Normal brain development requires continuous communication between developing neurons and their environment filled by a complex network referred to as extracellular matrix (ECM). The ECM is divided ...into distinct families of molecules including hyaluronic acid, proteoglycans, glycoproteins such as tenascins, and link proteins. In this study, we characterize the temporal and spatial distribution of the extracellular matrix molecules in the embryonic and postnatal mouse hindbrain by using antibodies and lectin histochemistry. In the embryo, hyaluronan and neurocan were found in high amounts until the time of birth whereas versican and tenascin-R were detected in lower intensities during the whole embryonic period. After birth, both hyaluronic acid and neurocan still produced intense staining in almost all areas of the hindbrain, while tenascin-R labeling showed a continuous increase during postnatal development. The reaction with WFA and aggrecan was revealed first 4th postnatal day (P4) with low staining intensities, while HAPLN was detected two weeks after birth (P14). The perineuronal net appeared first around the facial and vestibular neurons at P4 with hyaluronic acid cytochemistry. One week after birth aggrecan, neurocan, tenascin-R, and WFA were also accumulated around the neurons located in several hindbrain nuclei, but HAPLN1 was detected on the second postnatal week. Our results provide further evidence that many extracellular macromolecules that will be incorporated into the perineuronal net are already expressed at embryonic and early postnatal stages of development to control differentiation, migration, and synaptogenesis of neurons. In late postnatal period, the experience-driven neuronal activity induces formation of perineuronal net to stabilize synaptic connections.
The development of neurons is regulated by several spatiotemporally changing factors, which are crucial to give the ability of neurons to form functional networks. While external physical stimuli may ...impact the early developmental stages of neurons, the medium and long-term consequences of these influences have yet to be thoroughly examined. Using an animal model, this study focuses on the morphological and transcriptome changes of the hippocampus that may occur as a consequence of fetal ultrasound examination. We selectively labeled CA1 neurons of the hippocampus with in-utero electroporation to analyze their morphological features. Furthermore, certain samples also went through RNA sequencing after repetitive ultrasound exposure. US exposure significantly changed several morphological properties of the basal dendritic tree. A notable increase was also observed in the density of spines on the basal dendrites, accompanied by various alterations in individual spine morphology. Transcriptome analysis revealed several up or downregulated genes, which may explain the molecular background of these alterations. Our results suggest that US-derived changes in the dendritic trees of CA1 pyramidal cells might be connected to modification of the transcriptome of the hippocampus and may lead to an increased dendritic input.
The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, ...typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021,
https://doi.org/10.3389/fnana.2021.652422
). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.
Extracellular matrix (ECM) became an important player over the last few decades when studying the plasticity and regeneration of the central nervous system. In spite of the established role of ECM in ...these processes throughout the central nervous system (CNS), only few papers were published on the ECM of the olfactory system, which shows a lifelong plasticity, synaptic remodeling and postnatal neurogenesis. In the present study, we have described the localization and organization of major ECM molecules, the hyaluronan, the lecticans, tenascin-R and HAPLN1 link protein in the olfactory bulb (OB) of the rat. We detected all of these molecules in the OB showing differences in the molecular composition, staining intensity, and organization of ECM between the layers and in some cases within a single layer. One of the striking features of ECM staining pattern in the OB was that the reactions are shown dominantly in the neuropil, the PNNs were found rarely and they exhibited thin or diffuse appearance Similar organization was shown in human and mice samples. As the PNN limits the neural plasticity, its rare appearance may be related to the high degree of plasticity in the OB.
Gaucher disease is a lysosomal storage disease characterized by the malfunction of glucocerebrosidase resulting in the accumulation of glucosylceramide and other sphingolipids in certain cells. ...Although the disease symptoms are usually attributed to the storage of undigested substrate in lysosomes, here we show that glycosphingolipids accumulating in the plasma membrane cause profound changes in the properties of the membrane. The fluidity of the sphingolipid-enriched membrane decreased accompanied by the enlargement of raft-like ordered membrane domains. The mobility of non-raft proteins and lipids was severely restricted, while raft-resident components were only mildly affected. The rate of endocytosis of transferrin receptor, a non-raft protein, was significantly retarded in Gaucher cells, while the endocytosis of the raft-associated GM1 ganglioside was unaffected. Interferon-γ-induced STAT1 phosphorylation was also significantly inhibited in Gaucher cells. Atomic force microscopy revealed that sphingolipid accumulation was associated with a more compliant membrane capable of producing an increased number of nanotubes. The results imply that glycosphingolipid accumulation in the plasma membrane has significant effects on membrane properties, which may be important in the pathogenesis of Gaucher disease.
Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory ...dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that blocking histone H3.1 phosphorylation at position serine 10 (S10) in spinal Pdyn neurons significantly increases the thermal nociceptive threshold in mice. In contrast, neither mechanosensation nor acute chemonociception was affected by the transgenic manipulation of histone H3.1. These results suggest that blocking rapid epigenetic tagging of S10H3 in spinal Pdyn neurons alters acute thermosensation and thus explains the involvement of Pdyn cells in the immediate response to burn-injury-associated tissue damage.
All known biological functions of the pro-inflammatory cytokine interleukin-1β (IL-1β) are mediated by type 1 interleukin receptor (IL-1R1). IL-1β-IL-1R1 signaling modulates various neuronal ...functions including spinal pain processing. Although the role of IL-1β in pain processing is generally accepted, there is a discussion in the literature whether IL-1β exerts its effect on spinal pain processing by activating neuronal or glial IL-1R1. To contribute to this debate, here we investigated the expression and cellular distribution of IL-1R1 in the superficial spinal dorsal horn in control animals and also in inflammatory pain.
Experiments were performed on rats and wild type as well as IL-1R1-deficient mice. Inflammatory pain was evoked by unilateral intraplantar injection of complete Freund adjuvant (CFA). The nociceptive responsiveness of control and CFA-treated animals were tested daily for withdrawal responses to mechanical and thermal stimuli before and after CFA injection. Changes in the expression of 48 selected genes/mRNAs and in the quantity of IL-1R1 protein during the first 3 days after CFA injection were measured with the TaqMan low-density array method and Western blot analysis, respectively. The cellular localization of IL-1R1 protein was investigated with single and double staining immunocytochemical methods.
We found a six times and two times increase in IL-1R1 mRNA and protein levels, respectively, in the dorsal horn of CFA-injected animals 3 days after CFA injection, at the time of the summit of mechanical and thermal allodynia. Studying the cellular distribution of IL-1R1, we found an abundant expression of IL-1R1 on the somatodendritic compartment of neurons and an enrichment of the receptor in the postsynaptic membranes of some excitatory synapses. In contrast to the robust neuronal localization, we observed only a moderate expression of IL-1R1 on astrocytes and a negligible one on microglial cells. CFA injection into the hind paw caused a remarkable increase in the expression of IL-1R1 in neurons, but did not alter the glial expression of the receptor.
The results suggest that IL-1β exerts its effect on spinal pain processing primarily through neuronal IL-1R1, but it can also interact in some extent with IL-1R1 expressed by astrocytes.
Neuronal differentiation and synaptogenesis are regulated by precise orchestration of intrinsic and extrinsic chemical and mechanical factors throughout all developmental steps critical for the ...assembly of neurons into functional circuits. While ultrasound is known to alter neuronal migration and activity acutely, its chronic effect on neuronal behavior or morphology is not well characterized. Furthermore, higher-frequency (3-5 MHz) ultrasound (HFU) is extensively used in gynecological practice for imaging, and while it has not been shown harmful for the developing brain, it might be associated with mild alterations that may have functional consequences. To shed light on the neurobiological effects of HFU on the developing brain, we examined cortical pyramidal cell morphology in a transgenic mouse model, following a single and short dose of high-frequency ultrasound. Layer V neurons in the retrosplenial cortex of mouse embryos were labeled with green and red fluorescent proteins by
electroporation at the time of their appearance (E14.5). At the time of their presumptive arrival to layer V (E18.5), HFU stimulation was performed with parameters matched to those used in human prenatal examinations. On the third postnatal day (P3), basic morphometric analyses were performed on labeled neurons reconstructed with Neurolucida. Low-intensity HFU-treated cells showed significantly increased dendritic branching compared to control (non-stimulated) neurons and showed elevated c-fos immunoreactivity. Labeled neurons were immunopositive for the mechanosensitive receptor TRPC4 at E18.5, suggesting the role of this receptor and the associated signaling pathways in the effects of HFU stimulation.
Endocannabinoids are pleiotropic lipid messengers that play pro-homeostatic role in cellular physiology by strongly influencing intracellular Ca
concentration through the activation of cannabinoid ...receptors. One of the best-known endocannabinoid '2-AG' is chemically unstable in aqueous solutions, thus its molecular rearrangement, resulting in the formation of 1-AG, may influence 2-AG-mediated signaling depending on the relative concentration and potency of the two isomers. To predict whether this molecular rearrangement may be relevant in physiological processes and in experiments with 2-AG, here we studied if isomerization of 2-AG has an impact on 2-AG-induced, CB1-mediated Ca
signaling
. We found that the isomerization-dependent drop in effective 2-AG concentration caused only a weak diminution of Ca
signaling in CB1 transfected COS7 cells. We also found that 1-AG induces Ca
transients through the activation of CB1, but its working concentration is threefold higher than that of 2-AG. Decreasing the concentration of 2-AG in parallel to the prevention of 1-AG formation by rapid preparation of 2-AG solutions, caused a significant diminution of Ca
signals. However, various mixtures of the two isomers in a fix total concentration - mimicking the process of isomerization over time - attenuated the drop in 2-AG potency, resulting in a minor decrease in CB1 mediated Ca
transients. Our results indicate that release of 2-AG into aqueous medium is accompanied by its isomerization, resulting in a drop of 2-AG concentration and simultaneous formation of the similarly bioactive isomer 1-AG. Thus, the relative concentration of the two isomers with different potency and efficacy may influence CB1 activation and the consequent biological responses. In addition, our results suggest that 1-AG may play role in stabilizing the strength of cannabinoid signal in case of prolonged 2-AG dependent cannabinoid mechanisms.
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