Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Rett's Syndrome (RTT) is a severe neurodevelopmental disorder, characterized by cognitive disability that appears in the first months/years of life. Recently, mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been detected in RTT patients characterized by early-onset seizures. CDKL5 is highly expressed in the brain starting from early postnatal stages to adulthood, suggesting the importance of this kinase for proper brain maturation and function. However, the role/s of CDKL5 in brain development and the molecular mechanisms whereby CDKL5 exerts its effects are still largely unknown. In order to characterize the role of CDKL5 on brain development, we created a mice carrying a targeted conditional knockout allele of Cdkl5. A first behavioral characterization shows that Cdkl5 knockout mice recapitulate several features that mimic the clinical features described in CDKL5 patients and are a useful tool to investigate phenotypic and functional aspects of Cdkl5 loss. We used the Cdkl5 knockout mouse model to dissect the role of CDKL5 on hippocampal development and to establish the mechanism/s underlying its actions. We found that Cdkl5 knockout mice showed increased precursor cell proliferation in the hippocampal dentate gyrus. Interestingly, this region was also characterized by an increased rate of apoptotic cell death that caused a reduction in the final neuron number in spite of the proliferation increase. Moreover, loss of Cdkl5 led to decreased dendritic development of new generated granule cells. Finally, we identified the Akt/GSK3-beta signaling as a target of Cdkl5 in the regulation of neuronal precursor proliferation, survival and maturation. Overall our findings highlight a critical role of CDKL5/AKT/GSK3-beta signaling in the control of neuron proliferation, survival and differentiation and suggest that CDKL5-related alterations of these processes during brain development underlie the neurological symptoms of the CDKL5 variant of RTT.

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, è coinvolto nella mediazione delle proprietà di rinforzo e nella dipendenza da diverse sostanze d’abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticità, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un’interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilità di un’interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l’attivazione dei recettori CB1 e 5-HT2 e l’apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l’applicazione esogena di 5-HT (5 M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l’attivazione dei medesimi recettori e l’apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l’influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l’eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d’abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc. / The nucleus accumbens (NAc), a major component of the mesolimbic system, is involved in the mediation of reinforcing and addictive properties of many dependence-producing drugs. Glutamatergic synapses within the NAc can express plasticity, including a form of endocannabinoid (eCB)-long-term depression (LTD). Recent evidences demonstrate cross-talk between eCB signaling pathways and those of other receptor systems, including serotonin (5-HT); the extensive co-localization of CB1 and 5-HT receptors within the NAc suggests the potential for interplay between them. Here, we found that 20 min low-frequency (4 Hz) stimulation (LFS-4Hz) of glutamatergic afferences in rat brain slices induces a novel form of eCB-LTD in the NAc core, which requires 5-HT2 and CB1 receptors activation and L-type voltage-gated Ca2+ channels opening. Moreover, we found that exogenous 5-HT application (5 μM, 20 min) induces an analogous LTD (5HT-LTD) at the same synapses, requiring the activation of the same receptors and the opening of the same Ca2+ channels; LFS-4Hz-LTD and 5-HT-LTD were mutually occlusive. Present results suggest that LFS-4Hz induces the release of 5-HT, which acts at 5-HT2 postsynaptic receptors increasing Ca2+ influx through L-type voltage-gated channels and 2-arachidonoyl-glycerol production and release; the eCB travels retrogradely and binds to presynaptic CB1 receptors, causing a long-lasting decrease of glutamate release resulting in LTD. These observations might be helpful to understand the neurophysiological mechanisms underlying drug addiction, major depression and other psychiatric disorders characterized by dysfunction of 5-HT neurotransmission in the NAc.

Obiettivo della tesi è stato quello di studiare il ruolo svolto dall’ipotalamo laterale (LH) nella regolazione dei processi di integrazione dell’attività autonomica e termoregolatoria con quella degli stati di veglia e sonno. A questo scopo, l’attività dell’LH è stata inibita per 6 ore (Esperimento A) mediante microiniezioni locali dell’agonista GABAA muscimolo nel ratto libero di muoversi, nel quale sono stati monitorati in continuo l’elelttroencefalogramma, l’elettromiogramma nucale, la pressione arteriosa (PA) e la temperatura ipotalamica (Thy) e cutanea. Gli animali sono stati studiati a temperatura ambientale (Ta) di 24°C e 10°C. I risultati hanno mostrato che l’inibizione acuta dell’LH riduce l’attività di veglia e sopprime la comparsa del sonno REM. Ciò avviene attraverso l’induzione di uno stato di sonno NREM caratterizzato da ipersincronizzazione corticale, con scomparsa degli stati transizionali al sonno REM. Quando l’animale è esposto a bassa Ta, tali alterazioni si associano a un ampio calo della Thy, che viene compensato da meccanismi vicarianti solo dopo un paio d’ore dall’iniezione. Sulla base di tali risultati, si è proceduto ad un ulteriore studio (Esperimento B) volto ad indagare il ruolo del neuropeptide ipocretina (prodotto in modo esclusivo a livello dell’LH) nei processi termoregolatori, mediante microiniezioni del medesimo nel bulbo rostrale ventromediale (RVMM), stazione cruciale della rete nervosa preposta all’attivazione dei processi termogenetici. La somministrazione di ipocretina è stata in grado di attivare la termogenesi e di potenziare la comparsa della veglia, con concomitante lieve incremento della PA e della frequenza cardiaca, quando effettuata alle Ta di 24°C o di 10°C, ma non alla Ta di 32°C. In conclusione, i risultati indicano che l’LH svolge un ruolo cruciale nella promozione degli stati di veglia e di sonno REM e, per tramite dell’ipocretina, interviene in modo coplesso a livello del RVMM nella regolazione dei processi di coordinamento dell'attività di veglia con quella termoregolatoria. / Aim of my thesis was to investigate the lateral hypothalamus (LH) role in the regulation of autonomic and thermoregulatory integration processes with wakefulness and sleep states. For this purpose, LH was inhibited for up to 6 hours ( Experiment A) by local microinjections of the GABAA agonist muscimol in free behaving rats, implanted with electrodes for chronic electroencephalogram, nuchal electromyogram, arterial pressure (PA ), hypothalamic (Thy ) and skin temperature. The animals were exposed to 24 ° C and 10 ° C ambient temperature ( Ta ). The results showed that the acute inhibition of LH reduces wakefulness and suppresses the onset of REM sleep. This occurs through the induction of NREM sleep state, characterized by high cortical synchronization levels, with REM sleep transitional states disappearance. At low Ta, these results were associated with a large Thy decrease, reversed by compensatory systems a couple of hours after the injections. Starting from these results, we proceeded to investigate (Experiment B) the role of the hypocretin neuropeptide (produced exclusively in the LH ) in thermoregulatory processes, microinjecting hypocretin in the Rostral Ventromedial Medulla ( RVMM ), a crucial center of the neural network responsible for the thermogenetic processes activation. The hypocretin administration was able to activate thermogenesis and enhance wakefulness, with concomitant slight blood pressure and heart rate increase, at Ta of 24 ° C and 10 ° C, but not at Ta of 32 ° C. In conclusion, the results indicate that the LH plays a crucial role in wakefulness and REM sleep promotion and, through hypocretin, it intervenes in a complex way in regulating the wakefulness coordination processes, at the RVMM level.

No description available.

Autism spectrum disorders (ASDs) represent a heterogeneous group of neurodevelopmental disorders characterised by deficits in social interaction and communication, and by restricted and stereotyped behaviour. The diagnosis of autism is based on behavioural observation of the subject as research has not yet identified specific markers. Today, several studies show that disturbances in sensory processing are a crucial feature of autism. Indeed, around 90% of individuals diagnosed with autism show atypical responses to various sensory stimuli. These sensory abnormalities (described as hyper- or hypo-reactivity to sensory stimulation) are currently recognised as diagnostic criteria for autism. Among the sensory defects, tactile abnormalities represent a very common finding impacting the life of autistic individuals. It has been shown how abnormal responses to tactile stimuli not only correlate with the diagnosis of autism but also predict its severity. Indeed hypo-responsiveness to tactile stimuli is associated with greater severity of the main symptoms of autism. To date, the neural substrates of these behaviours are still poorly understood. Over the years, the use of genetically modified animal models has enabled a major step forward in the study of the aetiology of autism spectrum disorders. Interestingly, several animal models that carry autism-related mutations also show deficits of a sensory nature. This is the case with the Shank3b-/- and Cntnap2-/- mouse models, strains in which the expression of the gene in question is suppressed. The SHANK3 gene encodes for a crucial protein in the structure of the postsynaptic density of glutamatergic synapses. In humans, haploinsufficiency of SHANK3 causes the Phelan-McDermid syndrome, a neurodevelopmental disorder characterised by ASD-like behaviour, developmental delay, intellectual disability and absent or severely delayed speech. Individuals with Phelan-McDermid syndrome often show dysfunctions in somatosensory processing, including disturbances in tactile sensitivity. CNTNAP2 codes for CASPR2, a transmembrane protein of the neurexin superfamily involved in neuron-glia interactions and clustering of potassium channels in myelinated axons. Missense mutation in CNTNAP2 is causative of cortical dysplasia-focal epilepsy syndrome (CDFE), a rare disorder characterized by epileptic seizures, language regression, intellectual disability, and autism. Following these findings, mice lacking the Shank3b isoform (Shank3b-/-) and Cntnap2 gene (Cntnap2-/-) show autistic-like behaviours. In this study, we used an interdisciplinary approach (behavioural, molecular, and imaging techniques) to study the neuronal substrates of whisker-mediated behaviours in genetic mouse models of ASD. We performed two behavioural tests, namely the textured novel object recognition test (tNORT) and the whisker nuisance test (WN) to have in-depth insight in whisker dependent behaviours. Following behavioural assessment, through a molecular approach, we investigated the neural underpinnings of this aberrant behaviour. We evaluated neuronal activation in key brain areas involved in the processing of sensory stimuli via c-fos mRNA in situ hybridization. Finally, using a seed-based approach in resting-state functional magnetic resonance imaging (rsfMRI) we probed the functional connectivity phenotype of these mutant mice. The contribution of the peripheral nervous system to sensory processing was also assessed via RT-qPCR at the level of the trigeminal ganglion. Sensory abnormalities that characterize ASDs represent a symptom of primary relevance in the life of autistic individuals. Scientific research has only recently addressed this important aspect and animal models represent a useful preclinical tool to investigate the causal role of genetic mutations in the aetiology of ASDs. In such context, the complementary approach used in this work represents a crucial step to the understanding of sensory-related deficits which characterize ASD.

The last decade has seen an increasing interest in bio-mathematical modelling and scientific computing, resulting in new applications to relevant physiological phenomena and to a better understanding of the origin of various diseases. A topic of great interest to several degenerative diseases is filtration across microvessel walls. The role of the microvessel wall is to let oxygen and nutrients contained in the blood stream to reach the interstitium, and ultimately the surrounding cells, while blocking macromolecules. An understanding of these processes is important in preventing and curing neuro-degenerative diseases, as well as for exploring possible mechanisms to make drug delivery more efficient. This work presents a one-dimensional, time dependent mathematical model describing transport of blood plasma and macromolecules across blood vessel walls. The model takes into account the heterogeneous microvessel wall composition, in order to accurately describe trans-vascular flow. This results in a multi-layered domain, accounting for variable physical properties across the layers forming the micro-vascular wall. In particular, the glycocalyx and endothelium, accounted for in many biological studies, are represented in our model. This micro-structural, yet simplified description of the vascular wall, allows us to simulate the effect of glycocalyx damage and of other pathologies, such as hypertension, hemorrhage and hypovolemia, both in steady and time-dependent states. Due to the simplicity, and thus efficiency of the proposed model, simulations are fast and provide results which are in line with published experimental studies. Furthermore, the simulation tool may be useful for practical applications in physiological and medical studies, by evaluating the possible consequences of pathological conditions.

Resting-state fMRI (rsfMRI) is widely used to map brain network organization in health and disease. However, the neural underpinnings and significance of interregional coupling as assessed with rsfMRI remain unclear. Neocortical Excitatory/Inhibitory (E/I) balance critically affects local and long-range information processing and as such can conceivably bias large-scale interareal functional connectivity and fMRI coupling. This notion would be consistent with the concomitant presence of neocortical E/I imbalance and atypical rsfMRI connectivity in multiple brain disorders, such as autism, or schizophrenia. Here, we combine chemogenetic manipulations, rsfMRI, electrophysiology in the mouse to causally probe how alteration in regional neocortical E/I balance affects interareal neural and rsfMRI coupling. We used DREADD-based chemogenetics to remotely alter the E/I balance of the mouse medial prefrontal cortex (PFC) by increasing pyramidal neuron excitability (↑Excitation), or by reducing the activity of fast spiking parvalbumin positive (PV+) inhibitory interneurons (↓Inhibition). For each of the employed manipulations, we recorded both locally elicited fMRI network activity, as well as large scale functional connectivity as measured with rsfMRI. To uncover the neural rhythms associated with our manipulations, we carried out corresponding in-vivo multielectrode electrophysiology in a sperate cohort of animals. Chemogenetic activation of pyramidal neurons (↑Excitation) or inhibition of PV+ interneurons (↓Inhibition) resulted in increased neural firing and evoked-BOLD activity in the targeted area. Both manipulations also produced socio-behavioral impairments in a three-chamber social-interaction test. However, the two manipulations were associated with different spectral signatures as probed with local-field potential measurements (LFP). Specifically, ↑Excitation produced largely decreased broad-band oscillatory power, while ↓Inhibition led to a robust increase in local oscillatory activity. Notwithstanding these spectral differences, both manipulations produced analogous patterns of fMRI hypoconnectivity in the mouse default mode network. To understand how these different local spectral activity may produce converging patterns of rsfMRI dysconnectivity, we turned to multielectrode electrophysiology and measured interareal coupling between the manipulated region (PFC) and its anatomical targets (retrosplenial cortex). These investigations revealed that fMRI hypoconnectivity produced by ↑Excitation or ↓Inhibition was associated with dissociable LFP coherency signatures. Specifically, chemogenetic activation of pyramidal neurons (↑Excitation) produced decreased coherence in slow-δ range (0.1-4 Hz), while inhibition of PV+ activity (↓Inhibition) produced a composite response, comprising reductions in slow frequency coherence along with a largely increased coherence in theta to beta bands. By relating fMRI changes to corresponding LFP coherency differences, we found that neural coupling in slow (0.1-4 Hz), but not faster frequency bands, significantly predicted the fMRI connectivity changes produced by all the employed manipulations. These results suggest that large-scale fMRI connectivity is primarily supported by electrophysiological coherence in infraslow/slow rhythms, and it is disproportionately less sensitive to high frequency coupling. Importantly, this relationship also held when recordings obtained upon chemogenetic silencing of the PFC (i.e. producing a reduced ↓Excitation) were included. Collectively, our results shed light on the general principles underlying macro-scale fMRI network organization in the mammalian brain with a number of important implications for the interpretation of rsfMRI connectivity in health and disease. First, they suggest that the relationship between interareal neural activity and fMRI connectivity is critically biased by local Excitatory/Inhibitory ratio. Second, our findings also support a simple framework whereby interareal patterns of hyper- and hypo-connectivity observed in brain disorders may counterintuitively reflect reduced or increased neural excitability of afferent systems, respectively. Third, these observations point at a possible unifying mechanistic link between E/I imbalance and connectivity disruption in brain disorders, suggesting that these two phenotypes may be the result of a unique etiopathological insult. Future extensions of this framework may offer opportunities to model the local contribution of regional E/I balance in affecting fMRI connectivity, and to physiologically decode fMRI dysconnectivity in human disorders

Successfully acting largely depends on moving at the right time. Consider a member of an orchestra just few instants before starting to play her piece. She should be ready not only to launch the planned movements when appropriate, but also to stop them if required. Action initiation and control are characteristic features of many of our daily life actions. There is a large amount of evidence in monkeys and humans suggesting that the dorsal premotor cortex (PMD) and the supplementary motor areas (SMA) might be critically involved in these features. However, the distinctive role of these areas is still matter of controversy. The aim of the present thesis is to provide some preliminary steps toward a comprehension of whether and how the human dorsal precentral areas may selectively contribute to action initiation and control. In doing this we shall introduce and discuss a series of transcranial magnetic stimulation (TMS) experiments carried out with two different paradigms, namely dual-coil TMS and single pulse TMS paradigm. These experiments were primarily devoted to explore the structural and functional properties of PMD. They also allowed us to assess whether PMD and SMA may be differentially and selectively involved in action control. In more detail, we first investigated the structural connectivity between PMD and the ipsilateral orofacial M1, introducing a novel dual-coil TMS approach. Results displayed the existence of short-latency influences of the left PMD on the ipsilateral orofacial M1, measured by recording motor evoked potentials (MEPs) in the orofacial muscles. Then, taking advantage of this novel approach, we started to explore the functional PMD-M1 connectivity. We tested the short-latency effects of TMS, as measured by changes in orofacial MEPs, during a delayed motor task. The results showed an inhibitory activity in the PMD-M1 module during the SET-period. We also manipulated the duration of the SET-period, to establish whether the effects were time-locked to the start of the delay period or rather time-locked to the predicted GO-signal. Hence, the investigation of the PMD-M1 connectivity paved us the way to explore, first, the role of PMD in initiating action and, then, the differential role of PMD and SMA in controlling and inhibiting action. Indeed, we run a further study, in which we carried out two single pulse TMS experiments. We first stimulated PMD during a stop-signal task, then we contrasted the PMD stimulation with SMA stimulation when participants underwent the same stop-signal task. There are five chapters to come. In Chapter 1 we shall review some key studies exploring anatomical and functional properties of PMD and SMA in both monkeys and humans, with particular emphasis on their putative role in action initiation and control. In Chapter 2 we shall focus on the methodological aspects of our experimental studies. In particular, we shall introduce the so-called twin- or dual-coil TMS paradigm, discuss its main approaches present in the literature and propose a variant of them. In Chapter 3 we shall present and discuss our first dual-coil TMS study exploring, for the first time, the ipsilateral PMD-corticofacial system connectivity. In Chapter 4 we shall examine three dual-coil TMS studies investigating the functional connectivity between PMD and ipsilateral M1 during a motor delayed task. Finally, in Chapter 5 we shall scrutinize two single pulse TMS studies capitalizing on a stop-signal task in order to assess the role of PMD and SMA in action control. Results and future lines of research will be sketched in the Concluding remarks.

The honeybee, Apis mellifera, is an established model for the study of olfactory processing, olfactory learning and memory, and the related plasticity. The primary centre for olfactory processing in the bee brain, the antennal lobe, has a very important function in odour coding and odour discrimination. Nevertheless, both its structure and its function are plastic. In this thesis, I analysed the structural antennal lobe plasticity related to associative learning, and that related to a non-associative experience, i.e. prolonged odour exposure, in the adult honeybee. Subsequently, I analysed the functional modification taking place in the latter case within the output units of the antennal lobe, showing that parallel structural and functional changes occur. In the last part of the thesis, I focused on the effects of a common neonicotinoid pesticide, imidacloprid, on antennal lobe function and the discrimination abilities of honeybees. I demonstrated that both are strongly impaired in the acute treatment of the brain with such substance.