Autoradiographic analysis of the incorporation of uridine-H3 into hyperchromic motoneurons of mouse spinal cord]

Under conditions of local administration of uridine-H3 solution a study was made of the dynamics of the isotope incorporation into the RNA of the nuclei and the cytoplasm of the perikarion of the hyperchromic motor neurons of the ventro-lateral nucleus of mouse spinal cord. An increase in the content of hyperchromic neurocytes in the population under study was reached in mice as a result of swimming for four hours. A different character of the label regression curves for the hyperchromic and normochromic motor neurons was shown.     

Method of quantitative assessment of morphological changes in the central nervous system]

The author present a variant of quantitative assessment of the degree of morphological changes in the central nervous system neurons. It is suggested that the following groups of the changed neurons should be distinguished: swollen neuron with the initial manifestations of breaking large tigroid lumps into smaller ones; swollen neuron with marked phenomena of breaking large tigroid lumps into smaller ones and initial phenomena of hypochromatosis; swollen neuron with total tigrolysis with hyper- and hypochromatosis; dehydrated hyperchromic neuron; vacuolized neuron; contracted atrophic neuron; perished neuron. Each of the mentioned groups is given a mark, characterizing the degree of morphological changes. A formula for the assessment of the degree of morphological changes for individual formations and zones of the central nervous system is suggested.     

Status of the DNA-RNA-protein synthesizing system in neurons of the sensomotor cortex of the brain of the 30-day-old rat

By means of the electron cytochemical method ribonucleoprotein (RNP) particles, condensed chromatin (CCh) and ribosomes of cytoplasm are describe in normo- and hyperchromic neurons (HChN) of the V and VI layers of the sensomotor cortex. The normochromic neurons are characterized by nearly a complete absence of CCh, a great number of fibrillar RNP particles. The ribosomes of cytoplasma are organized as polysomes. This demonstrates a high metabolic activity of the DNA-RNA-protein system in these cells. In nuclei of one HChN group numerous small CCh clumps are revealed, amount of RNP particles does not change noticeably, comparing the nuclei of the normochromic cells. In cytoplasm a partial dissociation of polysomes takes place. All this demonstrates a decreased RNA synthesis in the nucleus and protein in cytoplasm of the given cells. In another HChN group the nucleus is filled with large CCh clumps. The number of fibrillar RNP particles decreases noticeably, and the number of granular ones increases. A complete dissociation of polysomes occurs. This demonstrates that in the cells mentioned not only RNA and protein synthesis is decreased, but the processing and nucleo-cytoplasmic transport of RNA is disturbed. The presence of transitional forms between the neuronal forms described makes it possible to suppose certain cyclicity in the work of their plastic apparatus, the normo- and hyperchromic neurons being morphologic equivalents of certain phases of the cycle.     

Selective Destruction of Cultured Dopaminergic Neurons from Fetal Rat Mesencephalon by 1-Methyl-4-Phenylpyridinium: Cytochemical and Morphological Evidence

Dopaminergic neurons in cultures of dissociated cells from fetal rat mesencephalon were exposed to the principal metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+), and several of its structural analogues. At concentrations between 0.01 and 0.1 microM, MPP+ inhibited catecholamine accumulation as visualized by cytofluorescence. Between 0.1 and 10.0 microM, MPP+ resulted in disappearance of tyrosine hydroxylase immunoreactivity without affecting other cells in the cultures. At concentrations higher than 10 microM, MPP+ was toxic to all cells present in the cultures. The effect of low concentrations of MPP+ on catecholamine cytofluorescence of the dopaminergic neurons was partially reversible. The intermediate concentrations produced irreversible structural changes of tyrosine hydroxylase-positive cells, resulting in complete disappearance of these neurons. The morphological changes were specific to the dopaminergic neurons and were not evident in other cells viewed with phase contrast microscopy. Of the structural analogues tested, the 1-ethyl analogue of MPP+ was effective in selectively destroying dopaminergic neurons in our culture system. The antioxidants L-acetyl-carnitine, beta-carotene, and alpha-tocopherol failed to protect against MPP+ neurotoxicity when co-incubated with the toxin.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Electron-cytochemical study of the changes in the structure of hyperchromic neurons shown after chlorpromazine administration

By means of Bernhard's electron cytochemical method for revealing DNP and RNP, the hyperchromic neurons in the cerebral cortex have been investigated 3, 6, 24 and 48 hours after chlorpromazine hydrochloride administration (15 mg/kg). Development of hyperchromia in the neurons has been followed beginning from minimal changes in the nucleus and cytoplasm up to their maximal reconstruction and a subsequent restoration to the normal stage. The data obtained demonstrate that hyperchromia of the neuron during various phases of its development can be characterized both by a decreasing synthetic activity and by restorative processes during the last period when the functional state is normalized.     

Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

Formation of striatum structural and ultrastructural organization in rat postnatal ontogenesis at changes of conditions of their embryonic development

Using light microscopy (Nissl and Golgi techniques), electron microscopy and immunohistochemistry, formation of structure of the brain striatum dorsolateral part from birth to three month of age was studied in rats submitted to acute hypoxia at the period of embryogenesis. Hypoxia at the 13.5th day of pregnancy (E 13.5) was found to lead to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, and the majority of large neurons for this period were degenerated by the type of chromatolysis with swelling of the cell body and processes and lysis of cytoplasmic organelles. By the end of the third week, shrunken hyperchromic or pycnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was found in neuropil around the degenerating neurons. By the 30th day as well as in adult animals, destruction of mitochondrial apparatus, an increased number of lysosomes, and blade-shaped nuclei, which are characteristics of the apoptotic cell death, were observed. This is also confirmed by an increased expression of proapoptotic protein (p53) and its co-localization with caspase-3 in a part of neurons. Morphometric analysis showed a decrease of the cell distribution density in striatum and a change of ratio of different cell types in hypoxia-exposed rats as compared with control group. The most pronounced decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons (larger than 80 μm²) was revealed at early stages of postnatal ontogenesis. After 3 postnatal weeks, the number of middle-sized neurons (30–95 μm²) decreased (by 11.8–19.2% as compared with control, p < 0.05). The obtained data have shown that changes of embryogenesis conditions (hypoxia) at the period of the most intensive proliferation of telencephalon neuroblasts lead to impairment of the process of striatal nervous tissue formation. This might be the cause of delay of development and disturbances of behavior and learning, which are observed in rats exposed to prenatal hypoxia.     

A single neuron response to photodynamic effect of various aluminum and zinc phthalocyanines

The photodynamic effects of sulphonated zinc and aluminum phthalocyanine derivatives as well as phosphonated aluminum phthalOcyanine on the firing of isolated crayfish mechanoreceptor neurons were studied. After 30 min staining neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm2) and changes in neuron firing frequency were recorded. Neuron firing was found to be very sensitive to photodynamic effect and could serve as a sensitive indicator of cell photodamage. It changed the firing level and then died at nanomolar concentrations of phthalocyanines. The dynamics of the neuron responses to photodynamic effects included stages of firing activation and/or inhibition prior to irreversible firing abolition. The order of these stages depended on photosensitizer type and concentration. The comparison of the dependencies of neuron lifetime on photosensitizer concentrations showed ZnPcS2 to be the most effective photosensitizer.     

Changes in the ultrastructure of synapses and neurons of the ventral horns of the spinal cord in cats with tetany as a result of the removal of the parathyroid glands

An electron microscopic investigation on ultrastructure of synapses and ventral horns of the lumbar thickening in the spinal cord of cats with parathyroprival tetany has been performed. When motor disorders are clearly seen on the 2d - 4th days after removal of the parathyroid glands, together with the changes demonstrating functional activity of the synapses, certain irreversible degenerative changes of the "dark" type are seen in the axodendritic and axosomatic synapses. A complex of structural-metabolic changes has been revealed in the motor neurons, they have both functional-compensatory and degenerative character. It is possible to suggest that the changes revealed at parathyroprival tetany result from a disturbed metabolism and increased afferent influences.     

Use of critical point polyacrylamide sols in thermal denaturation experiments with chromatin at physiological ionic strength

Low percentage highly crosslinked polyacrylamide gels just above the critical point in the chemically polymerized sol to gel transition are used to generate polyacrylamide sols at critical point concentrations, 7.4 g liter-1, by mild heating. We find that chromatin samples mixed with these sols induce the sol to gel transition in a process of complex coacervation. In this state, salt insoluble chicken erythrocyte chromatin is stabilized against large scale aggregation and precipitation during thermal denaturation at physiological sodium ion concentrations. The hyperchromic melting behavior of DNA in polyacrylamide sols is reproducible and consistent throughout a wide range of sodium chloride concentrations. Empirical spectroscopic techniques are discussed which isolate temperature-dependent hyperchromic signals at 260 nm due to conformational changes of DNA in chromatin and local environmental changes which promote anomalous light scattering.     

Formation of structural and ultrastructural organization of striatum rat postnatal ontogenesis upon changes in their embryonal development

By light microscopy (by Nissl and Golgi), electron microscopy, and immunohistochemistry methods, formation of structure of the brain striatum dorsolateral part from birth to the 3-month age was studied in rats submitted to acute hypoxia at the period of embryogenesis. It has been established that hypoxia at the 13.5th day (E13.5) leads to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, while the majority of large neurons at this period are degenerated by the type of chromatolysis with swelling cell body and processes and lysis of cytoplasmic organoids. By the end of the 3rd week, shrunk hyperchromic or picnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was detected in neuropil around degenerating neurons. By the 30th day as well as in adult rats there was observed destruction of mitochondrial apparatus, an increase of the number of lysosomes, and the appearance of bladed nuclei - signs of apoptotic cell death, which was also confirmed by an increased expression of proapoptotic p53 protein and its colocalization with caspase-3 in a part of neurons. Morphometrical analysis has shown a decrease of density of striatum cell arrangement and a change of ratio of different cell types in the rats submitted to hypoxia as compared with control group. At early stages of postnatal ontogenesis there was the greatest decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons with the area more than 80 microm2. After 3 weeks of postnatal development the number of middlesize neurons (30-95 microm2) decreased (by 11.8-19.2%) as compared with control. The obtained data show that a change of conditions of embryogenesis (hypoxia) at the period of the most intensive proliferation of the forebrain neuroblasts leads to disturbances of the process of formation of the striatum nervous tissue. This can be the cause of delay of development and disturbances of behavior and learning observed in rats submitted to prenatal hypoxia.     

Differential effects of aging on fore- and hindpaw maps of rat somatosensory cortex

Getting older is associated with a decline of cognitive and sensorimotor abilities, but it remains elusive whether age-related changes are due to accumulating degenerational processes, rendering them largely irreversible, or whether they reflect plastic, adaptational and presumably compensatory changes. Using aged rats as a model we studied how aging affects neural processing in somatosensory cortex. By multi-unit recordings in the fore- and hindpaw cortical maps we compared the effects of aging on receptive field size and response latencies. While in aged animals response latencies of neurons of both cortical representations were lengthened by approximately the same amount, only RFs of hindpaw neurons showed severe expansion with only little changes of forepaw RFs. To obtain insight into parallel changes of walking behavior, we recorded footprints in young and old animals which revealed a general age-related impairment of walking. In addition we found evidence for a limb-specific deterioration of the hindlimbs that was not observed in the forelimbs. Our results show that age-related changes of somatosensory cortical neurons display a complex pattern of regional specificity and parameter-dependence indicating that aging acts rather selectively on cortical processing of sensory information. The fact that RFs of the fore- and hindpaws do not co-vary in aged animals argues against degenerational processes on a global scale. We therefore conclude that age-related alterations are composed of plastic-adaptive alterations in response to modified use and degenerational changes developing with age. As a consequence, age-related changes need not be irreversible but can be subject to amelioration through training and stimulation.     

Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification

Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loop-helix (bHLH) genes. However, the precise mechanism for specification of retinal neuronal subtypes, particularly horizontal neurons and photoreceptors, remains to be determined. Here, we examined retinas with three different combinations of triple bHLH gene mutations. In retinas lacking the bHLH genes Ngn2, Math3, and NeuroD, horizontal neurons as well as other neurons such as bipolar cells were severely decreased in number. In the retina lacking the bHLH genes Mash1, Ngn2, and Math3, horizontal and other neurons were severely decreased, whereas ganglion cells were increased. In the retina lacking the bHLH genes Mash1, Math3, and NeuroD, photoreceptors were severely decreased, whereas ganglion cells were increased. In all cases, glial cells were increased. The increase and decrease of these cells were the result of cell fate changes and cell death and seem to be partly attributable to the remaining bHLH gene expression, which also changes because of triple bHLH gene mutations. These results indicate that multiple bHLH genes cross-regulate each other, cooperatively specify neuronal subtypes, and regulate neuronal survival in the developing retina.     

The developmental expression of fluorescent proteins in organotypic hippocampal slice cultures from transgenic mice and its use in the determination of excitotoxic neurodegeneration

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.     

Dynamics of single unit responses of the rabbit visual cortex to repetitive photic stimulation

Single unit responses in the visual cortex of the waking rabbit to repetitive photic stimulation at a frequency of once every 2.5 sec were studied. Depending on the total number of spikes in the response, the dynamics of the responses could be divided into two types: "fast" and "slow." From 5 to 15 stimuli were required to establish a stable level of response with changes of the first type, but 50 to 100 stimuli were needed for the response with changes of the second type. About 50% of all neurons did not change the characteristics of response. In the group of neurons with changing responses, partial habituation was found in 55–59% of cells; there were 25% of neurons with sensitization of discharge and 17–20% with a humpbacked type of response dynamics. A "slow" dynamics of unit responses in most cases was accompanied by changes in the duration of inhibitory pauses in the response; negative correlation of these values was observed in 65% of neurons.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 10, No. 5, pp. 451–459, September–October, 1978.