Immediate early genes regulation in rat cerebellar cortex during long-term synaptic plasticity induction

The cerebellum is one of the brain areas involved in learning and memory formation. Long-term synaptic plasticity is thought to play a pivotal role in supporting these functions. Moreover Immediate Early Genes (IEGs) expression and de novo protein synthesis and/or modification have been strictly associated with maintenance of Long-Term Potentiation (LTP) as well as memory consolidation and storage. Two highly conserved signalling cascades, PKA and MAPK, seem to be involved in early- to late-LTP conversion; both pathway can activate CREB transcription factor through phosphorylation and P-CREB has been suggested to initiate the protein synthesis leading to late-LTP induction. The transcription factor c-fos is known to be rapidly and transiently induced in the Nervous System by a variety of stimuli and is thought to be directly involved in processes of neuronal plasticity including LTP. We used rat parasagittal cerebellar slices as a model system in which specific patterns of stimulation delivered to the mossy fibers can induce both Long-Term Potentiation and Long-Term Depression (LTD), depending on local inhibition and other regulating factors. Using Voltage Sensitive Dye (VSD) imaging we obtained high-resolution maps of the spatial distribution of LTP/LTD induced from a Teta Burst Stimulus (TBS) application. Control and stimulated slices were fixed at different times from the TBS application and processed for in situ hybridization or immunohystochemistry in order to detect IEGs mRNA expression patterns and protein expression/modifications. The expression pattern of c-fos and CREB mRNAs and their protein distribution and/or phosphorylation were then correlated with LTP/LTD maps generated by VSD imaging. Preliminary data indicate a significant increase of P-CREB in the granular layer suggesting that CREB phosphorylation is induced as early as 15 minutes post TBS application. In situ hybridization experiments indicate a good correlation between c-fos and CREB mRNAs up-regulation and LTP distribution at 120 minutes post TBS. At the protein level, the comparison of immunofluorescence signals and VSD immaging data indicate a clear correlation between c-Fos and P-CREB distribution and synaptic plasticity patterns. We are planning further experiments to confirm these data and to test our experimental system in the presence of drugs that could interfere with the transcription, translation or post-translational protein regulation

Immediate early genes expression in the cerebellar cortex correlates with LTP and LTD induction

The consolidation of changes following activity-dependent neural plasticity are believed to involve specific patterns of gene expression. In the hippocampus, immediate early genes are thought to contribute to long-term synaptic plasticity (LTP and LTD); this phenomenon may occur also in the cerebellum, in which the transcription factors c-Fos and P-CREB have been identified. The cerebellum granular layer (GL) can manifest both LTP and LTD following a Theta Burst Stimulus (TBS) delivered to the mossy fibers. We have employed VSD imaging in rat cerebellar slices (P18-24) in order to map the spatial distribution of LTP and LTD in the cerebellum GL. Fluorescence changes were correlated to LTP or LTD in two different post-TBS time ranges (15 and 120 min). Slices were then fixed and processed for immunohistochemistry in order to identify levels of c-Fos and P-CREB expression. The induction of long-term plasticity increased the average level of P-CREB both at 15 min (+39±4.9, p<0.01%) and 120 min (+24±7.2, p<0.05%) after TBS. The level of c-Fos was unaltered at 15 min, while it significantly increased at 120 min (+37±8.9, p<0.05%). By spatially correlating longterm synaptic plasticity with the corresponding variation of P-CREB and c-Fos, we observed that regions showing LTP well correlated (p<0.05) with positive variations of P-CREB and c-Fos. Conversely, areas showing LTD correlated exclusively (p<0.05) with negative variations of P-CREB. Slices were also evaluated by in situ hybridization and a similar analysis was performed. The levels of fos and CREB mRNA expression and their spatial correlation with the sign of long-term synaptic plasticity corresponded with the immunohistochemical results. As a further test, VSD recordings showed that the induction of granular layer LTP and LTD could be prevented by applying 50 mM D-APV, a selective NMDA receptor blocker. Moreover, in situ hybridization and immunohistochemistry analysis evidenced that in these conditions both mRNA and protein expression levels of c-fos and CREB were unchanged, confirming the involvement of these two transcription factors in cerebellar granular layer plasticity

Evaluation of the effects of a dynamic culture on osteogenic differentiation of oral-periosteal cells grown on PLGA sponges

Oral-periosteum derived stem cells represent an innovative cell source for bone tissue engineering applications in terms of accessibility and self-commitment towards osteogenic lineage [1]. In this scenario, biomaterials play a pivotal role in tissue engineering in supporting stem cells growth and regeneration of tissue defects [2]. Among these biomaterials, Fisiograft®, a synthetic co-polymer composed of polylactic and polyglycholic acids produced by Ghimas (Bologna, Italy), is highly biocom- patible and completely absorbed within 4-6 months. In particular, Fisiograft® sponges are normally used in dental applications to fix completely periodontal defects without damage Schneider’s membrane. We evaluated the osteogenic potential of Fisiograft® sponges on oral-periosteal cells derived from patients undergoing dental extractions. For this purpose, we created a dynamic culture based on a rotating apparatus in which we seeded periosteal cells with Fisiograft® sponges for 7, 14 and 21 days without adding osteogenic supplement in the medium. Osteoblast differen- tiation of cells was evaluated by Alizarin Red S staining and by qRT-PCR on genes involved in bone development. Results show that Fisograft® sponges promote greater osteogenic differentiation of cells in the dynamic culture with respect to standard condition already at 14 days, as demonstrated by Alizarin Red staining. BMP-2 and Osteoprotegerin genes are highly expressed by cells grown on Fisiograft® sponges in dynamic culture at 14 days with respect to plastic culture. Taken together, these results confirm the osteogenic potential of Fisiograft® sponges in accelerating the dif- ferentiation of cells to an osteoblast phenotype (already to 14 days of culture) without any osteogenic induction. The combination of this PLGA biomaterial and oral-peri- osteal cells could represent a promising bio-complex in maxillo-facial tissue repair

Study of the effects of different biomaterials on osteogenic differentiation of oral-periosteal cells

Bone regeneration is currently one of the most important challenges for regenerative medicine and it is considered an ideal clinical strategy in the maxillo-facial area [1]. Bone resorption of alveolar crest occurring after tooth extraction leads to several risks for future treatments, including dental implants. For this reason, alveolar ridge preservation (ARP) has become a key component of contemporary clinical dentistry. Several clinical techniques and bone substitute materials can be used to fill the socket after tooth extraction. For all of them, the principle aim is to keep the shape and the size of the bone socket of the extracted tooth allowing inserting the dental implants [2]. The goal of our study was to compare different biocompatible scaffolds based on PLGA (Fisiograft®), Bioglass (Activioss®) and collagen (Sombrero®) in an in vitro model of tissue engineering for dental applications. The cells used in our study derived from Periosteum obtained from four different patients that underwent socket preservation selected by the School of Dentistry of the University of Pavia, previous informed consent. We created bio-complexes constituted by mesenchymal-periosteal cells seeded on different types of biomaterials and we performed adhesion, morphological, proliferative and bone differentiation analyses at different time points (7, 14 and 28 days of culture) in proliferative and osteogenic conditions. Bone differentiation was evaluated by qRT-PCR on genes involved in osteoblast development, like BMP-2, Osteocalcin and Periostin. Our results demonstrated that Sombrero® enhanced adhesion and proliferation of periosteal cells, as highlighted by Haematoxylin-Eosin staining and XTT test (3 and 7 days). Long-term studies (14 and 28 days) demonstrated that periosteal differentiation is about the same among the different materials tested. From these preliminary studies we can conclude that it could be advantageous the clinical use of both collagenic and PLGA scaffolds in order to ameliorate initial colonization and subsequent mechanical support in maxillo-bone regeneration. This work was supported by grant from NATO 2016 (“RAWINTS” (G-984961): RApid Skin Wound healing by INtegrated Tissue engineering and Sensing)

Study of an intrinsically safe infrastructure for training and research on nuclear technologies

Within European Partitioning & Transmutation research programs, infrastructures specifically dedicated to the study of fundamental reactor physics and engineering parameters of future fast-neutron-based reactors are very important, being some of these features not available in present zero-power prototypes. This presentation will illustrate the conceptual design of an Accelerator-Driven System with high safety standards, but ample flexibility for measurements. The design assumes as base option a 70MeV, 0.75mA proton cyclotron, as the one which will be installed at the INFN National Laboratory in Legnaro, Italy and a Beryllium target, with Helium gas as core coolant. Safety is guaranteed by limiting the thermal power to 200 kW, with a neutron multiplication coefficient around 0.94, loading the core with fuel containing Uranium enriched at 20% inserted in a solid-lead diffuser. The small decay heat can be passively removed by thermal radiation from the vessel. Such a system could be used to study, among others, some specific aspects of neutron diffusion in lead, beam-core coupling, target cooling and could serve as a training facility

Expression of proteins involved in calcium homeostasis during crista ampullaris regeneration

Most of the events related to the mechano-transduction process in hair cell are regulated by cytoplasmic Ca++ concentrations. Ca++ influx trough the mechano-transduction channels regulates the adaptation process in the stereocilia, while Ca++ entering into the cell through the voltage gated channels regulates the neurotransmitter release. Moreover Ca++ activates the K+ channels that dominate the inhibitory postsynaptic potential at efferent synapses. Cytoplasmic Ca++ concentration is regulated by a complex system of proteins, including the Na+/Ca++ exchanger, Ca++ pumps (PMCAs, SERCAs) and a copious supply of diffusible proteins that buffers free Ca++ in specific cell compartments. These proteins intercept Ca++ near its sites of entry and restrict the spread of the free ion. Moreover this buffering limits the period during which Ca++ local concentration are high enough to trigger its physiological effects. In frog crista ampullaris PMCAs seems to be the most relevant mechanism of Ca++ extrusion since the lack of a functional Na+/Ca++ exchanger. We investigated the distribution of different subtypes of PMCAs finding that each isoform has its specific expression domain along the crista. We also demonstrate a similar compartmentalization for the IP3R, which is involved in Ca++ release from the IP3-sensitive intracellular stores, and for some buffering proteins. These data and the morphological identification of diverse hair cell types differentially distributed along the crista ampullaris, suggest the presence in the sensory epithelium of distinct functional domains where the mechanisms of Ca++ homeostasis regulation are not completely overlapping. This hypothesis is confirmed by the observation that the different regions of the crista ampullaris show a different sensibility to the ototoxic damage induced by gentamicin treatment: although the mechanism of actions of this drug has not jet completely understood, aminoglycosides are known to interfere with Ca++ homeostasis. Our studies on frog crista ampullaris regeneration showed that the complete crista functional activity is restored well afterwards its morphological recovery. Indeed, while the appearance of a stereociliary apparatus expressing a correct pattern of Ca++ pumps seems to be sufficient to restore hair cell basal activity, the recovery of the evoked sensory discharge seems to need also the expression of Ca++ buffering proteins. This study try to correlate the appearance of the different proteins involved in Ca++ homeostasis with the complete functional recovery of the sensory epithelium