Neonatal Neurons Research Articles

Dietary nucleotides drive changes in infant fecal microbiota in vitro and gut microbiota-gut-brain development in neonatal rats: A potential “nitrogen source” for early microbiota growth

Various dietary factors in human milk are important nutrients for the formation of the infant gut microbiota (GM). While promoting the growth of the GM, some human milk components that are difficult to absorb and utilize will be broken down by the GM, and converted into nutrients that the baby can use, such as breast milk oligosaccharides—the ‘carbon source’ for infant GM. This study reveals that nucleotides (NTs), significant non-protein nitrogen sources in human milk, can enhance the abundance of beneficial microbial genera such as g_Bifidobacterium, g_Bacteroides, and g_Blautia in in vitro fecal fermentation fluids of infants at low doses (2 mg/mL). Conversely, high doses of NTs (20 mg/mL) increased the abundance of g_Escherichia-Shigella. Furthermore, low-dose NTs fermentation broth significantly enhanced the expression of neurodevelopmental marker genes such as Tuj1, Sox2, Dcx, and NeuN in NE-4C neural stem cells, whereas a single NTs digestion broth did not exhibit significant activity. However, in vivo studies using neonatal rats as a model demonstrated that both low-dose NTs fermentation broth and NTs digestive juices promoted behavioral development in neonatal rats (PND 20) and neuron maturation in the prefrontal cortex and hippocampus. Non-targeted metabolomics results indicate that low-dose dietary NTs promote the production of certain neuroregulatory metabolites in infant fecal fermentation, such as uridine, L-tyrosine, L-glutamic acid, and succinic acid. These findings suggest that NTs may serve as an important “nitrogen source” during GM formation in early life and have a dose effect in driving the development of the microbiota-gut-brain axis in early life.

Cisplatin induced alterations in nociceptor developmental trajectory elicits a TrkA dependent platinum-based chemotherapy induced neuropathic pain

Cisplatin-based chemotherapy is a common treatment for paediatric cancer. Unfortunately, cisplatin treatment causes neuropathic pain, a highly prevalent adverse health related complication in adult childhood cancer survivors. Due to minimal understanding of this condition, there are currently no condition tailored analgesics available. Here we investigated an alteration in nociceptor maturation that results in neuronal sensitisation and manifestation of cisplatin induced survivorship pain in a TrkA dependent manner. Cisplatin was administered (i.p. 0.1 mg/kg Postnatal day 14 and 16) to neonatal male and female Wistar rats and nociceptive behavioural assays were performed. In vitro studies utilised isolated neonatal dorsal root ganglia sensory neurons treated with cisplatin (5 μg/ml) to elucidate impact upon nociceptor activation and neurite growth, in combination with TrkA inhibition (GW441756 10 nM and 100 nM). Cisplatin treated male and female neonatal Wistar rats developed a delayed but lasting mechanical and heat hypersensitivity. Cisplatin administration led to increased TrkA expression in dorsal root ganglia sensory neurons. Nerve growth factor (NGF) induced TrkA activation led to sensory neuritogenesis and nociceptor sensitisation, which could be prevented through pharmacological TrkA inhibition (GW441756 either s.c. 100 nM or i.p. 2 mg/kg). Administration of TrkA antagonist suppressed cisplatin induced TRPV1 mediated nociceptor sensitisation and prevented cisplatin induced neuropathic pain. These studies provide greater understanding of the underlying mechanisms that cause cisplatin induced childhood cancer survivorship pain and allowing identification of potential therapeutic targets.

Embryonically active piriform cortex neurons promote intracortical recurrent connectivity during development

Neuronal activity plays a critical role in the maturation of circuits that propagate sensory information into the brain. How widely does early activity regulate circuit maturation across the developing brain? Here, we used targeted recombination in active populations (TRAP) to perform a brain-wide survey for prenatally active neurons in mice and identified the piriform cortex as an abundantly TRAPed region. Whole-cell recordings in neonatal slices revealed preferential interconnectivity within embryonically TRAPed piriform neurons and their enhanced synaptic connectivity with other piriform neurons. Invivo Neuropixels recordings in neonates demonstrated that embryonically TRAPed piriform neurons exhibit broad functional connectivity within piriform and lead spontaneous synchronized population activity during a transient neonatal period, when recurrent connectivity is strengthening. Selectively activating or silencing these neurons in neonates enhanced or suppressed recurrent synaptic strength, respectively. Thus, embryonically TRAPed piriform neurons represent an interconnected hub-like population whose activity promotes recurrent connectivity in early development.

Obstructive sleep apnea affects cognition: dual effects of intermittent hypoxia on neurons.

Obstructive sleep apnea (OSA) is a common respiratory disorder. Multiple organs, especially the central nervous system (CNS), are damaged, and dysfunctional when intermittent hypoxia (IH) occurs during sleep for a long time. The quality of life of individuals with OSA is significantly impacted by cognitive decline, which also escalates the financial strain on their families. Consequently, the development of novel therapies becomes imperative. IH induces oxidative stress, endoplasmic reticulum stress, iron deposition, and neuroinflammation in neurons. Synaptic dysfunction, reactive gliosis, apoptosis, neuroinflammation, and inhibition of neurogenesis can lead to learning and long-term memory impairment. In addition to nerve injury, the role of IH in neuroprotection was also explored. While causing neuron damage, IH activates the neuronal self-repairing mechanism by regulating antioxidant capacity and preventing toxic protein deposition. By stimulating the proliferation and differentiation of neural stem cells (NSCs), IH has the potential to enhance the ratio of neonatal neurons and counteract the decline in neuron numbers. This review emphasizes the perspectives and opportunities for the neuroprotective effects of IH and informs novel insights and therapeutic strategies in OSA.

CNSC-06. REDIRECTING IMMUNOSUPPRESSIVE GLIOMA MICROENVIRONMENT THROUGH PHARMACOLOGICAL INTERVENTION TARGETING NEURONAL ACTIVITY

Abstract Recent single-cell RNA-sequencing analyses have revealed a strong association between glioma-neuronal circuit remodeling and regional immunosuppression. Subsequent preclinical investigations using syngeneic tumor models demonstrated that genetic disruption of circuit assembly genes, including Thrombospondin-1 (TSP-1/Thbs1) in GBM cells suppressed synaptogenesis, excitatory neurotransmitter ligand and receptor density in addition to neuronal activities while simultaneously restoring inflammatory responses, indicating glioma-neuron-immune cross talk. However, the underlying mechanisms and directions of causality remain unclear. Here we test the hypothesis that tumor-derived synaptogenic factors directly reprogram the tumor immune microenvironment (“model 1”). Alternatively, tumor intrinsic neuronal activity may indirectly regulate adaptive immunity (“model 2”). In this study, we performed in-vitro bone-marrow-derived macrophage (BMDM) reprogramming assays evaluating various stimulation conditions and in-vivo experiments evaluating the effect of neuronal activity-oriented therapy on the tumor-associated macrophages (TAMs). Our in-vitro assays demonstrated that, secreted factors from mouse neonatal cortical neurons (not tumor-derived synaptogenic factors) induced an anti-inflammatory, M2-like phenotype of BMDMs, corroborating the proposed model 2. This M2-inducing effect was inhibited when the neuronal activities were suppressed pharmacologically, using perampanel (PER), a noncompetitive inhibitor of neuronal AMPA-type glutamate receptors. Direct treatment of BMDMs with PER did not affect the polarization patterns, further supporting the M2-inducing effect of glutamatergic excitatory neuronal signals. In in-vivo syngeneic orthotopic tumor models, treatment with PER significantly prolonged survival compared to the non-treated group (27.5 [PER] vs. 23 days [control], p = 0.01). Flow cytometric analysis of brain-infiltrating leukocytes showed that TAMs isolated from PER-treated mice exhibited a more proinflammatory M1-like polarization compared to the control group (median M1/M2 ratio, 1.2 [PER] vs. 0.7 [control], p = 0.004), which explained the improved prognosis. Collectively, these findings unveil novel immunosuppression mechanisms induced by glioma-neuronal circuit remodeling and the resulting excitatory neuronal signals, and potential therapeutic vulnerabilities that could be targeted in combination with cancer immunotherapy.

Short-term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by in vivo Imaging with Improved Spatiotemporal Resolution.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon in vivo time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8-h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell-surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.Significance StatementThe formation of proper dendritic patterns during early postnatal development is essential for normal neuronal circuit function in adulthood. To elucidate the mechanisms responsible for this refinement, in vivo imaging with high spatiotemporal resolution is useful. Our previous long-term in vivo imaging studies have clarified aspects of dendritic refinement mechanism; however, due to the long intervals (8-h) between image acquisitions, rapid changes in dendritic morphology were missed. Here hourly in vivo time-lapse imaging of neonatal mouse barrel cortex over 8 h revealed the rapid changes in the dendrite morphology of layer 4 neurons, thereby providing a more comprehensive record of dendritic refinement during postnatal development for mechanistic analysis.

A novel de novo HCN2 loss-of-function variant causing developmental and epileptic encephalopathy treated with ketogenic diet.

Missense variants of HCN ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). While variants of HCN1 are an established cause of DEE, those of HCN2 have been reported in generalized epilepsies. Here, we describe the first case of DEE caused by the novel de novo heterozygous missense variant c.1379G>A (p.G460D) of HCN2. Functional characterization in transfected HEK293 cells and neonatal rat cortical neurons revealed that HCN2 p.G460D currents were strongly reduced compared to wild-type, consistent with a dominant negative loss-of-function effect. Immunofluorescence staining showed that mutant channels are retained within the cell and do not reach the membrane. Moreover, mutant HCN2 also affect HCN1 channels, by reducing the Ih current expressed by the HCN1-HCN2 heteromers. Due to the persistence of frequent seizures despite pharmacological polytherapy, the patient was treated with ketogenic diet, reporting a significant and long-lasting reduction of seizures. In vitro experiments conducted in a ketogenic environment demonstrated that the clinical improvement observed with this dietary regimen was not mediated by a direct action on HCN2 activity. These results expand the clinical spectrum related to HCN2 channelopathies, further broadening our understanding of the pathogenesis of DEE.

The neuroprotective mechanisms of naringenin: Inhibition of apoptosis through the PI3K/AKT pathway after hypoxic-ischemic brain damage

Ethnopharmacological relevanceNaringenin (NGN) is a widely distributed flavonoid with potent antioxidant and neuroprotective properties. Neuroprotective agents play a crucial role in the treatment of hypoxic-ischemic encephalopathy (HIE). It has shown potential therapeutic effects for neurological disorders. However, its efficacy on HIE is yet to be investigated. Aim of the studyThis study aims to investigate the potential neuroprotective effect of naringenin and its underlying molecular mechanisms in reducing oxidative stress, apoptosis, and improving brain outcomes following HIE. Additionally, the study aims to identify the potential targets, mechanisms, and functions of naringenin using network pharmacology analysis. Materials and methodsNeonatal mice were exposed to the hypoxic-ischemic brain damage (HIBD) model to determine brain water content, and brain tissue was subjected to hematoxylin and eosin (HE), immunohistochemistry (IHC), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Nissl staining to investigate its neuroprotective effects. Furthermore, the neonatal mouse primary neuron oxygen-glucose deprivation (OGD) model to measure reactive oxygen species (ROS) production in vitro. The protein levels were characterized by Western Blot, and mRNA levels were evaluated by a real-time quantitative PCR detecting system (qPCR). Transmission electron microscopy (TEM) and mitochondrial fluorescent staining were used to observe mitochondrial morphology. Neuronal nuclei (NeuN) and microtubule-associated protein 2 (MAP2) were detected by Immunofluorescence (IF). Finally, network pharmacology was employed to determine the common target of naringenin and HIE. The core genes were obtained via protein-protein interaction networks (PPI) analysis and molecular docking was examined, and the mechanism of action was explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Additionally, small interfering RNA (siRNA) was constructed for verification. ResultsNaringenin has a neuroprotective effect in HIBD by modulating Vegfa expression and activating the PI3K/AKT pathway to inhibit apoptosis. Furthermore, molecular docking results suggest that Vegfa is a potential binding target of naringenin, and silencing Vegfa partially reverses the pharmacological effects of NGN. ConclusionOur findings suggest that naringenin demonstrates potential clinical application for treating HIE as a novel neuroprotective agent.

MiR-10a induces inflammatory responses in epileptic hippocampal neurons of rats via PI3K/Akt/mTOR signaling pathway.

Epilepsy is a common chronic neurological disorder worldwide. MicroRNAs (miRNAs) play an important role in the pathogenesis of epilepsy. However, the mechanism of the regulatory effect of miR-10a on epilepsy is unclear. In this study, we investigated the effect of miR-10a expression on the PI3K/Akt/mTOR signaling pathway and inflammatory cytokines in epileptic hippocampal neurons of rats. The miRNA differential expression profile of rat epileptic brain was analyzed using bioinformatic approaches. Neonatal Sprague-Dawley rat hippocampal neurons were prepared as epileptic neuron models in vitro by replacing culture medium with magnesium-free extracellular solution. The hippocampal neurons were transfected with miR-10a mimics, and transcript levels of miR-10a, PI3K, Akt and mTOR were detected by quantitative reverse transcription-PCR, and PI3K, mTOR, Akt, TNF-α, IL-1β, IL-6 protein expression levels were detected by Western blot. Cytokines secretory levels were detected by ELISA. Sixty up-regulated miRNAs were identified in the hippocampal tissue of epileptic rats and might affect the PI3K-Akt signaling pathway. In the epileptic hippocampal neurons model, the expression levels of miR-10a were significantly increased, with decreasing levels of PI3K, Akt and mTOR, and increasing levels of TNF-α, IL-1β and IL-6. The miR-10a mimics promoted the expression of TNF-α, IL-1β and IL-6. Meanwhile, miR-10a inhibitor activated PI3K/Akt/mTOR pathway and inhibited cytokines secretion. Finally, cytokine secretion was increased by treated with PI3K inhibitor and miR-10a inhibitor. The miR-10a may promote inflammatory responses in rat hippocampal neurons by inhibiting the PI3K/Akt/mTOR pathway, suggesting that miR-10a may be one of the target therapeutic molecules for epilepsy treatment.

Timely insertion of AMPA receptor in developing vestibular circuits is required for manifestation of righting reflexes and effective navigation.

Vestibular information processed first by the brainstem vestibular nucleus (VN), and further by cerebellum and thalamus, underlies diverse brain function. These include the righting reflexes and spatial cognitive behaviour. While the cerebellar and thalamic circuits that decode vestibular information are known, the importance of VN neurons and the temporal requirements for their maturation that allow developmental consolidation of the aforementioned circuits remains unclear. We show that timely unsilencing of glutamatergic circuits in the VN by NMDA receptor-mediated insertion of AMPAR receptor type 1 (GluA1) subunits is critical for maturation of VN and successful consolidation of higher circuits that process vestibular information. Delayed unsilencing of NMDA receptor-only synapses of neonatal VN neurons permanently decreased their functional connectivity with inferior olive circuits. This was accompanied by delayed pruning of the inferior olive inputs to Purkinje cells and permanent reduction in their plasticity. These derangements led to deficits in associated vestibular righting reflexes and motor co-ordination during voluntary movement. Vestibular-dependent recruitment of thalamic neurons was similarly reduced, resulting in permanently decreased efficiency of spatial navigation. The findings thus show that well-choreographed maturation of the nascent vestibular circuitry is prerequisite for functional integration of vestibular signals into ascending pathways for diverse vestibular-related behaviours.

Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex

Neonatal hypoxia causes cytotoxic neuronal swelling by the entry of ions and water. Multiple water pathways have been implicated in neurons because these cells lack water channels, and their membrane has a low water permeability. NKCC1 and KCC2 are cation-chloride cotransporters (CCCs) involved in water movement in various cell types. However, the role of CCCs in water movement in neonatal neurons during hypoxia is unknown. We studied the effects of modulating CCCs pharmacologically on neuronal swelling in the neocortex (layer IV/V) of neonatal mice (post-natal day 8–13) during prolonged and brief hypoxia. We used acute brain slices from Clomeleon mice which express a ratiometric fluorophore sensitive to Cl− and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl−]i by multiphoton microscopy. Neurons were identified using a convolutional neural network algorithm, and changes in the somatic area and [Cl−]i were evaluated using a linear mixed model for repeated measures. We found that (1) neuronal swelling and Cl− accumulation began after OGD, worsened during 20 min of OGD, or returned to baseline during reoxygenation if the exposure to OGD was brief (10 min). (2) Neuronal swelling did not occur when the extracellular Cl− concentration was low. (3) Enhancing KCC2 activity did not alter OGD-induced neuronal swelling but prevented Cl− accumulation; (4) blocking KCC2 led to an increase in Cl− accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation; (5) blocking NKCC1 reduced neuronal swelling during early but not prolonged OGD and aggravated Cl− accumulation during prolonged OGD; and (6) treatment with the “broad” CCC blocker furosemide reduced both swelling and Cl− accumulation during prolonged and brief OGD, whereas simultaneous NKCC1 and KCC2 inhibition using specific pharmacological blockers aggravated neuronal swelling during prolonged OGD. We conclude that CCCs, and other non-CCCs, contribute to water movement in neocortical neurons during OGD in the neonatal period.

Parvalbumin interneuron loss mediates repeated anesthesia-induced memory deficits in mice.

Repeated or prolonged, but not short-term, general anesthesia during the early postnatal period causes long-lasting impairments in memory formation in various species. The mechanisms underlying long-lasting impairment in cognitive function are poorly understood. Here, we show that repeated general anesthesia in postnatal mice induces preferential apoptosis and subsequent loss of parvalbumin-positive inhibitory interneurons in the hippocampus. Each parvalbumin interneuron controls the activity of multiple pyramidal excitatory neurons, thereby regulating neuronal circuits and memory consolidation. Preventing the loss of parvalbumin neurons by deleting a proapoptotic protein, mitochondrial anchored protein ligase (MAPL), selectively in parvalbumin neurons rescued anesthesia-induced deficits in pyramidal cell inhibition and hippocampus-dependent long-term memory. Conversely, partial depletion of parvalbumin neurons in neonates was sufficient to engender long-lasting memory impairment. Thus, loss of parvalbumin interneurons in postnatal mice following repeated general anesthesia critically contributes to memory deficits in adulthood.

Fetuses and infants with Amyoplasia congenita in congenital Zika syndrome: The evidence of a viral cause. A narrative review of 144 cases.

Amyoplasia congenita is the most frequent type of arthrogryposis causing fetal hypokinesia, leading to congenital contractures at birth. The pathogenesis is thought to be impaired blood circulation to the fetus early in pregnancy, with hypotension and hypoxia damaging the anterior horn cells. In animal studies however a prenatal infection with a poliomyelitis-like viral agent was demonstrated. Congenital Zika virus syndrome (CZVS) has recently been described in infants with severe microcephaly, and in 10-25% of cases arthrogryposis. A search in PubMed for CZVS yielded 124 studies. After a selection for arthrogryposis, 35 papers were included, describing 144 cases. The studies were divided into two categories. 1) Those (87 cases) focussing on imaging or histological data of congenital brain defects, contained insufficient information to link arthrogryposis specifically to lesions of the brain or spinal motor neuron. 2) In the other 57 cases detailed clinical data could be linked to neurophysiological, imaging or histological data. In category 1 the most frequent brain abnormalities in imaging studies were ventriculomegaly, calcifications (subcortical, basal ganglia, cerebellum), hypoplasia of the brainstem and cerebellum, atrophy of the cerebral cortex, migration disorders and corpus callosum anomalies. In category 2, in 38 of 57 cases clinical data were indicative of Amyoplasia congenita. This diagnosis was confirmed by electromyographic findings (13 cases), by MRI (37 cases) or histology (12 cases) of the spinal cord. The latter showed small or absent lateral corticospinal tracts, and cell loss and degeneration of motor neuron cells. Zika virus-proteins and flavivirus-like particles were detected in cytoplasm of spinal neurons. The phenotype of arthrogryposis in CZVS is consistent with Amyoplasia congenita. These findings warrant search for an intrauterine infection with any neurotropic viral agent with affinity to spinal motor neurons in neonates with Amyoplasia.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Dexmedetomidine Attenuates Apoptosis and Neurological Deficits by Modulating Neuronal NADPH Oxidase 2-Derived Oxidative Stress in Neonates Following Hypoxic Brain Injury.

Hypoxic-ischemic brain injury is an important cause of neonatal neurological deficits. Our previous study demonstrated that dexmedetomidine (Dex) provided neuroprotection against neonatal hypoxic brain injury; however, the underlying mechanisms remain incompletely elucidated. Overactivation of NADPH oxidase 2 (NOX2) can cause neuronal apoptosis and neurological deficits. Hence, we aimed to investigate the role of neuronal NOX2 in Dex-mediated neuroprotection and to explore its potential mechanisms. Hypoxic injury was modeled in neonatal rodents in vivo and in cultured hippocampal neurons in vitro. Our results showed that pre- or post-treatment with Dex improved the neurological deficits and alleviated the hippocampal neuronal damage and apoptosis caused by neonatal hypoxia. In addition, Dex treatment significantly suppressed hypoxia-induced neuronal NOX2 activation; it also reduced oxidative stress, as evidenced by decreases in intracellular reactive oxygen species (ROS) production, malondialdehyde, and 8-hydroxy-2-deoxyguanosine, as well as increases in the antioxidant enzymatic activity of superoxide dismutase and glutathione peroxidase in neonatal rat hippocampi and in hippocampal neurons. Lastly, the posthypoxicneuroprotective action of Dex was almost completely abolished in NOX2-deficient neonatal mice and NOX2-knockdown neurons. In conclusion, our data demonstrated that neuronal NOX2-mediated oxidative stress is involved in the neuroprotection that Dex provides against apoptosis and neurological deficits in neonates following hypoxia.

Cytotoxic Effects of Cannabidiol on Neonatal Rat Cortical Neurons and Astrocytes: Potential Danger to Brain Development.

The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB1) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.

On the accuracy of cell-attached current-clamp recordings from cortical neurons.

Cell-attached current-clamp (CA/CC) recordings have been proposed to measure resting membrane potential and synaptic/agonist responses in neurons without disrupting the cell membrane, thus avoiding the intracellular dialysis that occurs in conventional whole-cell recordings (WC). However, the accuracy of CA/CC recordings in neurons has not been directly assessed. Here, we used concomitant CA and WC current clamp recordings from cortical neurons in brain slices. Resting membrane potential values and slow voltage shifts showed variability and were typically attenuated during CA/CC recordings by ~10–20% relative to WC values. Fast signals were slowed down and their amplitude was greatly reduced: synaptic potentials by nearly 2-fold, and action potentials by nearly 10-fold in CA/CC mode compared to WC. The polarity of GABAergic postsynaptic responses in CA/CC mode matched the responses in WC, and depolarising GABAergic potentials were predominantly observed during CA/CC recordings of intact neonatal CA3 hippocampal pyramidal neurons. Similarly, CA/CC recordings reliably detected neuronal depolarization and excitation during network-induced giant depolarizing potentials in the neonatal CA3 hippocampus, and revealed variable changes, from depolarization to hyperpolarization, in CA1 pyramidal cells during sharp wave ripples in the adult hippocampus. Thus, CA/CC recordings are suitable for assessing membrane potential but signal distortion, probably caused by leakage via the seal contact and RC filtering should be considered.

Light links neonatal neurons for learning

The newborn mouse's retina senses light even before the eye opens, informing the developing brain of the visual world. Without this information, the brain forms fewer connections and the adult mouse learns sluggishly.

Maternal opioids decrease mu‐opioid receptor expression in the neonatal preBötzinger Complex

An understudied population in the opioid crisis are infants exposed to in utero opioids experiencing severe respiratory deficits. Our recent work demonstrated maternal opioids destabilized neonatal breathing, increased apneas, and blunted opioid‐induced respiratory frequency depression; however, the mechanism(s) underlying these neonatal breathing deficits remains unclear. We hypothesized deficits in neonatal breathing after maternal opioids are due to downregulation of mu‐opioid receptors on key cell types (e.g. neurons and astrocytes), essential for respiratory rhythm generation and modulation in the preBötzinger Complex. While opioid receptor expression is downregulated in the whole brain after maternal opioids, it remains unclear if opioid receptor expression changes in the preBötzinger Complex after in utero opioids to induce neonatal breathing deficits. To assess the typical developmental changes in opioid receptors in the preBötzinger Complex, mu‐opioid receptor expression was first assessed using immunohistochemistry and confocal microscopy on neonatal neurons and astrocytes in the preBötzinger Complex at three ages: the day of birth (postnatal day 0, P0) when neonates must begin robust rhythmic breathing, an early neonatal age (postnatal day 4, P4), and during a critical developmental window (postnatal day 11, P11). At all ages, mu‐opioid receptors were expressed on both neurons and astrocytes in the preBötzinger Complex; however, mu‐opioid receptor expression decreased from P0 (n=6) to P4 (n=6) and was lowest at P11 (n=6). Preliminary data after maternal opioids suggest mu‐opioid receptor expression was lower in preBötzinger Complexes at P0 (n=2) and P4 (n=2), but returned to control levels by P11 (n=2). These results are further supported by in vitro brainstem‐spinal cord recordings of the cervical rootlets (C4) from neonates after maternal opioids, demonstrating decreased opioid sensitivity of isolated respiratory control circuitry after maternal opioids (n=2). Results from these studies will contribute to our understanding of the mechanisms contributing to impaired neonatal breathing after maternal opioids and may lead to better treatments for infants suffering breathing deficits after maternal opioids.

P-hydroxy benzaldehyde facilitates reprogramming of reactive astrocytes into neurons via endogenous transcriptional regulation

Background Cerebral ischemia leads to linguistic and motor dysfunction, as the death of neurons in ischemic core is permanent and non-renewable. An innovative avenue is to induce and/or facilitate reprogramming of adjacent astrocytes into neurons to replace the lost neurons and re-establish brain homeostasis. Purpose This study aimed to investigate whether the p-hydroxy benzaldehyde (p-HBA), a phenolic compound isolated from Gastrodia elata Blume, could facilitate the reprogramming of oxygen-glucose deprivation/reperfusion (OGD/R)-damaged astrocytes into neurons. Study Design/Methods The primary parenchymal astrocytes of rat were exposure to OGD and reperfusion with define culture medium. Cells were then incubated with different concentration of p-HBA (1, 10, 100, 400 μM) and collected at desired time point for reprogramming process analysis. Results OGD/R could elicit endogenous neurogenic program in primary parenchymal astrocytes of rat under define culture condition, and these so-called reactive astrocytes could be reprogrammed into neurons. However, the neonatal neurons produced by this endogenous procedure could not develop into mature neurons, and the conversion rate was only 1.9%. Treatment of these reactive astrocytes with p-HBA could successfully promote the conversion rate to 6.1%, and the neonatal neurons could develop into mature neurons within 14 days. Further analysis showed that p-HBA down-regulated the Notch signal component genes Dll1, Hes1 and SOX2, while the transcription factor NeuroD1 was up-regulated. Conclusion The results of this study demonstrated that p-HBA facilitated the astrocyte-to-neuron conversion. This chemical reprogramming was mediated by inhibition of Notch1 signaling pathway and transcriptional activation of NeuroD1.