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The oviduct plays a crucial role in the reproductive process, serving as the stage for fertilization and the early stages of embryonic development. When the environment of this organ has been mimicked, it has been shown to enhance in vitro embryo epigenetic reprogramming and to improve the yield of the system. This study explores the anatomical intricacies of two oviduct regions, the uterotubal junction (UTJ) and the ampullary-isthmic junction (AIJ) by using micro-computed tomography (MicroCT). In this study, we have characterized and 3D-reconstructed the oviduct structure, by measuring height and width of the oviduct's folds, along with the assessments of fractal dimension, lacunarity and shape factor. Results indicate distinct structural features in UTJ and AIJ, with UTJ displaying small, uniformly distributed folds and high lacunarity, while AIJ shows larger folds with lower lacunarity. Fractal dimension analysis reveals values for UTJ within 1.189-1.1779, while AIJ values range from 1.559-1.770, indicating differences in structural complexity between these regions. Additionally, blind sacs or crypts are observed, akin to those found in various species, suggesting potential roles in sperm sequestration or reservoir formation. These morphological differences align with functional variations and are essential for developing an accurate 3D model. In conclusion, this research provides information about the oviduct anatomy, leveraging MicroCT technology for detailed 3D reconstructions, which can significantly contribute to the understanding of geometric-morphological characteristics influencing functional traits, providing a foundation for a biomimetic oviduct-on-a-chip. [ABSTRACT FROM AUTHOR]

The oviduct plays a crucial role in the reproductive process, serving as the stage for fertilization and the early stages of embryonic development. When the environment of this organ has been mimicked, it has been shown to enhance in vitro embryo epigenetic reprogramming and to improve the yield of the system. This study explores the anatomical intricacies of two oviduct regions, the uterotubal junction (UTJ) and the ampullary-isthmic junction (AIJ) by using micro-computed tomography (MicroCT). In this study, we have characterized and 3D-reconstructed the oviduct structure, by measuring height and width of the oviduct’s folds, along with the assessments of fractal dimension, lacunarity and shape factor. Results indicate distinct structural features in UTJ and AIJ, with UTJ displaying small, uniformly distributed folds and high lacunarity, while AIJ shows larger folds with lower lacunarity. Fractal dimension analysis reveals values for UTJ within 1.189–1.1779, while AIJ values range from 1.559–1.770, indicating differences in structural complexity between these regions. Additionally, blind sacs or crypts are observed, akin to those found in various species, suggesting potential roles in sperm sequestration or reservoir formation. These morphological differences align with functional variations and are essential for developing an accurate 3D model. In conclusion, this research provides information about the oviduct anatomy, leveraging MicroCT technology for detailed 3D reconstructions, which can significantly contribute to the understanding of geometric-morphological characteristics influencing functional traits, providing a foundation for a biomimetic oviduct-on-a-chip.

Females of the solitary digger wasp tribe Philanthini, called the beewolves (Hymenoptera, Crabronidae), cultivate strains of symbiotic bacteria that belong to the genus Streptomyces in unique and highly specialized glands in their antennae. The glands consist of large reservoirs that are surrounded by numerous gland cell complexes (class III). The symbionts are cultivated inside the reservoirs and are probably provisioned with nutrients secreted from the surrounding glands and/or sequestered from the hemolymph. The wasp female delivers the bacteria into the subterranean brood cell prior to oviposition. Fully grown larvae take up the bacteria and apply them to their cocoon. There the bacteria produce several antibiotics that protect the wasp offspring against fungus infestation. Hitherto Streptomyces bacteria were detected in the antennae of 38 species of the Philanthini. However, a detailed morphological analysis of the antennal glands is only available for a few species. In order to shed light on the evolutionary history of the association between beewolf wasps and bacteria, we investigated the morphology of the antennal glands of another 14 Philanthus species from the Palearctic, Paleotropic, and Nearctic. We generated 3D-models of the glands based on serial semithin sections and/or micro-CT (μCT). Despite broad similarities in number and structure of antennal glands, the results revealed interspecific differences with regard to overall shape, complexity, and relative size of the reservoirs as well as the number of the surrounding gland cell units. Mapping the morphology of all species studied so far on the phylogeny (that parallels geographical distribution) revealed that related species share similarities in gland morphology, but there are notable differences between lineages. In particular, compared to the North American species the European and African species possess more complex gland structures with a higher number of gland cells. We discuss morphological, ecological, and physiological aspects and provide scenarios for the evolution of the antennal glands of the Philanthini as symbiont cultivation organs.

Females of the solitary digger wasp tribe Philanthini , called the beewolves (Hymenoptera, Crabronidae), cultivate strains of symbiotic bacteria that belong to the genus Streptomyces in unique and highly specialized glands in their antennae. The glands consist of large reservoirs that are surrounded by numerous gland cell complexes (class III). The symbionts are cultivated inside the reservoirs and are probably provisioned with nutrients secreted from the surrounding glands and/or sequestered from the hemolymph. The wasp female delivers the bacteria into the subterranean brood cell prior to oviposition. Fully grown larvae take up the bacteria and apply them to their cocoon. There the bacteria produce several antibiotics that protect the wasp offspring against fungus infestation. Hitherto Streptomyces bacteria were detected in the antennae of 38 species of the Philanthini. However, a detailed morphological analysis of the antennal glands is only available for a few species. In order to shed light on the evolutionary history of the association between beewolf wasps and bacteria, we investigated the morphology of the antennal glands of another 14 Philanthus species from the Palearctic, Paleotropic, and Nearctic. We generated 3D-models of the glands based on serial semithin sections and/or micro-CT (μCT). Despite broad similarities in number and structure of antennal glands, the results revealed interspecific differences with regard to overall shape, complexity, and relative size of the reservoirs as well as the number of the surrounding gland cell units. Mapping the morphology of all species studied so far on the phylogeny (that parallels geographical distribution) revealed that related species share similarities in gland morphology, but there are notable differences between lineages. In particular, compared to the North American species the European and African species possess more complex gland structures with a higher number of gland cells. We discuss morphological, ecological, and physiological aspects and provide scenarios for the evolution of the antennal glands of the Philanthini as symbiont cultivation organs. [ABSTRACT FROM AUTHOR]

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution. [ABSTRACT FROM AUTHOR]

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Serotonin (5-HT)-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a pervasive innervation of the central nervous system (CNS) and are involved in the modulation of a plethora of functions in both developing and adult brain. Classical studies have described the post-natal development of serotonergic axons as a linear process of terminal field innervation. However, technical limitations have hampered a fine morphological characterization. With the advent of genetic mouse models, the possibility to label specific neuronal populations allowed the rigorous measurement of their axonal morphological features as well as their developmental dynamics. Here, we used the Tph2GFP knock-in mouse line, in which GFP expression allows punctual identification of serotonergic neurons and axons, for confocal microscope imaging and we performed 3-dimensional reconstruction in order to morphologically characterize the development of serotonergic fibers in specified brain targets from birth to adulthood. Our analysis highlighted region-specific developmental patterns of serotonergic fiber density ranging from a linear and progressive colonization of the target (Caudate/Putamen, Basolateral Amygdala, Geniculate Nucleus and Substantia Nigra) to a transient increase in fiber density (medial Prefrontal Cortex, Globus Pallidus, Somatosensory Cortex and Hippocampus) occurring with a region-specific timing. Despite a common pattern of early post-natal morphological maturation in which a progressive rearrangement from a dot-shaped to a regular and smooth fiber morphology was observed, starting from post-natal day 28 serotonergic fibers acquire the region specific morphological features present in the adult. In conclusion, we provided novel, target-specific insights on the morphology and temporal dynamics of the developing serotonergic fibers.

Serotonin (5-HT)-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a pervasive innervation of the central nervous system (CNS) and are involved in the modulation of a plethora of functions in both developing and adult brain. Classical studies have described the post-natal development of serotonergic axons as a linear process of terminal field innervation. However, technical limitations have hampered a fine morphological characterization. With the advent of genetic mouse models, the possibility to label specific neuronal populations allowed the rigorous measurement of their axonal morphological features as well as their developmental dynamics. Here, we used the Tph2GFP knock-in mouse line, in which GFP expression allows punctual identification of serotonergic neurons and axons, for confocal microscope imaging and we performed 3-dimensional reconstruction in order to morphologically characterize the development of serotonergic fibers in specified brain targets from birth to adulthood. Our analysis highlighted region-specific developmental patterns of serotonergic fiber density ranging from a linear and progressive colonization of the target (Caudate/Putamen, Basolateral Amygdala, Geniculate Nucleus and Substantia Nigra) to a transient increase in fiber density (medial Prefrontal Cortex, Globus Pallidus, Somatosensory Cortex and Hippocampus) occurring with a regionspecific timing. Despite a common pattern of early post-natal morphological maturation in which a progressive rearrangement from a dot-shaped to a regular and smooth fiber morphology was observed, starting from post-natal day 28 serotonergic fibers acquire the region specific morphological features present in the adult. In conclusion, we provided novel, target-specific insights on the morphology and temporal dynamics of the developing serotonergic fibers. [ABSTRACT FROM AUTHOR]

The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of spine development and unexpected features of synapse formation, including vacant and branched spines and presynaptic terminals establishing synapses with up to 10 spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3-4 and 8-9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner. [ABSTRACT FROM AUTHOR]

Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit α1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses.