Integrated laboratory classes to learn physiology in a psychology degree: impact on student learning and experience.

Physiology is a fundamental discipline to be studied in most Health Science studies including Psychology. Physiology content is perceived by students as rather difficult, who may lack vision on how to relate it with their professional training. Therefore, identifying novel active and more engaging pedagogical strategies for teaching physiology to psychology students may help to fill this gap. In this pilot study, we used the PBL methodology developed around a clinical case to evaluate psychology students' experience and learning in two laboratory classes modalities. The aim of this study was to compare the undergraduates' preference for laboratory classes taught either independently (cohort 1, n = 87 students) or integrated into the PBL-oriented clinical case (cohort 2, n = 92 students) for which laboratory classes were transformed into Integrated Laboratory Classes (ILCs). The students' academic performance was also evaluated to look for quantitative differences between cohorts. We found similar overall academic scores for the Physiology course between cohorts. Interestingly, when we compared the academic scores obtained in the theoretical content from each cohort, we found a significant improvement (p < 0.05) in cohort 2 where the students achieved better results as compared to cohort 1. A subset of students was asked to fill a questionnaire assessment on their experience and found that 78.9% of them preferred integrated laboratory classes over laboratory classes alone. They consistently reported a better understanding of the theoretical content and the value they gave to ILCs for learning. In conclusion, our pilot study suggests that integrating laboratory classes into PBL-oriented clinical contexts help to retain core physiology contents and it can be considered as an engaging learning activity worth implementing in Psychology teaching.

Laser-Capture Microdissection for the Analysis of Rat and Human Spinal Cord Ependyma by qPCR.

In the last few decades many efforts have been dedicated to decipher the nature and regenerative potential of neurogenic niches and endogenous stem cells after damage of the central nervous system. In the spinal cord, it has been largely focused on the ependymal region, which hosts neural precursors/stem cells (NSC) in rodents but differs between species and ages. In the current chapter, we detail our protocol to study the gene expression profile of this region using fresh frozen blocks of rat and human post-mortem spinal cords. We describe how to prepare and process those tissues, how to identify and dissect the ependymal region using Laser-Capture Microdissection (LCMD), and how to isolate and amplify RNA with different integrity states to finally obtain enough material for performing gene expression assays using Taqman® Low Density Arrays. LCMD technique maintains tissue integrity allowing for subsequent analysis without manipulation steps that may alter molecular properties of cells and the eventual loss of delicate cell types in comparison with other approaches that require previous disaggregation of the tissue and cell manipulation before isolation.

Neuroimmmune interactions of cannabinoids in neurogenesis: focus on interleukin-1ß (IL-1ß) signalling.

Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Activation of cannabinoid receptors suppresses chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the endocannabinoid system directs cell fate specification of NSCs (neural stem cells) in the CNS (central nervous system). The aim of our work is to understand better the relationship between the endocannabinoid and the IL-1ß (interleukin-1ß) associated signalling pathways and NSC biology, in order to develop therapeutical strategies on CNS diseases that may facilitate brain repair. NSCs express functional CB1 and CB2 cannabinoid receptors, DAGLα (diacylglycerol lipase α) and the NSC markers SOX-2 and nestin. We have investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators [IL-1ß and IL-1Ra (IL-1 receptor antagonist)] that control NSC fate decisions. Pharmacological blockade of CB1 and/or CB2 cannabinoid receptors abolish or decrease NSC proliferation, indicating a critical role for both CB1 and CB2 receptors in the proliferation of NSC via IL-1 signalling pathways. Thus the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by IL-1 signalling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult NSCs, and this may be of therapeutic interest in the emerging field of brain repair.

The endocannabinoid system modulates a transient TNF pathway that induces neural stem cell proliferation.

Evidence is emerging that the tumour necrosis factor (TNF-alpha) is a potent signal that induces neural stem cell proliferation and migration. We show that NSC self-renewal is controlled by bi-directional cross-talk between the endocannabinoid system and the TNF signalling pathway. By blocking endogenous TNF-alpha activity, we demonstrate that the TNF system is critical for the proliferation of NSC. Furthermore, we show that pharmacological blockade of the CB1/CB2 cannabinoid receptors dramatically suppresses TNF-alpha-induced NSC proliferation. Interestingly, we found that CB1 or CB2 agonists induce NSC proliferation coupled to a significant increase in both TACE/ADAM 17 and TNF-alpha levels. Overall these data suggest a novel mode of action for the endocannabinoid system in NSC proliferation that is coupled to TNF signalling and that may be of therapeutic interest in the emerging field of brain repair.

Gene marking of human neural stem/precursor cells using green fluorescent proteins.

BACKGROUND: Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. METHODS: hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. RESULTS: Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. CONCLUSIONS: Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of hNSCs, and is highly useful in vivo. The nature, properties, and possible side effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.

Long-term molecular and cellular stability of human neural stem cell lines.

Human Neural Stem Cells (hNSCs) are excellent candidates for in vitro and in vivo molecular, cellular, and developmental research, and also for ex-vivo gene transfer and cell therapy in the nervous system. However, hNSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. Here, we show that long-term cultured (up to 4 years) v-myc perpetuated hNSC lines do preserve short but stable and homogeneous telomeres (TRF and Q-FISH determinations). hNSC lines (but not strains) express high levels of telomerase activity, which is activated by v-myc, as demonstrated here. Telomerase activity is not constitutive, becoming non-detectable after differentiation (in parallel to v-myc down-regulation). hNSC lines also maintain a stable cell cycle length, mitotic potential, differentiation and neuron generation capacity, and do not express senescence-associated beta-galactosidase over years, as studied here. These data, collectively, help to explain the immortal nature of v-myc-perpetuated hNSC lines, and to establish them as excellent research tools for basic and applied neurobiological and translational studies.