Beside its unique nutritional content breast milk also contains live cells from the mother. Fate of these cells in the offspring has not been adequately described. In this study, we aimed to detect and identify maternal cells in the suckling’s blood and the brain. Green fluorescent protein expressing transgenic female mice (GFP+) were used as foster mothers to breastfeed wildtype newborn pups. One week and two months after the birth, blood samples and brains of the sucklings were analyzed to detect presence of GFP+ cells by fluorescence activated cell sorting, polymerase chain reaction and immunohistochemistry on the brain sections and optically cleared brains. The tests confirmed that maternal cells were detectable in the blood and the brain of the pups and that they differentiated into both neuronal and glial cell types in the brain. This phenomenon represents breastfeeding – induced microchimerism in the brain with functional implications remain to be understood.

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H₂O₂) in multiple cell locales (nucleus, cytosol, mitochondria) using the H₂O₂ biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H₂O₂ signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H₂O₂ levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H₂O₂, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H₂O₂ accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

Background/aim: Dorsal root ganglia (DRG) are structures containing primary sensory neurons. Intraganglionic (IG) and intrathecal (IT) applications are the most common methods used for viral vector transfer to DRG. We aim to compare the efficiencies and pathological effects of IT and IG viral vector delivery methods to DRG, through in vivo imaging. Materials and methods: Mice were divided into four groups of six each: IT, IG, IT-vehicle, and IG-vehicle. Adeno-associated virus (AAV) injection was performed for EGFP expression in IT/IG groups. DRGs were made visible through vertebral window surgery and visualized with multiphoton microscopy. After imaging, spinal cords and DRGs were removed and cleared, then imaged with light sheet microscopy. Results: No neuronal death was observed after IT injection, while the death rate was 17% 24 h after IG injection. EGFP expression efficiencies were 90%?95% of neurons in both groups. EGFP expression was only observed in targeted L2 DRG after IG injection, while it was observed in DRGs located between L1-L5 levels after IT injection. Conclusion: IT injection is a more suitable method for labeling DRG neurons in neurodegenerative injury models. However, when the innervation of DRG needs to be specifically studied, IT injection reduces this specificity due to its spread. In these studies, IG injection is the most suitable method for labeling single DRG neurons.

Parkinson’s disease (PD) is the second most common neurodegenerative disease with an estimation of 10 million people living with the disease and it is increasing in prevalence every year. Familial cases of PD are source of valuable information to determine genetical risk factors yet sporadic cases can emerge from distinct mechanisms so, identifying common pathways of familial and sporadic cases of PD may provide worthwhile insights to determine underlying mechanisms through the progression. LRRK2 mutations are the most common indicators of both sporadic and familial cases of PD and α-synuclein aggregation is one of the hallmarks of both cases of PD as well as in other synucleinopathies. As in the case of most neurological diseases, human studies addressing the molecular basis of pathology are generally restricted to post-mortem materials. For this reason, cell culture systems and animal models are widely used. There are two main approaches for modelling PD: genetically constructed PD models and neuroxin-based models. In this study, we aim to construct and compare both approaches by overexpressing wild-type (WT) and A53T mutant α-synuclein and treating cells with well-known neurotoxin 6-hydroxidopamine (6-OHDA) using dopaminergic human neuroblastoma SH-SY5Y cell line. Our findings suggest that WT or A53T α-synuclein overexpression by itself is not sufficient to cause significant toxicity in SH-SY5Y cells in the presented time scale. As expected, 6-OHDA treatment caused toxicity with an IC50 value of ~100 µM. In addition, 6-OHDA treatment causes 3- and 2.5-fold increase in SNCA and LRRK2 expression respectively.

Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.