Miquel Bosch

Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.

Acetylcholine and the cholinergic system are crucial to brain function, including functions such as consciousness and cognition. Dysregulation of this system is implicated in the pathophysiology of neurological conditions such as Alzheimer's disease. For this reason, cholinergic neuromodulation is relevant in both basic neuroscience and clinical neurology. In this study, we used photopharmacology to modulate neuronal activity using the novel selective type-1 muscarinic (M1) photoswitchable drugs: the agonist benzyl quinolone carboxylic acid-azo-iperoxo (BAI) and the antagonist cryptozepine-2. Our aim was to investigate the control over these cholinergic receptors using light and to investigate the effects of these drugs on physiological spontaneous slow waves and on epileptic activity in the cerebral cortex. First, we used transfected HEK cell cultures and demonstrated BAI’s preferential activation of M1 muscarinic acetylcholine receptors (mAChRs) compared with M2 mAChRs. Next, we found that white-light illumination of BAI increased the frequency of spontaneous slow-wave activity in brain cortical networks of both active slices and anesthetized mice, through M1-mAChRs activation. Illumination of cryptozepine-2 with UV light effectively suppressed not only the muscarinic-induced increase in slow-wave frequency, but also muscarinic-induced epileptiform discharges. These findings not only shed light on the role of M1 acetylcholine receptors in the cortical network dynamics but also lay the groundwork for developing advanced light-based pharmacological therapies. Photopharmacology offers the potential for high-precision spatiotemporal control of brain networks with high pharmacological specificity in both healthy and pathological conditions.

Graphene-based transistors (gSGFETs) enabled stable full-band brain recordings for 5+ months, allowing precise brain state identification and prediction, which is critical both in brain science and neurology.

Adult neurogenesis persists in mammals in the neurogenic zones, where newborn neurons are incorporated into preexisting circuits to preserve and improve learning and memory tasks. Relevant structural elements of the neurogenic niches include the family of cell adhesion molecules (CAMs), which participate in signal transduction and regulate the survival, division, and differentiation of radial glial progenitors (RGPs). Here we analyzed the functions of neural cell adhesion molecule 2 (NCAM2) in the regulation of RGPs in adult neurogenesis and during corticogenesis. We characterized the presence of NCAM2 across the main cell types of the neurogenic process in the dentate gyrus, revealing different levels of NCAM2 amid the progression of RGPs and the formation of neurons. We showed that Ncam2 overexpression in adult mice arrested progenitors in an RGP-like state, affecting the normal course of young-adult neurogenesis. Furthermore, changes in Ncam2 levels during corticogenesis led to transient migratory deficits but did not affect the survival and proliferation of RGPs, suggesting a differential role of NCAM2 in adult and embryonic stages. Our data reinforce the relevance of CAMs in the neurogenic process by revealing a significant role of Ncam2 levels in the regulation of RGPs during young-adult neurogenesis in the hippocampus.

Adult neurogenesis persists in mammals in the neurogenic zones where newborn neurons are incorporated into existing neuronal circuits. Relevant molecular elements of the neurogenic niches include the family of Cell Adhesion Molecules (CAM), which participate in signal transduction and regulate radial glial progenitor's (RGPs) survival, division and differentiation. The Neural Cell Adhesion Molecule 2 (NCAM2) is expressed in brain development and in adult stages, and controls dendrite arborisation and synaptic formation and maintenance during development. Nevertheless, the role of NCAM2 in neurogenesis and lineage progression is not well understood. Here we analyse the functions of NCAM2 in the regulation of RGPs in adult neurogenesis in the dentate gyrus and during corticogenesis, by using different lentiviral-mediated genetic approaches to modulate its expression, both in vivo and in vitro. First, we characterized the expression of NCAM2 among the main actors of the neurogenic process revealing different levels of NCAM2 amid the progression of RGPs and the formation of juvenile neurons. Further, we show that overexpression of NCAM2 arrest infected cells in a RGP-like state, with characteristic morphological, immunocytochemical and electron microscopy features. In contrast, NCAM2 overexpression in embryonic cortical progenitors does not seems to alter cell fate, but causes transient migration deficits. These results reveal a differential role of NCAM2 in the regulation of adult and embryonic RGPs, and specifically, a significant implication of NCAM2 in the regulation and progression of RGPs during adult neurogenesis in the hippocampus.

Epithelial cell divisions must be tightly coordinated with cell loss to preserve epithelial integrity. However, it is not well understood how the rate of epithelial cell division adapts to changes in cell number, for instance during homeostatic turnover or upon wounding of epithelia. Here, we show epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell cycle progression. We demonstrate that tensile forces on E-cadherin adhesions are reduced as local cell density increases, which prompts the accumulation of the G2 checkpoint kinase Wee1. This elevated abundance of Wee1 results in inhibitory phoshorylation of Cdk1, and thereby establishes a pool of cells that is temporarily halted in G2-phase. Importantly, these cells are readily triggered to divide upon epithelial wounding, due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data thus demonstrate that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.

Phosphorylation of eEF1A2 coordinates a transient pause in protein synthesis with cytoskeletal remodeling for synaptic plasticity.

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.