In vivo two-photon imaging of the embryonic cortex reveals spontaneous ketamine-sensitive calcium activity.

Prior to sensory experience spontaneous activity appears to play a fundamental role in the correct formation of prominent functional features of different cortical regions. The use of anaesthesia during pregnancy such as ketamine is largely considered to negatively affect neuronal development by interfering with synaptic transmission. Interestingly, the characteristics of spontaneous activity as well as the acute functional effects of maternal anaesthesia remain largely untested in the embryonic cortex in vivo. In the present work, we performed in vivo imaging of spontaneous calcium activity and cell motility in the marginal zone of the cortex of E14-15 embryos connected to the mother. We made use of a preparation where the blood circulation from the mother through the umbilical cord is preserved and fluctuations in intracellular calcium in the embryonic frontal cortex are acquired using two-photon imaging. We found that spontaneous transients were either sporadic or correlated in clusters of neuronal ensembles at this age. These events were not sensitive to maternal isoflurane anaesthesia but were strongly inhibited by acute in situ or maternal application of low concentration of the anaesthetic ketamine (a non-competitive antagonist of NMDA receptors). Moreover, simultaneous imaging of cell motility revealed a correlated strong sensitivity to ketamine. These results show that anaesthetic compounds can differ significantly in their impact on spontaneous early cortical activity as well as motility of cells in the marginal zone. The effects found in this study may be relevant in the etiology of heightened vulnerability to cerebral dysfunction associated with the use of ketamine during pregnancy.

Active diffusion of nanoparticles of maternal origin within the embryonic brain.

AIM: To investigate porous silicon (PSi) nanoparticles (NPs) behavior in the embryonic brain. MATERIALS & METHODS: Fluorescently labeled PSi NPs were injected into the embryonic brains intraventricularly and to the mother intravenously (iv.). Brain histology from different time points up to 3 days was analyzed and live brains imaged with two-photon microscopy. RESULTS: PSi NPs were able to penetrate 80% of the embryonic cortical depth. Particle motility was confirmed in real-time in vivo. PSi NPs were able to penetrate the embryonic cortex after either iv. maternal or intraventricular injection. No developmental of macromorphological changes or increased cell apoptosis was observed. CONCLUSION: PSi NPs penetrate deep in the brain tissues of embryos after intraventricular injection and after iv. injection to the mother.

In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex.

The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.