Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well-established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.

Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication.

Although urotensin II (UII) and somatostatin 1 (SS1) exhibit some structural similarities, their precursors do not show any appreciable sequence identity and, thus, it is widely accepted that the UII and SS1 genes do not derive from a common ancestral gene. The recent characterization of novel isoforms of these two peptides, namely urotensin II-related peptide (URP) and somatostatin 2 (SS2)͞ cortistatin (CST), provides new opportunity to revisit the phylogenetic relationships of UII and SS1 using a comparative genomics approach. In the present study, by radiation hybrid mapping and in silico sequence analysis, we have determined the chromosomal localization of the genes encoding UII-and somatostatin-related peptides in several vertebrate species, including human, chicken, and zebrafish. In most of the species investigated, the UII and URP genes are closely linked to the SS2͞CST and SS1 genes, respectively. We also found that the UII-SS2͞CST locus and the URP͞SS1 locus are paralogous. Taken together, these data indicate that the UII and URP genes, on the one hand, and the SS1 and SS2͞CST genes, on the other hand, arose through a segmental duplication of two ancestral genes that were already physically linked to each other. Our results also suggest that these two genes arose themselves through a tandem duplication of a single ancestral gene. It thus appears that the genes encoding UII-and somatostatinrelated peptides belong to the same superfamily.duplication ͉ multigenic family ͉ neuropeptides ͉ radiation hybrid mapping

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo, in vivo) and the clinical considerations will also be discussed.

During early postnatal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer (IGL) while basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML).We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissuetype plasminogen activator (tPA) from GN but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL and IGL. Interestingly, plasminogen activator inhibitor 1 (PAI-1) reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through extracellular matrix (EM) degradation.Keywords: PACAP, tPA, granule neurons, basket and stellate cells, interneuron migration, cerebellum Raoult et al, page 4 4 During brain development, the migration of immature neurons from germinative zones to their final destination is essential for the establishment of proper neuronal circuits (Rakic 1990;Hatten 1999; Yacubova 2003, Evsyukova et al. 2013).Impairment of this process results in either cell death or misplacement of the neurons, leading to deficiency of diverse brain functions (Rakic 1988;Flint and Kriegstein 1997;Gressens 2006). In the developing cerebellum, immature granule neurons (GN; excitatory interneurons) and basket/stellate cells (B/SC; inhibitory interneurons) originate from two separate germinative zones, and exhibit distinct modes of migration over the same developmental period i.e. the first 3 postnatal weeks (Komuro and Yacubova 2003; Consalez and Hawkes 2013). Thus, upon completion of their final mitosis, GN migrate tangentially within the external granular layer (EGL) and then change direction to migrate radially along the processes of Bergmann glial cells through the molecular layer (ML) (Komuro et al. 1998(Komuro et al. , 2001. When entering the Purkinje cell layer (PCL), GN detach from glial cells and slow down (Komuro and Rakic 1998). Two hours later, GN resume their migration and cross the border between the PCL and the internal granular layer (IGL). Within the IGL, GN migrate radially until reaching their final position at the bottom of the IGL (Komuro and Rakic 1998). In contrast to GN, less is known for B/SC migration but they exhibit a centrifugal move during early post-natal development from the deep white matter to the ML (Zhang and Goldman 1996;Milosevic and Goldman 2004) where they complete their migration in four phases (Cameron et al. 2009a). Raoult et a...

During cortex development, fine interactions between pyramidal cells and migrating GABA neurons are required to orchestrate correct positioning of interneurons, but cellular and molecular mechanisms are not yet clearly understood. Functional and age-specific expression of NMDA receptors by neonate endothelial cells suggests a vascular contribution to the trophic role of glutamate during cortical development. Associating functional and loss-of-function approaches, we found that glutamate stimulates activity of the endothelial proteases MMP-9 and t-PA along the pial migratory route (PMR) and radial cortical microvessels. Activation of MMP-9 was NMDAR-dependent and abrogated in t-PA −/− mice. Time-lapse recordings revealed that glutamate stimulated migration of GABA interneurons along vessels through an NMDAR-dependent mechanism. In Gad67-GFP mice, t-PA invalidation and in vivo administration of an MMP inhibitor impaired positioning of GABA interneurons in superficial cortical layers, whereas Grin1 endothelial invalidation resulted in a strong reduction of the thickness of the pial migratory route, a marked decrease of the glutamate-induced MMP-9-like activity along the PMR and a depopulation of interneurons in superficial cortical layers. This study supports that glutamate controls the vessel-associated migration of GABA interneurons by regulating the activity of endothelial proteases. This effect requires endothelial NMDAR and is t-PA-dependent. These neurodevelopmental data reinforce the debate regarding safety of molecules with NMDA-antagonist properties administered to preterm and term neonates.

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders.

Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.