Biology subjects

Masseck, O. A.

Publications and source records attributed to Masseck, O. A..

8 recordsLinked to original sources

bioRxiv2026

Serotonergic innervation and cortical progenitor regulation in human brain assembloids

Neuromodulatory signaling is classically associated with mature neural circuits, yet serotonergic projections reach the developing cortex prior to circuit formation, suggesting a role in early human cortical development. Here, we establish a human raphe-cortical assembloid platform by generating iPSC-derived hindbrain-patterned raphe organoids that produce serotonergic neurons and form projections into fused cortical organoids, exhibiting endogenous serotonin release within developing cortical tissue. Using this system, we find that serotonergic innervation is associated with a shift toward progenitor-enriched states, accompanied by increased proliferative activity, activation of developmental transcriptional programs, and predicted signaling interactions targeting cortical progenitors and neurons. Consistent with these findings, cortical regions receiving serotonergic projections exhibit increased mitotic activity, and pharmacological modulation demonstrates selective proliferative responses in basal progenitor populations, consistent with observations in human fetal tissue. Together, this system provides a framework to investigate how early neuromodulatory input shapes human cortical development and developmental vulnerability.

neuroscience↗

bioRxiv2025

Uncovering Hidden Phenotypes in NEX-Cre Mice: Behavioral and Cellular Alterations Demand Re-Evaluation of a Widely Used Transgenic Line

Transgenic mouse strains are essential tools in neuroscience, enabling targeted genetic manipulations to investigate brain function and neurological diseases. The NEX-Cre mouse line, which targets glutamatergic principal neurons in the neocortex and hippocampus by expressing Cre-recombinase under the NEX (NeuroD6) promotor, has been widely used for conditional gene manipulation. Contrary to previous reports suggesting no behavioral and histological abnormalities in NEX-Cre mice, our study reveals distinct behavioral and cellular phenotypes. Behavioral analyses indicate reduced anxiety-like behavior, anhedonia, and increased locomotor activity in NEX-Cre (-/-) mice. Additionally, SVM analysis uncovered subtle strain-specific and genotype-specific behavioral traits across all NEX-Cre genotypes relative to the commonly used C57BL/6J mouse strain. Histological analyses of Golgi-Cox-stained brain slices revealed alterations in dendritic spine density across key brain regions, including the caudate putamen, hippocampal CA1, nucleus accumbens core region, lateral septum, and medial prefrontal cortex. These findings highlight significant inter-and intra-strain variability, emphasizing the importance of careful characterization of transgenic models and the need for appropriate control groups and experimental designs to ensure the reliability and validity of studies utilizing Cre-Driver lines.

animal behavior and cognition↗

bioRxiv2024

PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities

Genetically encoded calcium (Ca2+) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.

neuroscience↗

bioRxiv2024

Fluorescence lifetime imaging of sDarken as a tool for the evaluation of serotonin levels

Recent advances in the development of genetically encoded biosensors have resulted in a variety of different neurotransmitter sensors for the precise measurement of the dynamics of neurotransmitters, neuromodulators, peptides and hormones in real time. However, intensity-based measurements of fluorescent biosensors are limited by their dependence on the expression level of the sensor, the intensity of the excitation light, and photobleaching overtime. Here, we show that the FLIM of sDarken (a GPCR-based genetically encoded sensor for serotonin) decreases with increasing serotonin concentrations. Different members of the sDarken family, with different affinities for serotonin, show concentration-dependent changes in fluorescence lifetime according to their dynamic range. We believe that this feature of sDarken is a value-adding complement to intensity-based information and may lead to a better understanding of serotonin dynamics in health and disease.

neuroscience↗

bioRxiv2023

Linking serotonergic median raphe input to dorsal CA1 with mnemonic functions

The role of serotonergic signaling within the hippocampus and its role in mnemonic function is still not well understood. We used optogenetics to specifically alter median raphe serotonin input to the dorsal CA1 subfield to investigate its role in hippocampus-dependent behavior. Unexpectedly, neither activation nor inhibition of serotonin release at dCA1 fiber terminals significantly modulated object recognition, spatial memory, or anxiety behavior. Nevertehless, we observed opposite effects of increased and decreased serotonergic input on spatial learning, and a marked reduction in the use of a direct search strategy in spatial navigation following activation of serotonergic terminals in dCA1, i.e. release of serotonin.Furthermore, since the 5-HT1A receptor is one of the most abundant serotonergic receptors in CA1, we also optogenetically activated 5-HT1A pathways specifically in pyrdamidal neurons of dCA1. Activation of 5-HT1A signaling significantly improved spatial memory without affecting object recognition or avoidance behavior. In conclusion, our data reveal modulatory effects of serotonin specifically on the acquisition of spatial memory.

neuroscience↗

bioRxiv2022

sDarken: Next generation genetically encoded fluorescent sensors for serotonin

We developed a new family of genetically encoded serotonin (5-HT) sensors (sDarken) on the basis of the native 5-HT1A receptor and circularly permuted GFP. sDarken 5-HT sensors are bright in the unbound state and diminish their fluorescence upon binding of 5-HT. Sensor variants with different affinities for serotonin were engineered to increase the versatility in imaging of serotonin dynamics. Experiments in vitro and in vivo showed the feasibility of imaging serotonin dynamics with high temporal and spatial resolution. As demonstrated here, the designed sensors showed excellent membrane expression, have high specificity, a superior signal-to-noise ratio, detect the endogenous release of serotonin and are suitable for two-photon in vivo imaging.

neuroscience↗

bioRxiv2021

Improved two-photon imaging of GPCR-based optogenetic neurotransmitter sensors using orthogonally polarized excitation

Fluorescent proteins such as GFP are best excited by light that is polarized parallel to the dipole axis of the fluorophore. In most cases, fluorescent proteins are randomly oriented, resulting in unbiased images even when polarized light is used for excitation, e.g. in two-photon microcopy. Here we reveal a surprisingly strong polarization sensitivity in a class of GPCR-based neurotransmitter sensors where the fluorophore is anchored on both ends. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. Our data reveal a major problem for two-photon measurements of neurotransmitter concentration that has not been recognized by the neuroscience community. To remedy the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization, removing the orientation bias from images. The passive device, which we inserted in the beam path of an existing two-photon microscope, also removed the strong direction bias in second harmonic generation (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.

neuroscience↗

bioRxiv2021

A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

plant biology↗