Resuscitation of Brain Neurons in the Presence of Ca2+ After Toxic NMDA-Receptor Activity

Abstract: Cultured cerebellar granule cells were subjected to toxic activation of the NMDA receptor that was terminated by MK-801. Subsequent resuscitation experiments were mostly conducted in the presence of a physiological concentration of Ca²⁺. Addition of pyruvate and inorganic phosphate, in addition to glucose, which was always present, rescued ∼40% of the dying neurons. La³⁺ and ruthenium red were also effective resuscitating agents. The combination of pyruvate, inorganic phosphate, and ruthenium red rescued 65% of the dying neurons. Parallel studies with ⁴⁵Ca indicated that La³⁺ and ruthenium red facilitated the decrease of ⁴⁵Ca in the neurons, whereas inorganic phosphate, supported by energy-yielding pyruvate, formed perhaps, a less harmful Ca complex inside the neurons.     

Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

Influences of cerebellar interpositus nucleus and fastigial nucleus on neuronal activity of lateral hypothalamic area

Stimulation of cerebellar interpositus nucleus and (astigial nucleus could influence the neuronal activi-ty of lateral hypothalamic area in the cat, and some of the neurons which respond to the cerebellar stimulations are glucose-sensitive neurons. These results suggest that the cerebellum is involved not only in motor control, but also in the regulation of non-somatic functions through the cerebello-hypothalamic pathways.     

NA和5－HT对小脑脑片浦肯野细胞自发及诱发电活动的影响

在大鼠小脑脑片上观察了ＮＡ和５－ＨＴ对浦肯野细胞（ＰＣ）的自发放电活动及由白质刺激所引起的诱发放电活动的影响。结果表明：（１）ＮＡ使ＰＣ产生抑制、兴奋和双相反应,以抑制反应为主（７９．８％）;５－ＨＴ引起ＰＣ兴奋和抑制反应,以兴奋反应略多（５７．８％）。（２）先后灌流ＮＡ和５－ＨＴ对同一个ＰＣ自发放电的影响主要为抑制（ＮＡ）－兴奋（５－ＨＴ）（５３．８％）。（３）ＮＡ对ＰＣ的诱发复杂锋电位（ＣＳ）和简单锋电位（ＳＳ）反应,主要产生增强效应（５７．１％和６２．８％）;５－ＨＴ对ＰＣ诱发ＣＳ和ＳＳ反应则主要产生压抑作用（６０．０％和６８．２％）。（４）先后灌流ＮＡ和５－ＨＴ对同一个ＰＣ的诱发ＣＳ和ＳＳ反应,主要表现为ＮＡ对这两种诱发反应的增强和５－ＨＴ的压抑效应（６０．０％和５２．９％）。这些结果提示,ＮＡ能和５－ＨＴ能传入纤维可以通过释放ＮＡ和５－ＨＴ调节ＰＣ的兴奋性水平并改变ＰＣ对爬行纤维和苦状纤维突触传入的反应敏感性,影响小脑皮层神经元网络的感觉运动整合过程。     

Acetyl-L-Carnitine Arginine Amide Prevents β 25–35-Induced Neurotoxicity in Cerebellar Granule Cells

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with 25–35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KC1 in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later -amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with 25–35 was able to rescue neurons from 25–35-induced neurotoxicity. We also studied the changes in Ca²⁺ homeostasis following glutamate stimulation, in control and -amyloid treated single cells, either in presence or in absence of ST857. 25–35 did not affect basal [Ca²⁺]_i, while modified glutamate-induced [Ca²⁺]_i increase, causing a sustained plateau phase of [Ca²⁺]_i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with 25–35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca²⁺ homeostasis.     

Morphological and Biochemical Changes During Programmed Cell Death of Rat Cerebellar Granule Cells

Cultured cerebellar granule cells deprived of depolarizing concentrations of KCl and serum die by programmed cell death. Recently, it was shown that serum removal by itself can lead to oxidative stress and DNA fragmentation in these cells. We have modified the protocol which initiates cell death in such a way that only the effect of KCl withdrawal-induced cell death was observed. We have performed a series of experiments to correlate the structural and biochemical changes in this process of cell death. Significant morphological alterations occur in cell bodies and neurites during a 48-hour period of KCl removal. Cell viability dropped to 53%, 34% or 10% of control levels, respectively, as a result of 1-, 2-, or 3-day KCl removal. A series of experiments was conducted to determine the change of total protein level, protein synthesis rate, RNA synthesis rate, and mitochondrial activity during the first 48 hours of KCl removal. These studies not only provide a picture correlating the morphological and biochemical changes in the process of programmed cell death, but also serve as a reference for future studies of this complex phenomenon.     

Monoallelic and Biallelic Variants in EMC1 Identified in Individuals with Global Developmental Delay,Hypotonia, Scoliosis,and Cerebellar Atrophy

The paradigm of a single gene associated with one specific phenotype and mode of inheritance has been repeatedly challenged. Genotype-phenotype correlations can often be traced to different mutation types, localization of the variants in distinct protein domains, or the trigger of or escape from nonsense-mediated decay. Using whole-exome sequencing, we identified homozygous variants in EMC1 that segregated with a phenotype of developmental delay, hypotonia, scoliosis, and cerebellar atrophy in three families. In addition, a de novo heterozygous EMC1 variant was seen in an individual with a similar clinical and MRI imaging phenotype. EMC1 encodes a member of the endoplasmic reticulum (ER)-membrane protein complex (EMC), an evolutionarily conserved complex that has been proposed to have multiple roles in ER-associated degradation, ER-mitochondria tethering, and proper assembly of multi-pass transmembrane proteins. Perturbations of protein folding and organelle crosstalk have been implicated in neurodegenerative processes including cerebellar atrophy. We propose EMC1 as a gene in which either biallelic or monoallelic variants might lead to a syndrome including intellectual disability and preferential degeneration of the cerebellum.     

An enigmatic hypoplastic defect of the deciduous canine

A roughly circular hypoplastic defect restricted to the labial enamel surface of the deciduous canine is described. This pathology is quite common in available samples of Upper Paleolithic and Neolithic children and a cadaver sample of recent Calcuttans, affecting 44% to 70% of individuals. It is rare in a Neanderthal sample and in children from a clinical practice in Vancouver. The lesion occurs twice as commonly in the lower jaw. The defect appears to commence at or after birth owing to localized pressure on thin or nonexistent alveolar bone overlying the bulging crypt of the deciduous canine. Population differences in the incidence of the pathology probably reflect innate and acquired variation in hard and soft tissue thicknesses in this region.     

Rat cerebellar granule cells in culture associate and metabolize differently exogenous GM1 ganglioside molecular species containing a C18 or C20 long chain base

A study has been made of the association properties of the two GM1 ganglioside molecular species GM1-C₁₈ and GM1-C₂₀ (containing C₁₈ and C₂₀ long chain bases, respectively) to rat cerebellar granule cells in culture. Both gangliosides recognized, to the same extent, and associated with them to give a form of association, the trypsin-labile form. This form was removed by treatment with trypsin enzyme. Both gangliosides associated stably with the cells to become components of the cell membranes. Although similar amounts of the two gangliosides entered the cells, being then metabolized, the time course of the association was different for the two gangliosides: after 15 h of ganglioside-cell incubation the amount of GM1-C₁₈ inserted into the cell membrane was 2.43 times higher than that of GM1-C₂₀.