizzo in biomedicina.Quantum Dots (QDs) are fluorescent semiconductor nanocrystals that have a narrow and tunable emission wavelengths across the visible and infrared spectrum. This makes them a promising tool for in vivo, in vitro and ex vivo imaging (Smith et al., 2000). However, the assessment of biocompatibility and biosafety of QDs is a critical issue for further applications as diagnostic and imaging tools on humans. The work of this thesis focuses on non modified QTracker® 800 QDs, which are a commercial type of QDs with a heavy metals core of CdSeTe, specifically designed for in vivo vascular and tumour imaging, for their emission wavelength in the near infrared spectrum (800nm). The biodistribution of QTracker QDs is assessed in Balb-c mice (40pM, 10μl/g) during 3 weeks following an i.v. injection. Accumulation is monitored using inductively coupled plasma mass spectroscopy, optical imaging, confocal microscopy and electron microscopy, and revealed a major accumulation of QDs in liver and spleen, specifically in the mononuclear phagocytic system. The accumulation in the brain is observed over 3 weeks, suggesting a long biological half-life, in the scale of weeks or months. It is in particularly observed around blood vessels, in different brain areas. QDs are found in neurons, glia and epithelial cells, both in cytosol and nucleus. Animal’s behaviour is investigated and novel object recognition test shows that QDs can significantly impair recognition memory at 3 weeks after treatment (with no locomotory and coordination alterations observed). Such impairment is possibly due to the alteration of physiological conditions in the anatomical regions that encode for recognition memory, such as enthorinal and perirhinal cortices, dentate gyrus and areas CA1-CA3 of hippocampus. Therefore, an increased inflammatory response in terms of microglia and astrocytes activation is observed in all these regions. However, it do not have an effect on hippocampal plasticity, which is investigated by measuring long term potentiation (LTP) at 1 and 3 weeks after treatment. Taken together, the data presented in this thesis suggest that due to long term accumulation, induction of neuroinflammation and behavioural changes, the application of QDs in human should be cautious

Serotonergic modulation of the ventral pallidum by 5HT1A, 5HT5A, 5HT7 AND 5HT2C receptors

Introduction: Serotonin's involvement in reward processing is controversial. The large number of serotonin receptor sub-types and their individual and unique contributions have been difficult to dissect out, yet understanding how specific serotonin receptor sub-types contribute to its effects on areas associated with reward processing is an essential step. Methods: The current study used multi-electrode arrays and acute slice preparations to examine the effects of serotonin on ventral pallidum (VP) neurons. Approach for statistical analysis: extracellular recordings were spike sorted using template matching and principal components analysis, Consecutive inter-spike intervals were then compared over periods of 1200 seconds for each treatment condition using a student’s t test. Results and conclusions: Our data suggests that excitatory responses to serotonin application are pre-synaptic in origin as blocking synaptic transmission with low-calcium aCSF abolished these responses. Our data also suggests that 5HT1a, 5HT5a and 5HT7 receptors contribute to this effect, potentially forming an oligomeric complex, as 5HT1a antagonists completely abolished excitatory responses to serotonin application, while 5HT5a and 5HT7 only reduced the magnitude of excitatory responses to serotonin. 5HT2c receptors were the only serotonin receptor sub-type tested that elicited inhibitory responses to serotonin application in the VP. These findings, combined with our previous data outlining the mechanisms underpinning dopamine's effects in the VP, provide key information, which will allow future research to fully examine the interplay between serotonin and dopamine in the VP. Investigation of dopamine and serotonins interaction may provide vital insights into our understanding of the VP's involvement in reward processing. It may also contribute to our understanding of how drugs of abuse, such as cocaine, may hijack these mechanisms in the VP resulting in sensitization to drugs of abuse

Neurobiological mechanisms of heterogeneous nuclear ribonucleoprotein H1 in methamphetamine stimulant and addictive behaviors

Addiction to psychostimulants such as methamphetamine (MA) is a significant public health issue in the United States with no FDA-approved pharmacological interventions. MA addiction is a heritable neuropsychiatric disorder, however, its genetic basis is almost entirely unknown. Available human genome-wide association studies (GWAS) lack sufficient power to detect the influence of common genetic variation on the risk of addiction. Mammalian model organisms offer an attractive alternative to more rapidly uncover novel genetic factors that contribute to addiction-relevant neurobehavioral traits. Using quantitative trait locus (QTL) mapping in mice, we identified a locus on chromosome 11 that contributed to a decrease in sensitivity to the locomotor stimulant properties of MA. To fine map this QTL, we generated interval-specific congenic lines and deduced a 206 kb critical interval on chromosome 11 that contained only two protein coding genes (Rufy1 and Hnrnph1). Replicate mouse lines heterozygous for Transcription Activator-like Effector Nucleases (TALENs)-induced frameshift deletions in Hnrnph1 (Hnrnph1+/-), but not in Rufy1 (Rufy1+/-), recapitulated the decrease in MA sensitivity observed in congenic mice; thus, identifying Hnrnph1 as a novel quantitative trait gene for MA sensitivity. Hnrnph1, an RNA-binding protein, has not previously been identified in human GWAS of neuropsychiatric disorders but has been implicated in mu-opioid receptor splicing associated with heroin dependence. The primary objectives of this dissertation is to (1) detail the forward genetic and reverse genetic approaches taken to identify Hnrnph1 as a quantitative trait gene for MA sensitivity; (2) assess the MA addiction-relevant behaviors presented by Hnrnph1+/- mice through conditioned place preference (CPP) and oral self-administration procedures; and (3) identify the neurobiological mechanisms through which Hnrnph1 affects behavior via transcriptome, immunohistochemical and neurochemical assessments of the mesocorticolimbic dopamine circuit. Overall, Hnrnph1+/- mice display increased dopaminergic innervation and MA dose-dependent dopamine release in nucleus accumbens, which could underlie reduced drug sensitivity, reward, and reinforcement. The results of this thesis provide substantial evidence to implicate Hnrnph1 in MA addiction

Neuroprotection: Rescue from Neuronal Death in the Brain

Dear Colleagues, The brain is vulnerable to injury. Following injury in the brain, apoptosis or necrosis may occur easily, leading to various functional disabilities. Neuronal death is associated with a number of neurological disorders including hypoxic ischemia, epileptic seizures, and neurodegenerative diseases. The brain subjected to injury is regarded to be responsible for the alterations in neurotransmission processes, resulting in functional changes. Oxidative stress produced by reactive oxygen species has been shown to be related to the death of neurons in traumatic injury, stroke, and neurodegenerative diseases. Therefore, scavenging or decreasing free radicals may be crucial for preventing neural tissues from harmful adversities in the brain. Neurotrophic factors, bioactive compounds, dietary nutrients, or cell engineering may ameliorate the pathological processes related to neuronal death or neurodegeneration and appear beneficial for improving neuroprotection. As a result of neuronal death or neuroprotection, the brain undergoes activity-dependent long-lasting changes in synaptic transmission, which is also known as functional plasticity. Neuroprotection implying the rescue from neuronal death is now becoming one of global health concerns. This Special Issue attempts to explore the recent advances in neuroprotection related to the brain. This Special Issue welcomes original research or review papers demonstrating the mechanisms of neuroprotection against brain injury using in vivo or in vitro models of animals as well as in clinical settings. The issues in a paper should be supported by sufficient data or evidence. Prof. Bae Hwan Lee Guest Edito

Impact of environmental risk factors for schizophrenia on the developing brain, characterisation of the effects of polyIC and THC on functional neural systems and behaviour

Strathclyde theses - ask staff. Thesis no. : T13455Cannabis abuse can produce deficits in cognition and has been implicated as a 'late' environmental risk factor in the pathogenesis of the poly-factorial disorder schizophrenia. Evidence suggests an age-related susceptibility to the deleterious effects of cannabis as early onset of use may increase the vulnerability of the brain to the adverse consequences of cannabis abuse. Animal models are crucial for exploration of mechanistic and causative theories, and long-term behavioural consequences of adolescent cannabis abuse in a controlled experimental environment. This thesis evaluates the vulnerability of the adolescent/peripubertal brain to Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, and explores the potential interplay between this schizophrenia-related 'late' environmental risk factor and an 'early' environmental risk factor (prenatal infection - maternal immune activation (MIA)) on functional neural systems and behaviours relevant to schizophrenia. Cannabinoid CB1 receptor ontogeny (activated in the brain by the receptor ligand THC) within important cognitive substrates, the prefrontal cortex (PFC) and hippocampus, was investigated to delineate a period of neurodevelopmental vulnerability for peripubertal THC treatment. CB1 receptor ligand binding revealed that the PFC and hippocampus follow differential late maturational trajectories throughout the peripubertal period. The 'vulnerability window' for peripubertal THC treatment was defined as post-natal day (PD) 35-56 to encompass the dynamic peripubertal ontogenetic patterns of the CB1 receptor in both these regions. Furthermore, age-related alterations in cerebral metabolism and regional functional connectivity profiles were evident in the hippocampus and important neuromodulatory nuclei including the ventral tegmental area, dorsal raphe, locus coeruleus and the diagonal band of Broca.;Acute THC administration (5mg/kg) produced hypometabolism in the thalamus and an altered functional connectivity profile between thalamic nuclei and the PFC, hippocampus and the nucleus accumbens. THC-induced anomalistic neural activity was evident in key neuromodulatory nuclei and produced perturbed functional connectivity within acetylcholine, noradrenaline, and dopamine neural pathways. Acute THC treatment resulted in alterations in cerebral metabolism in the amygdala and aberrant functional connectivity profiles between amygdaloid nuclei and the hippocampus, PFC and nucleus accumbens. There appeared to be an age-related sensitivity to THC in several thalamic, neuromodulatory and amygdaloid nuclei. Peripubertal low-dose intermittent THC (3.5mg/kg, 3 times a week), mimetic of light, recreational adolescent cannabis use, produced long-term cognitive inflexibility, as measured by the attentional-set shifting task, perturbed cerebral metabolism in the dorsolateral orbital cortex and the nucleus accumbens core and altered functional coupling between both these regions and neural substrates subserving reward-related learning including prefrontal, septal and amygdala subfields. High-dose daily THC (7mg/kg) throughout the peripubertal period, mimetic of heavy daily cannabis abuse, did not precipitate any schizophrenia-related behaviours in adulthood. MIA induced by prenatal exposure to the immune-stimulating agent polyriboinosinic-polyribocytidilic acid (PolyIC) did not produce any schizophrenia-related phenotypes in adulthood. However, prenatal PolyIC exposure produced residual hypermetabolism within discrete components of the prefrontal cortex dorsolateral orbital and cingulate cortices and hypometabolism within the CA3 subfield of the hippocampus. The functional connectivity signatures of all these regions indicated a unified MIA effect of aberrant mesocorticolimbic functional coupling in adulthood. Furthermore, chronic intermittent treatment with low-dose THC during the peripubertal period caused an increase in sensitivity to amphetamine (indicative of aberrant mesolimbic dopamine transmission) in PolyIC-treated offspring compared to PBS-treated offspring, suggestive of a synergistic effect of these two environmental risk factors. In conclusion, the findings presented in this thesis have provided clear evidence of dose-specific detrimental effects of 'adolescent' THC exposure on behaviour and the functional neural systems that may underpin these deficits which impact on behaviour and neural systems into adulthood.Cannabis abuse can produce deficits in cognition and has been implicated as a 'late' environmental risk factor in the pathogenesis of the poly-factorial disorder schizophrenia. Evidence suggests an age-related susceptibility to the deleterious effects of cannabis as early onset of use may increase the vulnerability of the brain to the adverse consequences of cannabis abuse. Animal models are crucial for exploration of mechanistic and causative theories, and long-term behavioural consequences of adolescent cannabis abuse in a controlled experimental environment. This thesis evaluates the vulnerability of the adolescent/peripubertal brain to Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, and explores the potential interplay between this schizophrenia-related 'late' environmental risk factor and an 'early' environmental risk factor (prenatal infection - maternal immune activation (MIA)) on functional neural systems and behaviours relevant to schizophrenia. Cannabinoid CB1 receptor ontogeny (activated in the brain by the receptor ligand THC) within important cognitive substrates, the prefrontal cortex (PFC) and hippocampus, was investigated to delineate a period of neurodevelopmental vulnerability for peripubertal THC treatment. CB1 receptor ligand binding revealed that the PFC and hippocampus follow differential late maturational trajectories throughout the peripubertal period. The 'vulnerability window' for peripubertal THC treatment was defined as post-natal day (PD) 35-56 to encompass the dynamic peripubertal ontogenetic patterns of the CB1 receptor in both these regions. Furthermore, age-related alterations in cerebral metabolism and regional functional connectivity profiles were evident in the hippocampus and important neuromodulatory nuclei including the ventral tegmental area, dorsal raphe, locus coeruleus and the diagonal band of Broca.;Acute THC administration (5mg/kg) produced hypometabolism in the thalamus and an altered functional connectivity profile between thalamic nuclei and the PFC, hippocampus and the nucleus accumbens. THC-induced anomalistic neural activity was evident in key neuromodulatory nuclei and produced perturbed functional connectivity within acetylcholine, noradrenaline, and dopamine neural pathways. Acute THC treatment resulted in alterations in cerebral metabolism in the amygdala and aberrant functional connectivity profiles between amygdaloid nuclei and the hippocampus, PFC and nucleus accumbens. There appeared to be an age-related sensitivity to THC in several thalamic, neuromodulatory and amygdaloid nuclei. Peripubertal low-dose intermittent THC (3.5mg/kg, 3 times a week), mimetic of light, recreational adolescent cannabis use, produced long-term cognitive inflexibility, as measured by the attentional-set shifting task, perturbed cerebral metabolism in the dorsolateral orbital cortex and the nucleus accumbens core and altered functional coupling between both these regions and neural substrates subserving reward-related learning including prefrontal, septal and amygdala subfields. High-dose daily THC (7mg/kg) throughout the peripubertal period, mimetic of heavy daily cannabis abuse, did not precipitate any schizophrenia-related behaviours in adulthood. MIA induced by prenatal exposure to the immune-stimulating agent polyriboinosinic-polyribocytidilic acid (PolyIC) did not produce any schizophrenia-related phenotypes in adulthood. However, prenatal PolyIC exposure produced residual hypermetabolism within discrete components of the prefrontal cortex dorsolateral orbital and cingulate cortices and hypometabolism within the CA3 subfield of the hippocampus. The functional connectivity signatures of all these regions indicated a unified MIA effect of aberrant mesocorticolimbic functional coupling in adulthood. Furthermore, chronic intermittent treatment with low-dose THC during the peripubertal period caused an increase in sensitivity to amphetamine (indicative of aberrant mesolimbic dopamine transmission) in PolyIC-treated offspring compared to PBS-treated offspring, suggestive of a synergistic effect of these two environmental risk factors. In conclusion, the findings presented in this thesis have provided clear evidence of dose-specific detrimental effects of 'adolescent' THC exposure on behaviour and the functional neural systems that may underpin these deficits which impact on behaviour and neural systems into adulthood

PET-imaging in depression and antidepressant therapies : focus on the serotonin system and the cerebral glucose metabolism

The main scope of the research summarised in this dissertation comprises the use of positron emission tomography to investigate the role of the serotonin transporter in depression and antidepressant therapies. Hereby, several studies were performed using the PET radiotracer [11C]DASB, which specifically targets the serotonin transporter. To allow qualitative and safe research with this radiotracer, the first research topic focussed on the optimization of the radiotracer’s purification procedure and its quality control. Using this radiotracer, a first-in-dog study was carried out to investigate the radiotracer’s distribution and to define the appropriate image quantification methods. Subsequently, this radiotracer was used to perform a dose-occupancy in the dog to estimate the optimal dosing regimen to treat dogs with behavioural disorders with escitalopram. A second part of the dissertation focuses on rats and the current position of repetitive transcranial magnetic stimulation (rTMS) in the rat. Hereby, several additional objectives were put forward. The first objective comprised the evaluation of the accuracy of a for rodents adapted human neuronavigation system to perform rTMS in the rat. A second objective was the investigation of the construct validity of two depression models in terms of altered regional glucose metabolism. This was investigated via a PET study using the radiotracer [18F]FDG. Finally, for the preferred depression model, which was the one based on chronic corticosterone injections, the scope was extended from the serotonin transporter to the serotonin 5-HT1A and 5-HT2A receptors to explore the role of the serotonin system in the pathophysiology of this depression model in the rat. For this purpose, three radiotracers were applied: [11C]DASB, [18F]MPPF, and [18F]altanserin. This allowed to image the serotonin transporters, the 5-HT1A receptors, and the 5-HT2A receptors, respectively