Search Results (7)

Primary cultures of neural cells are important key tools for basic and translational neuroscience research. These primary cell cultures are classically generated from the rodent brain hippocampus or cortex and optimized for enrichment in neurons at the expense of glial cells. Importantly, considerable differences exist in neuronal cell populations and in glial cell contribution between different brain regions. Because many basic and translational research projects aim to identify mechanisms underlying brainstem neuronal networks that affect major vital functions, primary cultures representative of cell populations present in the hindbrain are required. However, the preparation of primary cultures of brainstem/hindbrain neurons is scarcely described in the literature, limiting the possibilities for studying the development and physiology of these brain regions in vitro. The present report describes a reliable protocol to dissociate and culture in vitro embryonic mouse fetal hindbrain neurons in a defined culture medium, while control of astrocytes’ expansion was attained by using a chemically defined, serum-free supplement, namely CultureOne™. The neuronal cells maintained according to this protocol differentiate and, by 10 days in vitro, they develop extensive axonal and dendritic branching. Using immunofluorescence, we further characterized the different cell populations and neuronal subtypes. Patch–clamp recordings demonstrate the excitable nature of these neurons, while colocalization of pre- and postsynaptic neuronal markers showed that neurons form mature synapses, suggesting the establishment of functional networks in vitro. The cultures produced by this method show excellent reproducibility and can be used for molecular, biochemical, and physiological analyses, as illustrated here for tamoxifen-induced Cre recombination in genetically-modified neural cells. Full article

Background/Objectives: Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, is a debilitating, incurable neurodegenerative disorder characterised by motor neuron death in the spinal cord, brainstem, and motor cortex. With an incidence rate of about 4.42 cases per 100,000 people annually, ALS severely impacts motor function and quality of life, causing progressive muscle atrophy, spasticity, paralysis, and eventually death. The cause of ALS is largely unknown, with 90% of cases being sporadic and 10% familial. Current research targets molecular mechanisms of inflammation, excitotoxicity, aggregation-prone proteins, and proteinopathy. Methods: This review evaluates the efficacy of three stem cell types in ALS treatment: mesenchymal stem cells (MSCs), neural stem cells (NSCs), and induced pluripotent stem cells (iPSCs). Results: MSCs, derived from various tissues, show neuroprotective and regenerative qualities, with clinical trials suggesting potential benefits but limited by small sample sizes and non-randomised designs. NSCs, isolated from the fetal spinal cord or brain, demonstrate promise in animal models but face functional integration and ethical challenges. iPSCs, created by reprogramming patient-specific somatic cells, offer a novel approach by potentially replacing or supporting neurons. iPSC therapy addresses ethical issues related to embryonic stem cells but encounters challenges regarding genotoxicity and epigenetic irregularities, somatic cell sources, privacy concerns, the need for extensive clinical trials, and high reprogramming costs. Conclusions: This research is significant for advancing ALS treatment beyond symptomatic relief and modest survival extensions to actively modifying disease progression and improving patient outcomes. Successful stem cell therapies could lead to new ALS treatments, slowing motor function loss and reducing symptom severity. Full article

Neurotrophins are proteins that mediate neuronal development using spatiotemporal signaling gradients. The chicken nucleus magnocellularis (NM), an analogous structure to the mammalian anteroventral cochlear nucleus, provides a model system in which signaling between the brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) is temporally regulated. In the NM, TrkB expression is high early in development (embryonic [E] day 9) and is downregulated until maturity (E18–21). It is currently unknown how BDNF–TrkB signaling affects neuronal properties throughout development and across a spatial (i.e., frequency) axis. To investigate this, we exogenously applied BDNF onto NM neurons ex vivo and studied intrinsic properties using whole-cell patch clamp electrophysiology. Early in development (E13), when TrkB expression is detectable with immunohistochemistry, BDNF application slowed the firing of high-frequency NM neurons, resembling an immature phenotype. Current measurements and biophysical modeling revealed that this was mediated by a decreased conductance of the voltage-dependent potassium channels. Interestingly, this effect was seen only in high-frequency neurons and not in low-frequency neurons. BDNF–TrkB signaling induced minimal changes in late-developing NM neurons (E20–21) of high and low frequencies. Our results indicate that normal developmental downregulation of BDNF–TrkB signaling promotes neuronal maturation tonotopically in the auditory brainstem, encouraging the appropriate development of neuronal properties. Full article

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out. Full article

A few prior animal studies have suggested the transplantation or protective effects of mesenchymal stem cells (MSCs) in noise-induced hearing loss. This study intended to evaluate the fates of administered MSCs in the inner ears and the otoprotective effects of MSCs in the noise-induced hearing loss of rats. Human embryonic stem cell-derived MSCs (ES-MSCs) were systematically administered via the tail vein in adult rats. Eight-week-old Sprague-Dawley rats were randomly allocated to the control (n = 8), ES-MSC (n = 4), noise (n = 8), and ES-MSC+noise (n = 10) groups. In ES-MSC and ES-MSC+noise rats, 5 × 10⁵ ES-MSCs were injected via the tail vein. In noise and ES-MSC+noise rats, broadband noise with 115 dB SPL was exposed for 3 h daily for 5 days. The hearing levels were measured using auditory brainstem response (ABR) at 4, 8, 16, and 32 kHz. Cochlear histology was examined using H&E staining and cochlear whole mount immunofluorescence. The presence of human DNA was examined using Sry PCR, and the presence of human cytoplasmic protein was examined using STEM121 immunofluorescence staining. The protein expression levels of heat shock protein 70 (HSP70), apoptosis-inducing factor (AIF), poly (ADP-ribose) (PAR), PAR polymerase (PARP), caspase 3, and cleaved caspase 3 were estimated. The ES-MSC rats did not show changes in ABR thresholds following the administration of ES-MSCs. The ES-MSC+ noise rats demonstrated lower ABR thresholds at 4, 8, and 16 kHz than the noise rats. Cochlear spiral ganglial cells and outer hair cells were more preserved in the ES-MSC+ noise rats than in the noise rats. The Sry PCR bands were highly detected in lung tissue and less in cochlear tissue of ES-MSC+noise rats. Only a few STEM121-positivities were observed in the spiral ganglial cell area of ES-MSC and ES-MSC+noise rats. The protein levels of AIF, PAR, PARP, caspase 3, and cleaved caspase 3 were lower in the ES-MSC+noise rats than in the noise rats. The systemic injection of ES-MSCs preserved hearing levels and attenuated parthanatos and apoptosis in rats with noise-induced hearing loss. In addition, a tiny number of transplanted ES-MSCs were observed in the spiral ganglial areas. Full article

Estradiol (E2) enhances the anorectic action of apolipoprotein A-IV (apoA-IV), however, the intracellular mechanisms are largely unclear. Here we reported that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway was significantly activated by E2 and apoA-IV, respectively, in primary neuronal cells isolated from rat embryonic brainstem. Importantly, the combination of E2 and apoA-IV at their subthreshold doses synergistically activated the PI3K/Akt signaling pathway. These effects, however, were significantly diminished by the pretreatment with LY294002, a selective PI3K inhibitor. E2-induced activation of the PI3K/Akt pathway was through membrane-associated ERα, because the phosphorylation of Akt was significantly increased by PPT, an ERα agonist, and by E2-BSA (E2 conjugated to bovine serum albumin) which activates estrogen receptor on the membrane. Centrally administered apoA-IV at a low dose (0.5 µg) significantly suppressed food intake and increased the phosphorylation of Akt in the nucleus tractus solitarius (NTS) of ovariectomized (OVX) rats treated with E2, but not in OVX rats treated with vehicle. These effects were blunted by pretreatment with LY294002. These results indicate that E2’s regulatory role in apoA-IV’s anorectic action is through the ERα-PI3K pathway in the NTS. Manipulation of the PI3K/Akt signaling activation in the NTS may provide a novel therapeutic approach for the prevention and the treatment of obesity-related disorders in females. Full article

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α₂ adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other’s axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats. Full article