Neonatal Neurons Research Articles

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous ‘spiking’ might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 μm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 μM) and activating μ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 μM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

Chloroquine inhibits autophagy and deteriorates the mitochondrial dysfunction and apoptosis in hypoxic rat neurons

AimsMitochondrial dysfunction (MD) and apoptosis in the neurons are associated with neonatal hypoxic–ischemic (HI) encephalopathy (HIE). The present study was to explore the influence of autophagy on the induction of MD and apoptosis in the neurons in a neonatal HIE rats and in hypoxia-treated neurons in vitro. Materials and methodsTen-day-old HI rat pups were sacrificed for brain pathological examination and immunohistochemical analysis. The induction of autophagy, apoptosis and MD were also determined in the neurons under hypoxia, with or without autophagy inhibitor, chloroquine (CQ) treatment. Key findingsHI treatment caused atrophy and apoptosis of neurons, with a significantly increased levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase 3 and the B subunit of autophagy-related microtubule-associated protein 1 light chain 3 (LC3-B). in vitro experiments demonstrated that the hypoxia induced autophagy in neurons, as was inhibited by CQ. The hypoxia-induced cytochrome c release, cleaved caspase 3 and cleaved caspase 9 were aggravated by CQ. Moreover, there were higher levels of reactive oxygen species, more mitochondrial superoxide and less mitochondrial membrane potential in the CQ-treated neurons under hypoxia than in the neurons singularly under hypoxia. SignificanceApoptosis and autophagy were induced in HI neonatal rat neurons, autophagy inhibition deteriorates the hypoxia-induced neuron MD and apoptosis. It implies a neuroprotection of autophagy in the hypoxic–ischemic encephalopathy. Administration of autophagy inducer agents might be promising in HIE treatment.

Dexmedetomidine reduces propofol-induced apoptosis of neonatal rat hippocampal neurons via up-regulating Bcl-2 expression

Background: The use of anesthetics propofol in certain pediatric age groups remains off-label, but it may cause neuronal apoptosis, long-term cognitive and neurological abnormalities during early postnatal period. Dexmedetomidine (Dex) can exert neuroprotective effects in rats. This study aimed to evaluate the effects of Dex and propofol on the apoptosis of neonatal rat hippocampal neurons. Methods: Primary hippocampal neurons of neonatal rats were isolated and identified by immunofluorescence. Neurons were then divided into normal control, propofol (12 μg/ml propofol), and propofol+Dex group (12 μg/ml propofol+0.25 μg/ml Dex). The cell morphology was detected by Hematoxylin-Eosin (HE) staining. The cell apoptosis was analysed by flow cytometry and Hoechst assay. The expression of Bcl-2 mRNA and protein was determined by quantitative reverse transcription PCR and Western blot. Results: The apoptosis rate and proportion of Hoechst-positive cells in propofol group was significantly higher compared with control group (p=0.023 and 0.036, respectively). Propofol+Dex group exhibited significantly lower apoptosis and Hoechst-positive rate compared with propofol group (p=0.016 and 0.028, respectively). The expression of Bcl-2 mRNA and protein in propofol group was significantly decreased compared with control group (p=0.012 and 0.043, respectively). The Bcl-2 mRNA and protein expression in propofol+Dex group was significantly higher than those in propofol group (p=0.033 and 0.026, respectively). Conclusion: Propofol inhibited the growth and induced apoptosis of neonatal rat hippocampal neurons, whereas Dex suppressed the propofol-induced apoptosis via increasing the expression of Bcl-2. Our findings indicate that Dex in conjunction with propofol might reduce the propofol-induced neurotoxicity in pediatric patients who undergo general anesthetics, and therefore might provide an effective clinical measure against neural damages associated with general anesthetics.

Effects of acidosis on neuronal voltage-gated sodium channels: Nav1.1 and Nav1.3

ABSTRACTVoltage-gated sodium channels are key contributors to membrane excitability. These channels are expressed in a tissue-specific manner. Mutations and modulation of these channels underlie various physiological and pathophysiological manifestations. The effects of changes in extracellular pH on channel gating have been studied on several sodium channel subtypes. Among these, Nav1.5 is the most pH-sensitive channel, with Nav1.2 and Nav1.4 being mostly pH-resistant channels. However, pH effects have not been characterized on other sodium channel subtypes. In this study, we sought to determine whether Nav1.1 and Nav1.3 display resistance or sensitivity to changes in extracellular pH. These two sodium channel subtypes are predominantly found in inhibitory neurons. The expression of these channels highly depends on age and the developmental stage of neurons, with Nav1.3 being found mostly in neonatal neurons, and Nav1.1 being found in adult neurons. Our present results indicate that, during extracellular acidosis, both channels show a depolarization in the voltage-dependence of activation and moderate reduction in current density. Voltage-dependence of steady-state fast inactivation and recovery from fast inactivation were unchanged. We conclude that Nav1.1 and Nav1.3 have similar pH-sensitivities.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Photopolymerized Microfeatures Guide Adult Spiral Ganglion and Dorsal Root Ganglion Neurite Growth.

Microtopographical patterns generated by photopolymerization of methacrylate polymer systems will direct growth of neurites from adult neurons, including spiral ganglion neurons (SGNs). Cochlear implants (CIs) provide hearing perception to patients with severe to profound hearing loss. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by spread of the electrical currents in the inner ear. Directing the regrowth of SGN peripheral processes towards stimulating electrodes could help reduce current spread and improve spatial resolution provided by the CI. Previous work has demonstrated that micro- and nano-scale patterned surfaces precisely guide the growth of neurites from a variety of neonatal neurons including SGNs. Here, we sought to determine the extent to which adult neurons likewise respond to these topographical surface features. Photopolymerization was used to fabricate methacrylate polymer substrates with micropatterned surfaces of varying amplitudes and periodicities. Dissociated adult dorsal root ganglion neurons (DRGNs) and SGNs were cultured on these surfaces and the alignment of the neurite processes to the micropatterns was determined. Neurites from both adult DRGNs and SGNs significantly aligned to the patterned surfaces similar to their neonatal counterparts. Further DRGN and SGN neurite alignment increased as the amplitude of the microfeatures increased. Decreased pattern periodicity also improved neurite alignment. Microscale surface topographic features direct the growth of adult SGN neurites. Topographical features could prove useful for guiding growth of SGN peripheral axons towards a CI electrode array.

Inhibition of Dopamine Receptor in Neonate Hippocampus: Immunolocalization of Post Synaptic Density Protein-95 and Dopamine Receptor in vivo

The effect of haloperidol on neonatal dopaminergic neurotransmission in the hippocampus of postnatal day 20 rats (P.20) was investigated in this study. Haloperidol blocked dopamine receptors (D2R) and inhibited D2R on the membrane of neonate neurons. For this study the 0.5 ml (20 mg/kg) of haloperidol was administered to pregnant female animals intraperitoneally a week before delivery. At day P.20, 5 control animals and 5 haloperidol treated animals were taken to the behavioral studies room for the Y maze and Novel object recognition test, which was done 7 am in the morning before mating. Electrophysiology was done with 2 control pups and 2 treated pups. Electrodes were implanted in the brain at the hippocampal region 2 mm beneath the bregma, 2 mm lateral to the midline. Anterior Posterior (AP=0), Medial Lateral (ML=2 mm). Also immunolocalization and immunofluorescence of post synaptic density protein (PSD-95), hippocampal morphology and hippocampal neurons have been done respectively. Results from this study showed a decline in memory index for the Y maze as a result of the effect of D2R blockade thereby inhibiting neurotransmission in newborns. Electrophysiology result in this study showed an increase in the root mean square (RMS) of control pups. The increase in RMS is equivalent to increase in wave burst pattern caused by neuronal excitation. Immunochemistry result showed an increase in the number of PSD-95 in the hippocampus of an increase in tyrosine hydroxylase in the hippocampus of the treated neonatal rats when compared to the control neonatal rats Immunofluorescence showed decline in the number of neurons in the haloperidol treated rats and it also caused hippocampal damage in terms of morphology. Furthermore, results from electrophysiology showed a statistical significant difference with P value 0.04229 (P<0.05) using the student t-test. These findings suggest that D2R inhibition may cause decline in memory function, impair learning in newborns and disrupt neonatal dopaminergic neurotransmission.

Current Overview of Neonatal Convulsions.

Neonatal convulsions are one of the most common emergency neurological events in the early period after birth. The frequency has been reported to be 1.5 to 3 in 1000 live births. It has been established that the convulsion threshold is lower in infants due to immature neonatal neurons and differences in neurotransmitters. Hypoxic ischemic encephalopathy is the most common etiology in neonatal convulsions. Other causes vary, and may be related to the level of development of the country. Convulsions are classified into 4 different types according to the clinical findings. The most common is the subtle (undefined) type of seizure; the other types are defined as clonic, tonic, and myoclonic seizures. Non-epileptic paroxysmal movements frequently seen in the neonatal period, should not be confused with seizures. The most common non-epileptic paroxysmal movements are jitteriness, benign neonatal sleep myoclonus, and hyperekplexia. A newborn that experiences convulsions should be hospitalized and monitored with continuous video electroencephalogram, if possible. If an initial rapid evaluation detects an acute metabolic disorder, treatment is provided, and, if warranted, it will be followed by a plan for further treatment with anticonvulsant drugs. Phenobarbital is still currently recommended as first-line therapy, though there are studies of other anticonvulsant drugs. Levetiracetam and phenytoin are commonly used as second-step anticonvulsant drugs. The aim of treatment should be not only to stop acute symptomatic seizures, but also to reduce the risk of brain damage and to minimize the possible negative effects of epilepsy and neurological deficits.

In vitro neurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons

Neurotoxicity of local anaesthetics has been alerted by more and more peoples. Cav3.1 and Cav3.2 T-type calcium channels were closely related with local anaesthetics toxicity. However, the role of Cav3.3, another subtype of the T-type calcium channel, on the neurotoxicity induced by local anaesthetics remains unclear. CaMKIIγ is a kind of multifunctional kinase and associated with a variety of physiological and pathological process. T-type calcium channel is closely related with CaMKIIγ. Up-regulation CaMKIIγ can increase T-type currents at the dorsal root ganglia (DRG). On the contrary, down-regulation results in the T-type currents decrease. Is the relation between Cav3.3 T-type channel calcium and CaMKIIγ involved with the ropivacaine hydrochloride neurotoxicity? In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulate and down-regulate Cav3.3 mRNA expression of the DRG. The cells treated or untreated with ropivacaine hydrochloride (3 mM) for 4 h were used to evaluate the neurotoxicity. Cell viability, cell death rate and apoptosis rate, Cav3.3 and CaMKIIγ expression were detected with MTT method, Hoechst–PI, flow cytometry, qRT-PCR and western blotting. Results showed that the cell viability of the DRG treated with ropivacaine hydrochloride markedly decreased, death rate and apoptosis rate, Cav3.3 and CaMKIIγ mRNA and protein expression significantly increased. Cav3.3 overexpression aggravated DRG injury induced by ropivacaine hydrochloride and inhibition of Cav3.3 expression improved the cell damages. Cav3.3 can regulate CaMKIIγ mRNA and protein expression. In conclusion, Cav3.3 regulated CaMKIIγ in DRG, which was involved with the cell injury induced by ropivacaine hydrochloride.

Fibrotic scar model and TGF-β1 differently modulate action potential firing and voltage-dependent ion currents in hippocampal neurons in primary culture.

Traumatic injury of the central nervous system is accompanied by various functional and morphological changes. Animal models of traumatic brain injury are commonly used to investigate changes in behaviour, morphology, in the expression of various proteins around the site of the injury, or the expression of diagnostically important biomarkers. Excitability of a single neuron at, or close to, the site of injury was rarely investigated. Several invitro models were developed which allow such investigation. In the present work, we employed a fibrotic scar model according to Kimura-Kuroda and coauthors to analyse altered excitability of rat hippocampal neurons under the conditions mimicking traumatic brain injury. Hippocampal neurons from newborn rats were cultured either on a fibrotic scar model or in the presence of TGF-β1, a cytokine secreted at a brain injury site that may have both neuroprotective and neurodegenerative function. Fibrotic scar facilitated ability of neonatal hippocampal neurons to fire action potential series by increasing the density of voltage activated sodium and potassium currents. Chondroitin sulphate proteoglycans played substantial role in these effects, as proven by their full reversion after administration of Chondroitinase ABC. In contrast, TGF-β1 did not contribute to them. An application of TGF-β1 itself attenuated generation of action potentials, inhibited sodium current and potentiated potassium currents. Main alteration of electrophysiological parameters of neonatal hippocampal neurons caused by a fibrotic scar model is enhanced excitability. TGF-β1 may have predominantly neuroprotective role in injured rat hippocampus.

Effect of ω-3PUFAs on cognitive deficit in rats with schizophrenia

Objective To investigate the antagonistic effect of ω-3PUFAs on cognitive impairment in MK-801-induced schizophrenia (SZ) rats and its mechanism. Methods Rat model of schizophrenia was induced by MK-801. Morris water maze was used to detect the change of cognitive function in rats. The number of neonatal neurons in hippocampus was detected by Brdu staining. CREB, p-CREB, BDNF, TrkB and p-TrkB levels were detected by Weston Blot. Results MK-801 induced schizophrenia-like cognitive impairment (the escape latency in the water maze test was (6.51±3.10)s for Ctr group, (15.27±6.20)s for Mod group; acrossing times was (4.63±1.06) times for Ctr group, (2.00±1.15) times for Mod group), reduced the number of neonatal neurons in hippocampus (the relative level of neonatal neuron number per unit area, Mod/Ctr was 0.656±0.066) and impaired the CREB/BDNF/TrkB pathway (the relative level of gray value, Mod/Ctr: CREB was 0.393±0.065, p-CREB was 0.591±0.015, BDNF was 0.716±0.115, TrkB was 0.787±0.029, p-TrkB was 0.586±0.013). ω-3PUFAs improved the CREB/BDNF/TrkB pathway activity by increasing CREB and TrKB level and their phosphorylation (the relative level of gray value, Pre/Ctr: CREB was 1.139±0.111, p-CREB was 0.845±0.243, BDNF was 0.864±0.133, TrkB was 0.916±0.022, p-TrkB was 0.952±0.047), and then recovered the number of neonatal neurons in hippocampus (the relative level of neonatal neuron number per unit area, Pre/ Ctr was 1.183±0.101), thereby reduced the cognitive dysfunction in schizophrenia rats(the escape latency in the water maze was (7.44±4.55)s for Pre; acrossing times was (3.86±1.68) times for Pre). Conclusion ω-3PUFAs can relieve the MK-801-induced schizophrenia-like cognitive impairment. Key words: Schizophrenia; MK-801; ω-3PUFAs; Cognition impairment

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Impaired autophagosome clearance contributes to neuronal death in a piglet model of neonatal hypoxic-ischemic encephalopathy

To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3–5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1–2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5–6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.

Neuroprotective Effect of Oxysophocarpine by Modulation of MAPK Pathway in Rat Hippocampal Neurons Subject to Oxygen-Glucose Deprivation and Reperfusion.

Oxysophocarpine (OSC), an alkaloid isolated from Sophora flavescens Ait, has been traditionally used as a medicinal agent based on the observed pharmacological effects. In this study, the direct effect of OSC against neuronal injuries induced by oxygen and glucose deprivation (OGD) in neonatal rat primary-cultured hippocampal neurons and its mechanisms were investigated. Cultured hippocampal neurons, which were exposed to OGD for 2h followed by a 24h reoxygenation, were used as an in vitro model of ischemia and reperfusion. 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay were used to confirm neural damage and to further evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca2+]i and mitochondrial membrane potential (MMP) were measured to determine the intracellular mechanisms and to further estimate the degree of neuronal damage. Changes in expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, p-ERK1/2, p-JNK1/2, and p-p38 MAPK were also observed in the in vitro model. It was shown that OSC (0.8, 2, or 5µmol/L) significantly attenuated the increased absorbance of MTT, and the release of LDH manifests the neuronal damage by the OGD/R. Meanwhile, the pretreatment of the neurons during the reoxygenation period with OSC significantly increased MMP; it also inhibited [Ca2+]i the elevation in a dose-dependent manner. Furthermore, the pretreatment with OSC (0.8, 2, or 5µmol/L) significantly down-regulated expressions of IL-1β, TNF-α, p-ERK1/2, p-JNK1/2, and p-p38 MAPK in neonatal rat primary-cultured hippocampal neurons induced by OGD/R injury. In conclusion, OSC displays a protective effect on OGD-injured hippocampal neurons by attenuating expression of inflammatory factors via down-regulated the MAPK signaling pathway.

Bilirubin augments Ca2+ load of developing bushy neurons by targeting specific subtype of voltage-gated calcium channels

Neonatal brain is particularly vulnerable to pathological levels of bilirubin which elevates and overloads intracellular Ca2+, leading to neurotoxicity. However, how voltage-gated calcium channels (VGCCs) are functionally involved in excess calcium influx remains unknown. By performing voltage-clamp recordings from bushy cells in the ventral cochlear nucleus (VCN) in postnatal rat pups (P4-17), we found the total calcium current density was more than doubled over P4-17, but the relative weight of VGCC subtypes changed dramatically, being relatively equal among T, L, N, P/Q and R-type at P4-6 to predominantly L, N, R over T and P/Q at P15-17. Surprisingly, acute administration of bilirubin augmented the VGCC currents specifically mediated by high voltage-activated (HVA) P/Q-type calcium currents. This augment was attenuated by intracellular loading of Ca2+ buffer EGTA or calmodulin inhibitory peptide. Our findings indicate that acute exposure to bilirubin increases VGCC currents, primarily by targeting P/Q-type calcium channels via Ca2+ and calmodulin dependent mechanisms to overwhelm neurons with excessive Ca2+. Since P/Q-subtype calcium channels are more prominent in neonatal neurons (e.g. P4-6) than later stages, we suggest this subtype-specific enhancement of P/Q-type Ca2+ currents likely contributes to the early neuronal vulnerability to hyperbilirubinemia in auditory and other brain regions.

MiR-9 Regulates the Expression of BACE1 in Dementia Induced by Chronic Brain Hypoperfusion in Rats

Background/Aims: MicroRNA-9 (miR-9) plays important roles in nervous system diseases such as glioblastoma and neurodegenerative disorders. However, how miR-9 contributes to dementia requires further study. In this study, we evaluated the role of miR-9 in dementia and the molecular mechanisms underlying its effects. Methods: A rat model of dementia was created by occlusion of the bilateral common carotid artery (2VO) for 8 weeks. Learning and memory were assessed using the Morris Water Maze (MWM). MicroRNA expression profiling was performed according to a protocol provided by LC Sciences, and quantitative real-time PCR (qRT-PCR) was used to detect the level of miR-9. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining were used to assess pathological changes in brain tissue. Western blot and immunofluorescence were employed to detect the expression of β-site APP cleaving enzyme 1 (BACE1) and c-AMP response element-binding protein (CREB). Results: Learning and memory were significantly impaired in 2VO rats, and these changes were accompanied by neuronal loss and glial activation in brain tissues. miR-9 was greatly upregulated in both the hippocampus and cortex of rats following 2VO. Knockdown of endogenous miR-9 via lentiviral vector-mediated delivery of its antisense molecule (lenti-pre-AMO-miR-9) reduced the vulnerability to dementia, reversed the increase in BACE1 expression, and ameliorated the reduction in CREB expression triggered by 2VO. BACE1 protein levels were significantly increased, but CREB protein levels were significantly decreased in the presence of miR-9 in cultured neonatal rat neurons (NRNs). AMO-miR-9 rescued the upregulation of BACE1 and downregulation of CREB elicited by miR-9 in rats. Dual luciferase assay experiments showed that overexpression of miR-9 inhibited the expression of CREB by targeting its 3’UTR domain. CREB protein was downregulated by miR-9 overexpression which was reversed by miR-9 inhibition in cultured NRNs. TEM imaging showed that miR-9 caused damage to NRNs, which was reversed by addition of AMO-miR-9. Conclusion: We conclude that miR-9 plays an important role in regulating the process of dementia induced by 2VO in rats by increasing BACE1 expression via downregulation of CREB.

Volatile Anesthetic Depression of Ca2+ Entry Into and Glutamate Release from Cultured Cerebellar Granule Neurons

Background: Volatile anesthetics (VAs) are known to have actions on a variety of ligand- and voltage gated ion channels, and thereby inhibit neuronal function. VA effects mediated by actions on voltage-gated Ca channels (VGCCs) were determined by studying their effects on the depolarization-induced rise in intracellular Ca2+ transients and the consequent glutamate release in cultured neonatal rat cerebellar granule neurons. Methods: Using a glutamate dehydrogenase-coupled assay for glutamate release, and fura-2 to measure intracellular [Ca2+] ([Ca2+]i), neurons at 37℃ were depolarized by a rapid increase in [K+]o from 5 to 55 mM. Actions of halothane, isoflurane, enflurane, and sevoflurane were compared with effects of altered [Mg2+]o, and by specific blockade of L-, P/Q- and/or N-type VGCC by nicardipine, ω-agatoxin IVA, and ω-conotoxin-GVIA, respectively. Whole-cell patch-clamp studies in these same neurons of VGCC Ba2+ currents were also performed at 22℃. Results: Clinical VA concentrations dose-dependently depressed both peak [Ca2+]i and glutamate release by 35-70%. With N- and/or L-type VGCC blockade, VAs caused a further marked decrease in [Ca2+]i transients. VAs depressed whole cell patch-clamped Ba2+ currents in these granule cell neurons by 35-40%. Conclusions: VAs depress Ca2+ entry by inhibiting a variety of VGCCs, and thereby reduce neuronal glutamate release. This action may contribute to the mechanism of anesthesia as well as provide protection during ischemic insults that cause neuronal injury. Citation: Carl Lynch III, Ning Miao, Kaoru Nagao, Joseph J. Pancrazio. Volatile anesthetic depression of Ca2+ entry into and glutamate release from cultured cerebellar granule neurons. J Anesth Perioper Med 2017; 4: 45-59. doi:10.24015/JAPM.2017.0016This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

A major blow to primate neonatal imitation and mirror neuron theory.

Keven & Akins' (K&A's) compelling new hypothesis explaining the developmental and neural basis of neonatal tongue protrusion has important implications for current understanding of primate imitation and the explanatory value of mirror neurons. If correct, this hypothesis eliminates a major source of evidence for neonatal imitation. I explore the implications this has for mirror neuron research and the arguments building upon them.

Characterization of dorsal root ganglion neurons cultured on silicon micro-pillar substrates.

Our study focuses on characterization of dorsal root ganglion (DRG) neurons cultured on silicon micro-pillar substrates (MPS) with the ultimate goal of designing micro-electrode arrays (MEAs) for successful electrophysiological recordings of DRG neurons. Adult and neonatal DRG neurons were cultured on MPS and glass coverslips for 7 days in vitro. DRG neuronal distribution and morphometric analysis, including neurite alignment and length, was performed on MPS areas with different pillar width and spacing. We showed that MPS provide an environment for growth of adult and neonatal DRG neurons as permissive as control glass surfaces. Neonatal DRG neurons were present on MPS areas with narrow pillar spacing, while adult neurons preferred wider pillar spacing. Compared to the control glass surfaces the neonatal and adult DRG neurons in regions with narrow pillar spacing range developed a smaller number of longer neurites. In the same area, neurites were preferentially oriented along three directional axes at 30°, 90° and 150°. MPS architecture influenced growth directionality of all main DRG neuronal subtypes. We can conclude that specific micro-pillar substrate topography affects the morphology of DRG neurons. This knowledge can enable development of MEAs with precisely defined physical features for various neuroscience applications.

Neonatal inhibition of Na+-K+-2Cl−-cotransporter prevents ketamine induced spatial learning and memory impairments

Prolonged ketamine exposure in neonates at anesthetic doses is known to cause long-term impairments of learning and memory. A current theoretical mechanism explains this phenomenon as being neuro-excitotoxicity mediated by compensatory upregulation of N-methyl-d-aspartate receptors (NMDARs), which then initiates widespread neuroapoptosis. Additionally, the excitatory behavior of GABAergic synaptic transmission mediated by GABAA receptors (GABAARs), occurring during the early neuronal development period, is proposed as contributing to the susceptibility of neonatal neurons to ketamine-induced injury. This is due to differential developmental expression patterns of Na+-K+-2Cl− co-transporter (NKCC1) and K+-Cl− co-transporter. Studies have shown that bumetanide, an NKCC1 inhibitor, allows neurons to become inhibitory rather than excitatory early in development. We thus hypothesized that bumetanide co-administration during ketamine treatment would reduce over excitation and protect the neurons from excitotoxicity. In this initial study, the Morris Water Maze test was used to assess the effects of co-administration of ketamine and bumetanide to neonatal Sprague-Dawley rats on long-term learning and memory changes seen later in life. It was revealed that bumetanide, when co-treated with ketamine neonatally, significantly impeded behavioral deficits typically seen in animals exposed to ketamine alone. Therefore, these findings suggest a new mechanism by which neonatal ketamine induced learning impairments can be prevented.