Caspase-3 immunoreactivity in different cortical areas of young and aging macaque (Macaca mulatta) monkeys

It has been shown that cytochrome-c-dependent caspase-3 activation is significantly elevated in the aging macaque brain. To assess the underlying age-related changes in the cellular distribution of caspase-3, we have examined the motor cortex, cerebellum and hippocampus of young (4-year-old, n = 4) and old (20-year-old, n = 4)rhesus monkeys by immunohistochemistry. Western blot analyses of brain homogenate showed that the antibody reacted only with inactive 32-kDa procaspase and its active 20- and 17-kDa subunits, formed after granzyme B exposure. In the motor cortex, pyramidal cells of layers III and V were moderately labeled; the underlying white matter contained weakly stained astrocytes. In the hippocampus, hilar neurons and pyramidal cells in CA3 showed the strongest immunoreaction, pyramidal cells in CA1 and granule cells of the dentate gyrus were also strongly labeled. In contrast, CA2 pyramidal cells were only weakly stained, and neurons of the molecular layer were unlabeled. Weak caspase-3 immunoreaction of CA2 neurons parallels known decreased susceptibility to apoptosis. In the cerebellar cortex, clusters of strongly labeled Purkinje cells were observed next to groups of weakly and unstained cells; granule cells were generally unstained. The brains of aging monkeys displayed a similar pattern of caspase-3 immunoreactivity. In neocortical layer V, however, scattered very strongly labeled pyramidal cells were regularly detected, which were not observed in younger animals. This clustering of caspase-3 indicates increased vulnerability of a subset of pyramidal cells in the aging brain.     

METABOLITE LEVELS IN BRAIN FOLLOWING EXPERIMENTAL SEIZURES: THE EFFECTS OF ISONIAZID AND SODIUM VALPROATE IN CEREBELLAR AND CEREBRAL CORTICAL LAYERS

Abstract— Levels of glucose, lactate, GABA and cyclic nucleotides were examined in discrete layers of the cerebellum and cerebral cortex of mice following treatment with the anticonvulsant, sodium valproate, and/or the convulsant, isoniazid. The concentrations of the metabolites were essentially uniform among the layers of each region, whether from control or from drug-treated mice. Metabolite concentrations in the isoniazid-treated mice were determined either 30 min after administration (preconvulsive state), or immediatley after the onset of seizures. Glucose and lactate, two markers of energy status in the brain, were only minimally affected by drug treatment. However, the levels of GABA and cyclic nucleotides were markedly different from control values in the drug-treated animals. In the preconvulsive state, GABA levels in cerebellar layers were depressed and the cyclic nucleotides were elevated in most layers of both regions. At the onset of seizures, the reduction of GABA and the elevation of cyclic AMP in both regions was more pronounced than during the preconvulsive state. While the concentration of cyclic GMP remained elevated in the cerebellar layers at the onset of seizures, the level in the cerebral cortex returned to control values. Valproate elevated GABA in all the layers of both regions and decreased the cyclic GMP in the cerebellar layers. Generally, when valproate was administered in combination with isoniazid, it dampened the isoniazid induced changes in the metabolites. The events leading up to a seizure as well as those that sustain it may be reflected by the disparate responses of the metabolites in the cerebellum and cerebral cortex.     

A computer model of generation of the motor cortex neuron processes seen in the course of execution of instrumental movement

A computer model of neuronal processes in the motor cortex column is presented. The model is consisted of two pyramidal cell layers with two groups of inhibitory interneurons, selectively controlling pyramidal cell soma and dendrite, in each. Active Na, Ca and K conductances are included in the model of a single neuron. Horizontal excitatory connections between pyramidal cells in the upper layer are largely of NMDA-receptor type, that in the lower layer--of non-NMDA-type. All inhibitory synapses are of GABA(A)-type. The model reproduces the main phenomenon observed in the motor cortex during the execution of conditioned movements. Consequent to an early excitation the upper layer pyramidal cells generate a late NMDA-dependent reflexive response to afferent conditional stimulation, which as in a real case is diminished by GABA(A)-type synaptic inhibition and afferent stimulus strength increase. The characteristic inverse relation between the late response manifestation and the stimulus strength observed in the real cortex can be reproduced in the model only if NMDA-glutamate receptors were preferentially localized in the terminals of pyramidal cell backward collaterals, not in the terminals of the afferent fibers on pyramidal neurons. The intended component of motor cortex neuronal activity is generated in NMDA-independent manner by the pyramidal cells of lower layer. The slow time coarse of intended component as compared with short duration of AMPA epsp's is due to a consecutive relay-race--like activation of pyramidal neurons with different dendrit-to-soma ratio.     

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II–III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.     

Glycogen, ammonia and related metabolities in the brain during seizures evoked by methionine sulphoximine

Abstract— The levels of ATP, P-creatine, glucose, glycogen, lactate, glutamate and ammonia were measured in mouse brain after administration of the convulsive agent methionine sulphoximine (MSO). No changes were observed in ATP and P-creatine levels either before or during the seizures. Lactate levels were unchanged until the onset of seizures (4–5 hr) at which time the levels increased an average of 65 per cent. Glucose and glycogen levels increased progressively. Just before the onset of seizures the levels had increased 95 and 62 per cent, respectively. During the seizures both substances had increased a total of 130 per cent. Comparable changes were found in cerebral cortex, cerebellum and subcortical forebrain. Through the use of quantitative histochemical methods it was found that the greatest increases in glycogen occurred in layers I and III (layers II and IV were not analysed). Progressively smaller changes were found in layers V and VI and no increase at all was found in the subjacent white matter. Glucose, in contrast to glycogen, increased to about the same degree in all cerebral layers and in subjacent white matter. The increase in glycogen after MSO administration may be related to the fact that MSO also causes an increase in the ratio of brain to serum glucose levels. This would indicate that an increase in intracellular glucose had occurred. Ammonia levels were increased 300–400 per cent in both cerebrum and cerebellum. A time study in cerebellum showed that the increase begins early and reaches maximal levels long before the onset of seizures. Glutamate levels were reduced by small but statistically significant amounts in both cerebrum and cerebellum. Administration of methionine sulphoximine completely prevented seizures and the increase in lactate, but did not prevent the increases in glycogen and glucose. The rise in ammonia was reduced but not prevented. During 20 sec of complete ischaemia (decapitation) ATP, P-creatine and glucose fell somewhat more rapidly than normal in brain of animals undergoing MSO seizures. From the changes it was calculated that the metabolic rate had been increased about 20 per cent by the seizure. A new sensitive and specific enzymic method for determination of tissue ammonia is presented together with evised enzymic procedures for lactate and glutamate.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

Cortico-cortical connections in the motor cortex of the brain; a study using the peroxidase method]

Using a method based on retrograde axonal transport of horse-radish peroxidase (HRP), the cortico-cortical afferents of the motor cortex were studied. After enzyme injection into the posterior sigmoideus gyrus, the HRP product was found in the first and the second somatic sensory areas and in parietal cortex (fields 5a, 5b). The HRP-positive neurons occurred in layers II, III and V of the cortex and belonged to the pyramidal cells.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Subthalamic Nucleus High-Frequency Stimulation Restores Altered Electrophysiological Properties of Cortical Neurons in Parkinsonian Rat

Electrophysiological recordings performed in parkinsonian patients and animal models have confirmed the occurrence of alterations in firing rate and pattern of basal ganglia neurons, but the outcome of these changes in thalamo-cortical networks remains unclear. Using rats rendered parkinsonian, we investigated, at a cellular level in vivo, the electrophysiological changes induced in the pyramidal cells of the motor cortex by the dopaminergic transmission interruption and further characterized the impact of high-frequency electrical stimulation of the subthalamic nucleus, a procedure alleviating parkinsonian symptoms. We provided evidence that a lesion restricted to the substantia nigra pars compacta resulted in a marked increase in the mean firing rate and bursting pattern of pyramidal neurons of the motor cortex. These alterations were underlain by changes of the electrical membranes properties of pyramidal cells including depolarized resting membrane potential and increased input resistance. The modifications induced by the dopaminergic loss were more pronounced in cortico-striatal than in cortico-subthalamic neurons. Furthermore, subthalamic nucleus high-frequency stimulation applied at parameters alleviating parkinsonian signs regularized the firing pattern of pyramidal cells and restored their electrical membrane properties.     

Compensatory processes in the motor cortex of the cerebral hemispheres in partial damage to the ventrolateral thalamic nucleus in the cat]

In cats alimentary instrumental reflex was elaborated of lever transference by the right forepaw in a definite zone--prior to and after the electrolytic damage of the left ventrolateral thalamic nucleus. It was found that even a slight damage led to disturbance of precise movements mostly expressed in the first two weeks; after that gradual normalization of the motor reactions took place. At morphological study of various pyramidal neurones and also satellite and general glia in the zone of presentation of the ventrolateral nucleus in the cerebral cortex a hypertrophy was discovered of the pyramidal cells and their processes in III and V layers, accompanied by active reaction of various glia forms. Morphometric processing and statistic analysis of the obtained data showed the dominance of structural compensatory changes observed in the cortex on the side of VL damage.     

Long-range neuronal circuits underlying the interaction between sensory and motor cortex

In the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.     

Changes in the mRNAs encoding subtypes I,II and III sodium channel alpha subunits following kainate-induced seizures in rat brain

Several lines of evidence underscore a possible role of voltage-gated Na+ channels (NaCH) in epilepsy. We compared the regional distribution of mRNAs coding for Na+ channel α subunit I, II and III in brains from control and kainate-treated rats using non-radioactive in situ hybridization with subtype-specific digoxigenin-labelled cRNA probes. Labelling intensity was evaluated by a densitometric analysis of digitized images. Heterogeneous distribution of the three Na+ channel mRNAs was demonstrated in brain from adult control rats, which confirmed previous studies. Subtype II mRNAs were shown to be abundant in cerebellum and hippocampus. Subtype I mRNAs were also detected in these areas. Subtype III mRNAs were absent in cerebellar cortex, but significantly expressed in neurons of the medulla oblongata and hippocampus. The three subtypes were differentially distributed in neocortical layers. Subtype II mRNAs were present in all of the layers, but mRNAs for subtypes I and III were concentrated in pyramidal cells of neocortex layers IV–V. During kainate-induced seizures, we observed an increase in Na+ channel II and III mRNA levels in hippocampus. In dentate gyrus, subtype III mRNAs increased 3 h after K A administration to a maximum at 6 h. At this latter time, a lower increase in NaCh III mRNAs was also recorded in areas CA1 and CA3. NaCh III overexpression in dentate gyrus persisted for at least 24 h. In the same area, NaCh II mRNAs were also increased with a peak 3 h after K A injection and a return to control levels by 24 h. No changes in NaCh I mR NAs were seen. The K A-induced up-regulation in NaCh mR NAs probably resulted in an increase in hippocampal neuronal excitability.     

Structural bases of sensomotor integration in the cat's cerebral cortex

Connections of the somatosensory cortex surrounding the postcruciate fossa with the lateral region of the motor area (in the cruciate sulcus) were established by the Nauta-Gygax and Fink-Heimer methods and also by the retrograde horse-radish peroxidase transport method. A high degree of differentiation was found in the organization of transcortical sensomotor projections. The pyramidal, stellate, and inverted pyramidal neurons in the third layer of the cortex were shown to take part in the formation of these pathways. Results obtained by the experimental degeneration method combined with electron microscopy showed that afferentation from the first somatosensory area of the cortex reaches mainly cells in layers III and V. It is suggested that the influence of the association fibers on projection neurons in the motor area is transmitted either directly or through interneurons.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 460–466, September–October, 1981.     

Interneurons of the motor region of the neocortex]

Interneurons of motor area in the brain cortex have been studied in cats and monkeys. The greatest attention has been paid to pyramidal interneurons, among which six cell types have been described according to their axonal composition. Unlike stellate interneurons, all types of pyramidal interneurons possess less developed axonal collaterals. Interneuronal contacts are situated on dendrites or cell bodies of middle and large long-axonal pyramids. Functional role of cortical interneurons seems to be different. Some of them are of inhibitory nature (basket cells and, perhaps, other types of long-axonal stellate neurons), others are exciting elements. The latter include short-axonal stellate neurons and, perhaps, pyramidal interneurons. While comparing the cortex in cats and monkeys, it is evident that the neocortex in monkeys, especially its lower layers, is rich in pyramidal interneurons.     

A metabolomic comparison of mouse models of the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6?month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2?months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models.     

Autoradiography of protein synthesis as a method of assessment of morphofunctional changes in brain structures]

A combination of two groups of autoradiography technique (for a hole brain and individual cells) was applied with using 3H-leucine to evaluate the changes of brain functional activity on the level as anatomical structures and as different type neurons. It was found that Wistar rats with lowered motor activity induced by 3-4 weeks treatment with L-DOPA 100 mg/kg displayed the motor nuclei of the brain stem the cerebellum as highly labelled structures and the motor cortex and n. caudatus as feebly ones in comparison with control. However, a quantitative assessment of silver grains over the neurons of layers III and V of motor cortex and n. caudatus showed not only a significant increase of labelling, especially in neurons of layer V on 174%, in comparison with control but revealed unequal labelling of different type neurons. It was concluded that the applied two groups of autoradiography technique can be a useful approach to assess the brain functional activity.     

Thoughts on the cerebral cortex

The cortex is often described as a network processing information in the direction from sensory to motor areas. However, the structure of the cortex is asymmetrical only in the vertical direction, suggesting an input-output transformation between layers rather than between areas. This operation must be a very generally applicable one, since the plan of the cortex is basically the same everywhere. In an attempt to understand it, a skeleton cortex of only pyramidal cells is considered. They are characterized by a double dendritic expansion, an apical one in the first layer, which is considered as the input layer, and a basal one which receives excitation from the axon collaterals of other pyramidal cells. If pyramidal cells learn (perhaps by growing dendritic spines) to respond to frequent constellations of activity in their afferents, each will learn a property of the input (through its apical dendrites) provided that it was preceded by other properties sensed by neighbouring pyramidal cells (which influences it through its basal dendrites). Thus the pyramidal cells will code the input in terms of properties which have a tendency to follow each other. This will be a coding which reflects the causal structure of the world. Various uses of a network embodying the conditional probabilities of events in the input are described, including recognition of familiar sequences and prediction. The local variation of fiber patterns in the cerebral cortex of man, described as myeloarchitectonics, is interpreted as a macroscopical expression of the different statistics of the set of conditional probabilities linking the events represented by individual pyramidal cells in different areas (in different functional contexts).     

Retrograde degeneration of the pyramidal cells in the motor cortex of apes (Macaca fascicularis)]

In three adult macaques the retrograde degeneration of cell bodies in the motor cortex was investigated 6 months after unilateral pyramidal tract section. Large and small Betz cells of the fifth layer were identified microscopically and counted. The analysis of the data reveals that after pyramidotomy, (1) contrary to our expectations from the extent of the pyramidal lesions, a surprising percentage of undegenerated Betz cells remains in the contralateral motor cortex, (2) a greater percentage of small rather than large cell survives, and (3) the greatest loss of cells is in the foot region, the smallest in the face region. The results are discussed in relation to the role of pyramidal axon collaterals in the survival of cell bodies, and the distinction between pyramidal cells and pyramidal tract cells.