Oxytocin Receptor Ligands Induce Changes in Cytoskeleton in Neuroblastoma Cells

Aim of the present study was to evaluate effects of ligands of oxytocin receptors on gene expression of neurofilament proteins (nestin and microtubule-associated protein 2 (MAP2)) associated with neuronal differentiation and growth factors (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) related to neuronal growth. Fluorescent staining of F-actin was used to observe morphology of cells. Co-treatment with oxytocin and oxytocin receptor antagonist—atosiban—resulted in significant increase of MAP2 gene expression in SK-N-SH cells. There was no effect of oxytocin on gene expression of growth factors BDNF and NGF. Surprisingly, oxytocin with atosiban significantly increased mRNA levels for both BDNF and NGF. Gene expression of vasopressin receptor (V1aR) significantly decreased in response to vasopressin. Atosiban decreased mRNA levels for oxytocin receptor (OXTR) and V1aR. Oxytocin significantly decreased OXTR and nestin mRNA levels and increased mRNA levels for BDNF and NGF in U-87 MG cells. The densest recruitment of F-actin filaments was observed in apical parts of filopodia in SK-N-SH cells incubated in oxytocin presence. Present data demonstrate complex role of ligands of oxytocin receptors in regulation of gene expression of intermediate filaments and thus, oxytocin might be considered as a growth factor in neuronal type of cells.     

Low Doses of the Glycine/NMDA Receptor Antagonist R-(+)-HA-966 but not d-Cycloserine Induce Paroxysmal Activity in Limbic Brain Regions of Kindled Rats

(+)-HA-966 [ R -(+)-3-amino-1-hydroxypyrrolid-2-one], a functional antagonist at the glycine modulatory site on the W-methyl-D-aspartate (NMDA) receptor/ion channel complex, was evaluated in amygdala-kindled rats, a model of epilepsy recently shown to exhibit enhanced susceptibility to the adverse effects of competitive and non-competitive NMDA receptor antagonists. Since (+)-HA-966 displays weak partial agonistic effects at the glycine site (−10% efficacy of glycine), D-cycloserine, a glycine ligand with much higher intrinsic activity, was evaluated in kindled rats for comparison. Following drug administration, electrographic activity was recorded from the basolateral amygdala (i.e. the focal site) as well as the ipsilateral piriform cortex, ventral hippocampus and nucleus accumbens. In addition to the evaluation of original recordings, power spectrum analysis was used to delineate drug effects. (+)-HA-966 (20–40 mg/kg i.p.) induced marked alterations in electrographic recordings, including increases in amplitude and isolated spiking, i.e. signs of paroxysmal activity. The severity or duration of fully kindled seizures was not changed by (+)-HA-966, but the drug dramatically increased the duration of immobilization and limbic seizure activity following a kindled motor seizure. In contrast to (+)-HA-966, D-cycloserine did not induce any electrographic changes, even when administered in much higher doses than (+)-HA-966. The changes in electrographic recordings seen after administration of (+)-HA-966 in kindled rats were almost absent in non-kindled rats, indicating that kindling had increased the sensitivity to the paroxysmal effects of the glycine/NMDA receptor ligand. The data indicate that functional glycine/NMDA antagonists with low intrinsic efficacy may bear the risk of proconvulsant activity.     

Androgen Receptor Function During Undernutrition

Hypothalamo-pituitary inhibition of reproductive function during undernutrition is well known, however, the physiological mechanisms leading to suppression of gonadotrophin secretion are not clear. A variety of studies have indicated that testicular negative feedback on LH secretion is enhanced during food restriction. To evaluate directly the suppression by endogenous androgens on hypothalamic GnRH pulse generator activity during food restriction and examine the mechanism underlying the increased testicular steroidal feedback, we examined (1) circulating bioactive LH (bLH) levels in response to selective cerebral androgen blockade by intraventricular administration of an androgen receptor antagonist (hydroxyflutamide, SCH 16423) and (2) the binding capacity and affinity of androgen receptors in medio-basal hypothalamus, pituitary and prostate during undernutrition of intact mature male rats. Hydroxyflutamide (20 μg in 10μl vehicle), but not vehicle alone, markedly increased bLH levels in both food restricted and ad-lib fed rats. However, the faster (geometric mean 11.4 vs 27.7 min) and greater (47.2 vs 21.9 ng/ml) increase in bLH level in food restricted compared with ad-lib fed controls demonstrates an enhanced sensitivity to blockade of androgenic negative feedback during undernutrition. Food restriction increased androgen receptor binding capacity in pituitary (3.36 vs 0.77fmol/mg protein) but not in medio-basal hypothalamus or prostate while binding affinity was unchanged by undernutrition in all 3 tissues. These studies reveal that undernutrition both enhances tonic, androgen receptor-mediated feedback suppression of GnRH secretion and increases in pituitary (but not hypothalamic) androgen receptor numbers to cause inhibition of LH secretion.     

Linking of Glycine Receptor Transmembrane Segments Three and Four Allows Assignment of Intrasubunit-Facing Residues

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that mediate inhibitory neurotransmission in the brain and spinal cord and are targets of alcohols and anesthetics. The transmembrane (TM) domain of GlyR subunits is composed of four α-helical segments (TM1-4), but there are conflicting data about the orientation of TM3 and TM4 and, therefore, also the proximity of residues (e.g., A288) that are important for alcohol and anesthetic effects. In the present study, we investigated the proximity of A288 in TM3 to residues in TM4 from M404 to K411. We generated eight double mutant GlyRs (A288C/M404C, A288C/F405C, A288C/Y406C, A288C/W407C, A288C/I408C, A288C/I409C, A288C/Y410C, and A288C/K411C), as well as the corresponding single mutants, and expressed them in Xenopus laevis oocytes. To measure glycine responses, we used two-electrode voltage clamp electrophysiology. We built homology models of the GlyR using structures of the nicotinic acetylcholine receptor (nAChR) and a prokaryotic ion channel (Gloeobacter violaceus, GLIC) as templates, and asked which model best fit our experimental data. Application of the cross-linking reagent HgCl(2) in the closed state produced a leftward shift in the glycine concentration-response curves of the A288C/W407C and A288C/Y410C mutants, suggesting they are able to form cross-links. In addition, when HgCl(2) was coapplied with glycine, responses were changed in the A288C/Y406C, A288C/I409C, and A288C/Y410C double mutants, suggesting that agonist-induced rotation of TM4 allows A288C/Y406C and A288C/I409C to cross-link. These results are consistent with a model of GlyR, based on nAChR, in which A288, Y406, W407, I409, and Y410 face into a four-helical bundle.     

Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity

NMDARs are ligand-gated ion channels that cause an influx of Na⁺ and Ca²⁺ into postsynaptic neurons. The resulting intracellular Ca²⁺ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca²⁺ (Ca²⁺ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca²⁺ challenge, it inhibited the responses by 62%; similarly, the Ca²⁺ challenge induced a 3.7-fold decrease in the steroid IC₅₀ on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca²⁺ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca²⁺ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.     

Including Distal Motor Function within the NIHSS: Correlation with Motor Arm Function and IV rt-PA Treatment Response

ObjectivesThe Distal Motor Function (DMF) sub-score of the NIH Stroke Scale (NIHSS) was measured in the NINDS rt-PA Stroke Trials but is currently not included in the NIHSS. The correlation of DMF with the NIHSS Motor Arm Function (MAF) sub-score, the effect of IV tPA treatment on DMF, and whether adding DMF changes the utility of the NIHSS have not been analyzed.Materials and methodsMAF and DMF sub-scores were retrieved from the original NINDS rt-PA Stroke Trials for both sides of the body at baseline, 2 hours, 24 hours, 7–10 days, and 3 months after IV tPA treatment. MAF and DMF scores were correlated using Spearman correlation. Clustering of DMF and MAF scores was determined using a Bentler Comparative Fit Index (CFI) to estimate variation in NIHSS when adding DMF. The effect of IV tPA on DMF and MAF was assessed using a linear model comparing changes in scores from baseline to 3 months.ResultsMAF and DMF were highly correlated (p < 0.0001) across all time points for both dichotomous and continuous data on both sides. Intravenous tPA accounted for 21% of the change in DMF (p < 0.014, R² = 0.0157, N = 423) and 39% of the change in MAF (p < 0.093, R² = 0.0125, N = 547) from 0 to 3 months. On adding DMF to NIHSS, CFI decreased from 0.98 to 0.80 and DMF clustered with MAF, indicating that addition of DMF is unlikely to produce any discrepancy to NIHSS.ConclusionsIncluding DMF to the NIHSS does not appear to be of additional value. After IV tPA treatment, proximal and distal motor function in upper extremity strongly correlate over time but greater improvement in MAF is noted. Further research is needed on the role of IV tPA on minor strokes with deficits of DMF.     

NMDA Receptor Antagonist MK801 Protects Against 1-Bromopropane-Induced Cognitive Dysfunction

Neuroscience Bulletin - Occupational exposure to 1-bromopropane (1-BP) induces learning and memory deficits. However, no therapeutic strategies are currently available. Accumulating evidence has...     

用AChR单克隆抗体建立大鼠重症肌无力被动转移模型的研究

目的利用乙酰胆碱受体(AChR)单克隆抗体建立重症肌无力(MG)被动转移模型。方法将AChR单克隆抗体mAb35注入3种品系大鼠腹腔，观察其临床症状并行药理学和电生理学及超微结构鉴定。结果被动转移mAb35后3种大鼠均可出现肌无力症状。其临床症状、药理学特点、电生理特点和超微结构变化均与MG患者相似。结论利用AChR单克隆抗体可在此3种品系大鼠成功建立获得性自身免疫性MG模型。     

Postsynaptic Serine Racemase Regulates NMDA Receptor Function

d-serine is the primary NMDAR coagonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic d-serine remains limited. Though early studies suggested d-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and d-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional KO mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at 1 month of age, which could be rescued by exogenous d-serine. Interestingly, there was a restoration of LTP by 2 months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode of d-serine action.SIGNIFICANCE STATEMENT NMDARs are key regulators of neurodevelopment and synaptic plasticity and are unique in their requirement for binding of a coagonist, which is d-serine at most forebrain synapses. However, our understanding of the mechanisms regulating synaptic d-serine availability remains limited. d-serine is synthesized in the brain by the neuronal enzyme serine racemase (SR). Here, we show dendritic and postsynaptic localization of SR and d-serine in CA1 pyramidal neurons. In addition, using single-neuron genetic deletion of SR, we establish a role of postsynaptic SR in regulating NMDAR function. These results support an autocrine mode of d-serine action at synapses.     

Striatal Ascorbate and its Relationship to Dopamine Receptor Stimulation and Motor Activity

We have used the techniques of microdialysis and in vivo voltammetry to monitor striatal dopamine and ascorbate, as well as motor activity in unanaesthetized, freely-moving rats. Systemic administration of the non-selective dopamine receptor agonist apomorphine (0.5 mg/kg, s.c.) caused a decrease in dopamine, an increase in ascorbate, stereotyped behaviour and a generalized increase in motor activity. Separate systemic applications of the D1 receptor agonist SKF 38393 (10 mg/kg, s.c.) and the D2 receptor agonist Quinpirole (0.1 mg/kg s.c.) caused a decrease in dopamine but had no effect on ascorbate or motor activity. After coadministration of these drugs, there was an increase in both ascorbate and motor activity. Local application of apomorphine (0.01 mM) caused a reduction in dopamine similar to that seen following systemic application but had no effect on ascorbate or motor activity. The present results demonstrate that dopamine, via D1 and D2 receptors outside the striatum, plays an important role in the control of ascorbate release. These results lend further support to the hypothesis that changes in ascorbate levels are an index of glutamatergic neurotransmission.     

Hammersmith Functional Motor Scale and Motor Function Measure-20 in non ambulant SMA patients

The aim of this prospective longitudinal multi centric study was to evaluate the correlation between the Hammersmith Functional Motor Scale and the 20 item version of the Motor Function Measure in non ambulant SMA children and adults at baseline and over a 12 month period. Seventy-four non-ambulant patients performed both measures at baseline and 49 also had an assessment 12 month later. At baseline the scores ranged between 0 and 40 on the Hammersmith Motor function Scale and between 3 and 45 on the Motor Function Measure 20. The correlation between the two scales was 0.733. The 12 month changes ranged between −11 and 4 for the Hammersmith and between −11 and 7 for the Motor Function Measure 20. The correlation between changes was 0.48. Our results suggest that both scales provide useful information although they appeared to work differently at the two extremes of the spectrum of abilities. The Hammersmith Motor Function Scale appeared to be more suitable in strong non ambulant patients, while the Motor Function Measures appeared to be more sensitive to capture activities and possible changes in the very weak patients, including more items capturing axial and upper limb activities. The choice of these measures in clinical trials should therefore depend on inclusion criteria and magnitude of expected changes.     

Cerebellar Motor Function in Spina Bifida Meningomyelocele

Spina bifida meningomyelocele (SBM), a congenital neurodevelopmental disorder, involves dysmorphology of the cerebellum, and its most obvious manifestations are motor deficits. This paper reviews cerebellar neuropathology and motor function across several motor systems well studied in SBM in relation to current models of cerebellar motor and timing function. Children and adults with SBM have widespread motor deficits in trunk, upper limbs, eyes, and speech articulators that are broadly congruent with those observed in adults with cerebellar lesions. The structure and function of the cerebellum are correlated with a range of motor functions. While motor learning is generally preserved in SBM, those motor functions requiring predictive signals and precise calibration of the temporal features of movement are impaired, resulting in deficits in smooth movement coordination as well as in the classical cerebellar triad of dysmetria, ataxia, and dysarthria. That motor function in individuals with SBM is disordered in a manner phenotypically similar to that in adult cerebellar lesions, and appears to involve similar deficits in predictive cerebellar motor control, suggests that age-based cerebellar motor plasticity is limited in individuals with this neurodevelopmental disorder.