Medium-sized neurofilament protein related to maturation of a subset of cortical neurons.

Neurofilaments are typical structures of the neuronal cytoskeleton and participate in the formation and stabilization of the axonal and dendritic architecture. In this study, we have characterized a murine monoclonal antibody, FNP7, that is directed against the medium-sized neurofilament subunit NF-M. This antibody identifies a subset of neurons in the cerebral cortex of various species including human and in organotypic cultures of rat cortex. In the neocortex of all species examined, the antibody labels pyramidal cells in layers III, V, and VI, with a distinctive laminar distribution between architectonic boundaries. In comparison with other antibodies directed against NF-M, the FNP7 antibody identifies on blots two forms of NF-M that appear relatively late during development, at the time when dynamic growth of processes changes to the stabilization of the formed processes. Dephosphorylation with alkaline phosphatase unmasks the site, making it detectable for the FNP7 antibody. The late appearance suggests that the site is present during early development in phosphorylated form and with increasing maturation becomes dephosphorylated, mainly in dendrites. This event may relate to changes in cytoskeleton stability in a late phase of dendritic maturation. Furthermore, mainly corticofugal projections and only few callosal axons are stained, suggesting a differential phosphorylation in a subset of axons. The antibody provides a useful marker to study subsets of pyramidal cells in vivo, in vitro, and under experimental conditions.

Sodium sensing in neurons with a dendrimer-based nanoprobe.

Ion imaging is a powerful methodology to assess fundamental biological processes in live cells. The limited efficiency of some ion-sensing probes and their fast leakage from cells are important restrictions to this approach. In this study, we present a novel strategy based on the use of dendrimer nanoparticles to obtain better intracellular retention of fluorescent probes and perform prolonged fluorescence imaging of intracellular ion dynamics. A new sodium-sensitive nanoprobe was generated by encapsulating a sodium dye in a PAMAM dendrimer nanocontainer. This nanoprobe is very stable and has high sodium sensitivity and selectivity. When loaded in neurons in live brain tissue, it homogenously fills the entire cell volume, including small processes, and stays for long durations, with no detectable alterations of cell functional properties. We demonstrate the suitability of this new sodium nanosensor for monitoring physiological sodium responses such as those occurring during neuronal activity.

Bradykinin, but not muscarinic, inhibition of M-current in rat sympathetic ganglion neurons involves phospholipase C-β4

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I-K(M)), which can be inhibited by activation of M-1 muscarinic receptors (M-1 mAChR) and bradykinin (BK) B-2 receptors. Inhibition by the M1 mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein G alpha (q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves G alpha (q) and/or G alpha (11) (Jones et al., 1995). G alpha (q) and G alpha (11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I-K(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, - beta2, - beta3, and - beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I-K(M) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M-1 mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M-1 mAChR, inhibition of I-K(M) involves PLC and extends this finding by indicating that PLC-beta4 is involved.

Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain.

Changes in expression and function of voltage-gated sodium channels (VGSC) in dorsal root ganglion (DRG) neurons may play a major role in the genesis of peripheral hyperexcitability that occurs in neuropathic pain. We present here the first description of changes induced by spared nerve injury (SNI) to Na(v)1 mRNA levels and tetrodotoxin-sensitive and -resistant (TTX-S/TTX-R) Na(+) currents in injured and adjacent non-injured small DRG neurons. VGSC transcripts were down-regulated in injured neurons except for Na(v)1.3, which increased, while they were either unchanged or increased in non-injured neurons. TTX-R current densities were reduced in injured neurons and the voltage dependence of steady-state inactivation for TTX-R was positively shifted in injured and non-injured neurons. TTX-S current densities were not affected by SNI, while the rate of recovery from inactivation was accelerated in injured neurons. Our results describe altered neuronal electrogenesis following SNI that is likely induced by a complex regulation of VGSCs.

Quantitative distribution of parvalbumin, calretinin, and calbindin D-28k immunoreactive neurons in the visual cortex of normal and Alzheimer cases.

The distribution of parvalbumin (PV), calretinin (CR), and calbindin (CB) immunoreactive neurons was studied with the help of an image analysis system (Vidas/Zeiss) in the primary visual area 17 and associative area 18 (Brodmann) of Alzheimer and control brains. In neither of these areas was there a significant difference between Alzheimer and control groups in the mean number of PV, CR, or CB immunoreactive neuronal profiles, counted in a cortical column going from pia to white matter. Significant differences in the mean densities (numbers per square millimeter of cortex) of PV, CR, and CB immunoreactive neuronal profiles were not observed either between groups or areas, but only between superficial, middle, and deep layers within areas 17 and 18. The optical density of the immunoreactive neuropil was also similar in Alzheimer and controls, correlating with the numerical density of immunoreactive profiles in superficial, middle, and deep layers. The frequency distribution of neuronal areas indicated significant differences between PV, CR, and CB immunoreactive neuronal profiles in both areas 17 and 18, with more large PV than CR and CB positive profiles. There were also significantly more small and less large PV and CR immunoreactive neuronal profiles in Alzheimer than in controls. Our data show that, although the brain pathology is moderate to severe, there is no prominent decrease of PV, CR and CB positive neurons in the visual cortex of Alzheimer brains, but only selective changes in neuronal perikarya.

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic.

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) still carries a high burden by its mortality and long-term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes. METHODS: Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic-ischemic rats (Rice-Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase-3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy). RESULTS: Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10-fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats. INTERPRETATION: These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches. Ann Neurol 2014;76:695-711.

Fate of rubrospinal neurons after unilateral section of the cervical spinal cord in adult macaque monkeys: effects of an antibody treatment neutralizing Nogo-A.

The present study describes in primates the effects of a spinal cord injury on the number and size of the neurons in the magnocellular part of the red nucleus (RNm), the origin of the rubrospinal tract, and evaluates whether a neutralization of Nogo-A reduces the lesioned-induced degenerative processes observed in RNm. Two groups of monkeys were subjected to unilateral section of the spinal cord affecting the rubrospinal tract; one group was subsequently treated with an antibody neutralizing Nogo-A; the second group received a control antibody. Intact animals were also included in the study. Counting neurons stained with a monoclonal antibody recognizing non-phosphorylated epitopes on neurofilaments (SMI-32) indicated that their number in the contralesional RNm was consistently inferior to that in the ipsilesional RNm, in a proportion amounting up to 35%. The lesion also induced shrinkage of the soma of the neurons detected in the contralesional RNm. Infusing an anti-Nogo-A antibody at the site of the lesion did not increase the proportion of SMI-32 positive rubrospinal neurons in the contralesional RNm nor prevent shrinkage.

Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

CO-EXPRESSION OF GCDH AND OAT1 IN NEURONS AND PROXIMAL TUBULE CELLS

Tissue-specific expression studies of Glutaryl-CoA dehydrogenase (Gcdh) in adult rats revealed expression in the whole rat brain, almost exclusively in neurons, and surprisingly high expression in the juxtamedullar cortex of the kidney. The organic anion transporter 1 (OAT1) mediates basolateral uptake of glutarate derivatives from proximal tubule cells and contributes to their renal clearance. In brain, OAT1 is expressed at the choroid plexus, in neurons of cortex and hippocampus. We hypothesized that Gcdh and Oat1 are co-expressed in the same cells in kidney and brain and analyzed their mRNA expression by in situ hybridization on cryosections of adult rat brain, kidney and liver. In brain, Gcdh and Oat1 were found co-expressed in most neurons. Only the Purkinje neurons of the cerebellum were found to be Oat1 negative. In the kidney Gcdh and Oat1 are widely co-expressed with a specific high expression in proximal tubule cells. In conclusion there seems to be a functional coupling of Gcdh and Oat1 on a renal and neuronal level. Further studies are ongoing to confirm these findings in human tissues.

Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures.

The intravenous, short-acting general anesthetic propofol was applied to three-dimensional (aggregating) cell cultures of fetal rat telencephalon. Both the clinically used formulation (Disoprivan, ICI Pharmaceuticals, Cheshire, England) and the pure form (2,6-diisopropylphenol) were tested at two different periods of brain development: immature brain cell cultures prior to synaptogenesis and at the time of intense synapses and myelin formation. At both time periods and for clinically relevant concentrations and time of exposure (i.e., concentrations > or = 2.0 micrograms/ml for 8 hr), propofol caused a significant decrease of glutamic acid decarboxylase activity. This effect persisted after removal of the drug, suggesting irreversible structural changes in GABAergic neurons. The gamma-aminobutyric acid type A (GABAA) blocking agents bicuculline and picrotoxin partially attenuated the neurotoxic effect of propofol in cultures treated at the more mature phase of development. This protective effect was not observed in the immature brain cells. The present data suggest that propofol may cause irreversible lesions to GABAergic neurons when given at a critical phase of brain development. In contrast, glial cells and myelin appeared resistant even to high doses of propofol.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

A comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ1-42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value.OBJECTIVE This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers.METHODS The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2.RESULTS Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes.CONCLUSION M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.