Activation of calpains, calpastatin and spectrin cleavage in the brain during the pathology of fatal murine cerebral malaria

Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.     

Neuronal mechanisms of interaction of Deiters nucleus with the cerebral cortex.

The effects of stimulation of the vestibular nerve and five different cerebral cortex areas on the neuronal activity of the lateral vestibular nucleus of Deiters were studied. Stimulation of the cerebral cortex is shown to lead to antidromic and synaptic activation of Deiters neurons. The synaptic potentials of Deiters neurons evoked from the cerebral cortex were of mono- and polysynaptic origin. In particular, stimulation of the cerebral cortex evoked in Deiters neurons mono- and polysynaptic excitatory postsynaptic potentials. Collaterals of vestibulospinal neurons reaching different cortex fields as well as convergence of influences from these cortex fields on Deiters neurons were revealed. Inhibitory effects of the cerebral cortex on Deiters neurons were of polysynaptic origin and occurred rarely. The topical correlation between Deiters nucleus and different areas of the cerebral cortex was found. The peculiarities and functional significance of the effects obtained are discussed.     

Electrophysiologic analysis of the conditioned reflex activity of cats against a background of thirst

EEG of the cerebral cortex and hypothalamic electrogram of cats during salt load and water deprivation were recorded at performance of conditioned running elicited by signals of insipid and salt food. The signal of insipid food presented during thirst was accompanied only by the cerebral cortex activation, while the salt signal involved in activation not only the cortex but the hypothalamus as well. In cases when the insipid signal was reinforced by salt food and the animal ate it (though during thirst it rejected the food), strong cortex activation was observed with the involvement of paraventricular parts of the hypothalamus. During intense thirst reversal was obtained of the signal role of the conditioned cue in accordance with the new quality of the alimentary reinforcement. Hypothalamo-cortical mechanisms of dominant motivation and its conditioned provision are discussed.     

Mechanism of DNA Fragmentation During Hypoxia in the Cerebral Cortex of Newborn Piglets

We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets.     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Electroencephalogram and event-related potentials in the selective attention paradigm as related to the levels of some metabolites in the prefrontal cortex

The results of preliminary analysis at the first stage of the study have demonstrated significant relationships between electrophysiological parameters and the levels of a number of metabolites (measured by proton magnetic resonance spectroscopy) in the dorsolateral prefrontal cortex of the left cerebral hemisphere. The observed relationships are assumed to be mediated by individual-specific characteristics of activation of this cerebral region and its contribution to information processing. The neurophysiological markers of the weakened functional state of the brain are associated with decreased levels of N-acetyl aspartate and choline-containing compounds and an increased level of creatin/phosphocreatin in the tested area of the left prefrontal cortex.     

Glutamate-induced activation of nitric oxide synthase is impaired in cerebral cortex in vivo in rats with chronic liver failure

It has been proposed that impairment of the glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in hepatic encephalopathy. The aims of this work were to assess whether the function of this pathway and of nitric oxide synthase (NOS) are altered in cerebral cortex in vivo in rats with chronic liver failure due to portacaval shunt (PCS) and whether these alterations are due to hyperammonemia. The glutamate-nitric oxide-cGMP pathway function and NOS activation by NMDA was analysed by in vivo microdialysis in cerebral cortex of PCS and control rats and in rats with hyperammonemia without liver failure. Similar studies were done in cortical slices from these rats and in cultured cortical neurons exposed to ammonia. Basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These increases seem due to increased inducible nitric oxide synthase expression. NOS activation by NMDA is impaired in cerebral cortex in both animal models and in neurons exposed to ammonia. Chronic liver failure increases basal NOS activity, nitric oxide and cGMP but reduces activation of NOS induced by NMDA receptors activation. Hyperammonemia is responsible for both effects which will lead, independently, to alterations contributing to neurological alterations in hepatic encephalopathy.     

黑龙江省齐齐哈尔医学院第三附属医院神经内科

脑卒中后运动功能恢复的机制尚未完全阐明。研究表明中风后功能的恢复与大脑可塑性有关,本文旨在阐述近年对一侧脑缺血后双侧大脑半球的活动的研究成果。     

Brain regional alterations in the modulation of the glutamate-nitric oxide-cGMP pathway in liver cirrhosis. Role of hyperammonemia and cell types involved

Hepatic encephalopathy is a complex neuropsychiatric syndrome present in patients with liver disease that includes impaired intellectual function and alterations in personality and neuromuscular coordination. Hyperammonemia and liver failure result in altered glutamatergic neurotransmission, which contributes to hepatic encephalopathy. Alterations in the function of the glutamate-nitric oxide-cGMP pathway may be responsible for some of the neurological alterations found in hepatic encephalopathy. The function of this pathway is altered in brain from patients died with liver cirrhosis and one altered step of the pathway is the activation of soluble guanylate cyclase by nitric oxide, which is increased in cerebral cortex and reduced in cerebellum from these patients. Portacaval anastomosis and bile duct ligation plus hyperammonemia in rats reproduce the alterations in the activation of soluble guanylate cyclase by NO both in cerebellum and cerebral cortex. We assessed whether hyperammonemia is responsible for the region-selective alterations in guanylate cyclase modulation in liver cirrhosis and whether the alteration occurs in neurons or in astrocytes. Activation of guanylate cyclase by nitric oxide is lower in cerebellar neurons exposed to ammonia (1.5-fold) than in control neurons (3.3-fold). The activation of guanylate cyclase by nitric oxide is higher in cortical neurons exposed to ammonia (8.7-fold) than in control neurons (5.5-fold). The activation is not affected in cerebellar or cortical astrocytes. These findings indicate that hyperammonemia is responsible for the differential alterations in the modulation of soluble guanylate cyclase by nitric oxide in cerebellum and cerebral cortex of cirrhotic patients. Moreover, under the conditions used, the alterations occur selectively in neurons and not in astrocytes.     

Estradiol-induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: the roles of heat shock protein 90 (Hsp90) and MEK2.

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen-responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule-associated protein 2B. The intracellular distribution of the phospho-ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol-induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen-induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade.     

Dopamine D1-stimulated adenylyl cyclase activity in cerebral cortex of autopsied human brain.

Although the cerebral cortical dopamine D(1) receptor is considered to play a role in normal and abnormal brain function, little information is available on its characteristics in human brain. We compared dopamine-stimulated adenylyl cyclase (AC) activity in homogenates of cerebral cortex (frontal, temporal, parietal, occipital and cingulate cortex) of autopsied brain of neurologically normal subjects to that in striatum. Cerebral cortical AC activity was modestly and dose-dependently stimulated by dopamine (maximal 20-30%) with low microM EC50s and such stimulation was inhibited by the selective dopamine D1 receptor antagonist SCH23390. The magnitude of the maximal stimulation by dopamine was similar in autopsied and biopsied cerebral cortex. The extent of maximal stimulation was similar to that in dopamine-rich striatum (caudate, putamen and nucleus accumbens), despite much lower density of dopamine D1 receptors in cerebral cortex vs. striatum. The EC50 for dopamine stimulation in cerebral cortex (approximately 1 microM) was lower than that for caudate and putamen (approximately 3 microM). No detectable dopamine stimulation was observed in cerebellar cortex, thalamus or hippocampus. Dopamine stimulation in both cerebral cortex and striatum was independent of calcium activation. We conclude that dopamine stimulated AC can be measured in cerebral cortex of human brain allowing for the possibility that this process can be examined in human brain disorders in which dopaminergic abnormalities are suspected.     

Effect of serotonin and acetylcholine on the electrical activity of isolated cortex in the rabbit

Neuronal isolation of the rabbit's cerebral hemisphere shifts the EEG spectrum in direction of slower processes. Application of acetylcholine on the cortex elicits EEG activation and appearance of the theta-rhythm. Initially serotonin application is accompanied by the appearance of the theta-rhythm periods; in the process of subsequent administration of the drug these periods are gradually substituted by slow delta-waves. Combined application of serotonin and acetylcholine on isolated cortex elicits bursts of high-amplitude activity, abruptly substituted by "silence" phases. In contrast to the intact cortex where serotonin elicited prolonged and rhythmic alternation on EEG of phases of high-amplitude activity and of "silence" periods, in the isolated cortex the bursts of activity of about 1 min duration appeared only after application of the acetylcholine to serotonin-saturated cortex. Repeated phases of activation were either absent or were of short duration and extinguished rapidly.     

Expression and potential role of phospholipase D1 in cryoinjured cerebral cortex of rats

The expression and potential role of phospholipase D1 (PLD1) were studied in the cerebral cortex of rats after freeze injury. Histopathologically, cryoinjury, by exposing cerebral cortex to a prechilled rod for 1 minute, produced consistent pathological lesions, specifically neuronal death, infiltration of macrophages into the center of the cryoinjury, and reactive astrogliosis at the periphery, which caused the lesion site to become encased. Western blot analysis showed that PLD1 expression in the ipsilateral cerebral cortex increased significantly during days 1 to 3 after cryoinjury and declined slightly at post-injury day 7. PLD1 immunoreactivity was very low in the brains of sham-operated control adults. After cryoinjury, there was substantial PLD1 immunostaining of numerous inflammatory cells in the ipsilateral cortex, which were identical to ED1-positive macrophages. In addition, PLD1 immunoreactivity was increased in some neurons and astrocytes at the periphery of the cryoinjury at post-injury days 3 and 7. These findings suggest that cryoinjury by means of prechilled rods induced consistent histopathological changes in the cerebral cortex. In addition, expression of a cell activation signal, PLD1, was upregulated in macrophages and astrocytes in the ipsilateral cerebral cortex after cryoinjury.     

Formation of cortical inhibition in ontogenesis

Analysis of the literature on the development of cortical inhibition suggests that synaptic inhibition of cerebral cortical neurons arises almost simultaneously with the onset of their background activity. All types of cortical inhibition operate simultaneously since the emergence of inhibitory processes. Thus, the basic mechanisms of cortical inhibition in mature cerebral cortex begin to function since cortex activation at the earliest stages of ontogenesis.     

Arrangement and afferent connections of the neurons in the visual cortex differing in the size of the receptive fields

In the area 17 of the cerebral cortex of guinea pigs cells with small and medial receptive fields (RF) are concentrated at the sites of separate groupings of neurons excited by flashes of diffusion light. There are also cells with large RF here. At such sites of the cortex when specific afferents are electrically stimulated many cells exhibit monosynaptic activation irrespective of their RF size. The main part of the neurons with large RF is situated in the areas of the cortex between the excited grouping and monosynaptical excitation is not inherent in such neurons. Most cells of these areas are not excited by specific afferents.     

Three-dimensional electrophysiological topography of the rat corticostriatal system

Projections from the cerebral cortex are the major afferents of the caudoputamen and probably determine the functions subserved by each region of the nucleus. The corticostriatal system has been mapped using cytological techniques which give little information on the physiological importance of projections from individual cortical areas. The objective of this study was to characterize the three-dimensional topography of the corticostriatal system in the rat and to determine the physiological significance of these projections using electrophysiological techniques. Eight functionally distinct areas of the cerebral cortex (prefrontal, primary motor, rostral and caudal primary somatosensory, hindlimb, auditory, occipital and primary visual) were stimulated while recording the multiple unit activity in seven dorsal and seven ventral areas of the caudoputamen. Each stimulation site produced a distinctive pattern of activation within the caudoputamen. There was also a large site-dependent variation in electrophysiological activation produced by each stimulation. The motor and somatosensory areas produced the most powerful overall activation. In addition, a number of trends were obvious. There was a rostrocaudal topographical relationship between the site of stimulation and the area of the caudoputamen activated. Furthermore, more caudally and medially placed stimulation sites produced greater dorsal activation of the caudoputamen relative to ventral.     

Global relationship between anatomical connectivity and activity propagation in the cerebral cortex

Anatomical connectivity is a prerequisite for cooperative interactions between cortical areas, but it has yet to be demonstrated that association fibre networks determine the macroscopical flow of activity in the cerebral cortex. To test this notion, we constructed a large-scale model of cortical areas whose interconnections were based on published anatomical data from tracing studies. Using this model we simulated the propagation of activity in response to activation of individual cortical areas and compared the resulting topographic activation patterns to electrophysiological observations on the global spread of epileptic activity following intracortical stimulation. Here we show that a neural network with connectivity derived from experimental data reproduces cortical propagation of activity significantly better than networks with different types of neighbourhood-based connectivity or random connections. Our results indicate that association fibres and their relative connection strengths are useful predictors of global topographic activation patterns in the cerebral cortex. This global structure-function relationship may open a door to explicit interpretation of cortical activation data in terms of underlying anatomical connectivity.     

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for β-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_sα as well as G_iα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to G_sα, as well as affecting the G_i-mediated inhibitory pathway.     

Changes in the ultrastructure of the cerebral cortex in rats during learning and in disorders of higher nervous activity]

The results are outlined of a complex study of the ultrastructure of the rat cerebral cortex after learning and higher nervous activity disturbance. In the case of learning with preliminary stimulation with al-amphetamine or without it, the subcortical processes in the motor and auditory analysers of the cerebral cortex are characterized by sharp structural-functional activation of the apparatus of energy supply, of protein synthesis and of the synaptic zones. The length of the synaptic active zones increases, and extensive development of spine apparatuses is recorded in the postsynaptic area in the form of intricate membrane complexes. Quite the contrary, in cases of higher nervous activity disturbances, destruction of the organelles and desintegration of spine apparatuses is clearly pronounced. The question of the role of the latter in the memory processes is discussed.     

兔脑皮质静脉闭塞后脑组织Caspase-3活性的变化

目的观察兔脑皮质静脉闭塞后Caspase-3活性的变化。方法采用电凝法制作兔脑皮质引流静脉急性闭塞模型,琼脂糖凝胶电泳、荧光实时定量PCR和Western印迹检测Caspase-3表达。结果脑皮质静脉闭塞后8hCaspase-3活性已升高,24h达高峰,48h明显下降。结论细胞凋亡是脑缺血后脑损伤发生机制,Caspase-3蛋白参与了皮质静脉闭塞后脑缺血后神经元损伤的病理过程。