Human embryonic stem cells (hESCs) can exit the self‐renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self‐renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non‐neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca ²⁺ imaging and two-photon Na ⁺ imaging. GABA evoked Ca ²⁺ transients in astrocytes that persisted in the presence of GABA _A and GABA _B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca ²⁺ blocked GABA-induced Ca ²⁺ transients, and depletion of Ca ²⁺ stores with cyclopiazonic acid reduced Ca ²⁺ transients by approximately 90%. This indicates that the Ca ²⁺ transients depend on external Ca ²⁺ , but are mainly mediated by intracellular Ca ²⁺ release, conforming with Ca ²⁺ -induced Ca ²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca ²⁺ transients, whereas the InsP ₃ receptor blocker 2-APB inhibited the Ca ²⁺ transients. GABA also induced Na ⁺ increases in astrocytes, potentially reducing Na ⁺ /Ca ²⁺ exchange. To test whether reduction of Na ⁺ /Ca ²⁺ exchange induces Ca ²⁺ signaling, we inhibited Na ⁺ /Ca ²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca ²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca ²⁺ transients in astrocytes. The significance of GABAergic Ca ²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na ⁺ rises that reduce Na ⁺ /Ca ²⁺ exchange, thereby leading to a Ca ²⁺ increase sufficient to trigger Ca ²⁺ -induced Ca ²⁺ release via InsP ₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.

Neurotransmission at different synapses is highly variable, and cell-adhesion molecules like α-neurexins (α-Nrxn) and their extracellular binding partners determine synapse function. Although α-Nrxn affect transmission at excitatory and inhibitory synapses, the contribution of neurexophilin-1 (Nxph1), an α-Nrxn ligand with restricted expression in subpopulations of inhibitory neurons, is unclear. To reveal its role, we investigated mice that either lack or overexpress Nxph1. We found that genetic deletion of Nxph1 impaired GABA_B receptor (GABA_BR)-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami, a region where Nxph1 is normally expressed at high levels. To test the conclusion that Nxph1 supports presynaptic GABA_BR, we expressed Nxph1 ectopically at excitatory terminals in the neocortex, which normally do not contain this molecule but can be modulated by GABA_BR. We generated Nxph1-GFP transgenic mice under control of the Thy1.2 promoter and observed a reduced short-term facilitation at these excitatory synapses, representing an inverse phenotype to the knockout. Consistently, the diminished facilitation could be reversed by pharmacologically blocking GABA_BR with CGP-55845. Moreover, a complete rescue was achieved by additional blocking of postsynaptic GABA_AR with intracellular picrotoxin or gabazine, suggesting that Nxph1 is able to recruit or stabilize both presynaptic GABA_BR and postsynaptic GABA_AR. In support, immunoelectron microscopy validated the localization of ectopic Nxph1 at the synaptic cleft of excitatory synapses in transgenic mice and revealed an enrichment of GABA_AR and GABA_BR subunits compared with wild-type animals. Thus, our data propose that Nxph1 plays an instructive role in synaptic short-term plasticity and the configuration with GABA receptors. Significance Communication between neurons via synapses is essential for information processing and cognitive function in our brains and is found impaired in many neuropsychiatric disorders. Synaptic transmission is remarkably variable in strength, and cell-adhesion molecules as neurexins and their binding partners are candidates to regulate neurotransmission. This study changes our understanding of how neurotransmission can be adapted to local demands by investigating the previously undescribed functions of neurexophilins, arguably the most elusive ligands of α-neurexins. Neurexophilins are expressed only in subpopulations of synapses, and their presence is able to change short-term plasticity and molecular composition at these terminals.

Optogenetics has revolutionized neurobiological research by allowing to disentangle intricate neuronal circuits at a spatio-temporal precision unmatched by other techniques. Here, we review current advances of optogenetic applications in mammals, especially focusing on freely moving animals. State-of-the-art strategies allow the targeted expression of opsins in neuronal subpopulations, defined either by genetic cell type or neuronal projection pattern. Optogenetic manipulations of these subpopulations become particularly powerful when combined with behavioral paradigms and neurophysiological readout techniques. Thereby, specific roles can be assigned to identified cells. All-optical approaches with the opportunity to write complex three dimensional patterns into neuronal networks have recently emerged. While clinical implications of the new tool set seem tempting, we emphasize here the role of optogenetics for basic research.