Irradiation neurotoxicity assessed in organotypic cultures of rat hippocampus.

The neurotoxic effects of irradiation on the developing nervous system were studied in organotypic cultures of hippocampus prepared from newborn Sprague-Dawley rats. Hippocampus slices of 7-day-old rats were irradiated at the day of explantation at doses of 1, 2 and 4 Gy, and cultured in a roller drum for a fortnight. Light and electron microscopy showed remarkable damage to neuronal cells following irradiation, oligodendrocytes and myelogenesis being also affected. In contrast to alterations in neuronal perikarya, no morphological changes in synapses were obvious, though their number seemed to be reduced after irradiation with 4 Gy. These results confirm that low dose radiation produces damage to the central nervous system not only pre-natally, but even in post-natal periods of differentiation and development.     

Aluminum enhances glutamate-mediated neurotoxicity in organotypic cultures of rat hippocampus

Both, glutamate (GLU) and aluminum (Al) have been implicated in neuronal damage and/or death in certain human neurodegenerative disorders. Recent evidence suggests that aluminum (Al) may potentiate the increase in glutamate-induced intracellular calcium overload. The present ultrastructural study was undertaken to determine the effect of Al on the development of GLU-mediated neurotoxicity in tissue culture conditions. The experiments were performed on organotypic cultures of rat hippocampus treated with low, subtoxic concentration of GLU (50 microM) and AlCl3 (400 microM) added to the growth medium separately or in combination. The exposure of cultures to GLU in the presence of Al3+ ions for up to 24 hours resulted in the development of typical excitotoxic neuronal changes, whereas separate GLU treatment at subtoxic doses or single Al application did not produce any apparent tissue damage. The neuronal lesions resulting from the combined application of GLU plus Al consisted predominately of more or less pronounced mitochondrial abnormalities, which are characteristic for early excitotoxic events. Severe swelling of the mitochondria led to the disruption of their internal structure and finally resulted in an apparent microvacuolization of the perikaryal cytoplasm of some pyramidal neurons. The present morphological data evidenced that Al is capable to potentiate the GLU-induced degenerative changes in hippocampal neurons in vitro. This supports the view of a possible role of Al in the process of neurodegeneration and suggests that Al may participate in the development of glutamate-mediated excitotoxic neuronal injury under certain pathological conditions.     

Profile of phosphoprotein labelling in organotypic slice cultures of rat hippocampus

In recent years organotypic slice cultures of hippocampal tissue of rats have been widely used to study factors involved in neuronal death. Here we used 2D electrophoresis to study the phosphoprotein profile in such cultures and the effect of oxygen/glucose deprivation on this profile. Cultures were prepared from 7-day-old rats. After 14 days in culture the phosphorylation profile in the cultures, as shown by phospho-protein markers undergoing developmental change, closely resembled the profile of fresh tissue from 23-day-old rats. The results suggest that this model could be a good method to observe the development of the tissue and its response to an ischaemic lesion     

Postnatal effect of embryonic neurogenesis disturbance on reelin level in organotypic cultures of rat hippocampus

Despite a delayed emergence of the symptoms, schizophrenia is thought to be a late consequence of early disturbances during development. Several reports have found decreased levels of reelin in the cortex and the hippocampus of postmortem brains of schizophrenic patients. In the rat, intraperitoneal injection of the anti-mitotic agent methylazoxymethanol (MAM) during intra-uterine development (embryonic day 17) induces cytoarchitectural abnormalities in the hippocampus and the cortex and behavioural changes reminiscent of positive, negative and cognitive symptoms of schizophrenia. We aimed to examine whether a transient prenatal disturbance of neurogenesis induces postnatal changes in the expression of reelin in the hippocampus. Cellular modifications were explored using hippocampal organotypic slice cultures, which allow for conservation of the in vivo cytoarchitecture. MAM effect on hippocampal neurogenesis was confirmed by birthdating experiments. After 3 weeks in vitro, reelin was expressed by calretinin-negative cells. The number of reelin-positive neurons was increased whereas the total neuron number was decreased in the stratum oriens in the E17 MAM-exposed animals as compared to the control group. Not only an increase in the number of cells expressing reelin was observed, but there was also a slight increase in reelin mRNA levels in hippocampal pyramidal cells of MAM-exposed animals. In contrast, there was no significant change in the dentate gyrus. These results show that transient prenatal disturbance of neurogenesis induces long-term modifications in specific areas of the hippocampus and in particular in the number of neurons expressing reelin. They also confirm the value of organotypic slices to study postnatal maturation in the hippocampus.     

Cytoplasmic structure in organotypic cultures of rat hippocampus prepared by rapid freezing and freeze-substitution fixation

The functional consequences of chronic treatment with haloperidol (0.5 mg/kg s. c. for 21–23 days) on striatal extracellular levels of dopamine and excitatory amino acids, aspartate and glutamate, were examined using microdialysis techniques. Our studies indicate that, in both awake and anesthetized animals, chronic haloperidol treatment does not appear to change basal outflow of dopamine and its response to an exogenous antagonist (i. e., a challenge dose of haloperidol). Furthermore, in chronic haloperidol and vehicle-treated animals, extracellular dopamine levels were decreased below our limit of detection following perfusion of tetrodotoxin through the probe, or into the medial forebrain bundle, suggesting that in both groups of animals extracellular dopamine levels are neuronally derived and seemed to depend equally on impulse flow. However, some differences were observed between the vehicle and haloperidol-treated animals: the excitatory action of 30 mM K⁺ on extracellular dopamine levels was decreased, and extracellular levels of glutamate were significantly increased, in animals treated chronically with haloperidol. The alterations in extracellular glutamate levels suggests that events at the terminal may be involved in maintaining the “normal” extracellular dopamine levels. Furthermore, the decrease in response to stimulation by K⁺ suggests that chronic haloperidol treatment may decrease the responsivity of the striatal dopamine system to stimuli. © 1993 Wiley-Liss, Inc.     

A modified roller method for organotypic brain cultures: free-floating slices of postnatal rat hippocampus

We describe a novel procedure for organotypic cultivation of free-floating brain sections of postnatal rats with a modified roller technique. Three hundred to 350-microm-thick sections of hippocampus are cultured for 13-15 days at 35.5 degrees C in 10-15 ml of feeding medium in 50-100 ml bottles under constant rotation on a horizontal high-speed mini-roller (60 rpm). Histological analysis (paraffin sections, Nissl Cresyl Violet and Hematoxylin/Eosin staining) demonstrates good survival of neuronal and glial cells and complete preservation of the neuronal organization of cultivated hippocampus with minimal central necrosis. This novel protocol permits not only survival and development of long-term three-dimensional organotypic postnatal brain tissue but also allows simultaneous cultivation of any number of brain sections in one bottle (up to 50 and even more) and therefore is useful for high throughput study of neurocytotoxic and hypoxic/ischemic neuronal damage with subsequent histological, immunocytochemical, biochemical, and molecular analysis.     

Induction of cellular stress and chaperone activation in organotypic slice cultures of hippocampus

Neurodegenerative diseases are often associated with the occurrence of misfolded proteins preceding neuronal cell death. Accumulation of misfolded proteins in the endoplasmic reticulum induces ER stress, which in consequence enhances chaperone expression to restore protein homeostasis. Here we used organotypic hippocampal slice cultures to analyze the time course of chaperone expression and neuronal death after induction of ER stress by tunicamycin treatment. Shortly after explantation many cells stain positive for Fluoro Jade B demonstrating neuronal cell death. While in control cultures the number of Fluoro Jade B labeled cells remarkably decrease over the total period of cultivation, neuronal death remains elevated in ER-stressed slice cultures. Caspase-3 staining revealed that neuronal death is primarily due to apoptosis in tunicamycin-treated slice cultures. The chaperone GRP78/BiP is expressed at low levels in control sections. Its expression is largely restricted to hippocampal neurons. Tunicamycin treatment resulted in upregulation of GRP78/BiP in the neuronal cells. Double-immunolabeling for GFAP shows a concomitant de novo expression of GRP78/BiP in astrocytes. The astrocytic GRP78/BiP upregulation might reflect an early, neuroprotective response. The increase of GRP78/BiP in neurons and astrocytes show successful induction of the ER stress response. The hippocampal slice cultures are, thus, a useful tool to examine the process of neurodegeneration and to investigate neuroprotective devices in an ER stress paradigm.     

Ethanol-induced neuronal death in organotypic cultures of rat cerebral cortex

Ethanol can affect normal development of the cerebral cortex, e.g., it can disrupt cell migration and exacerbate cell death. In vitro studies using primary cultures or cell lines provide further evidence that cell migration and death are altered by ethanol exposure. Organotypic cultures are more complex than primary cell cultures, and maintain some normal connectivity, thus providing a "more in vivo-like" model of brain development. We predict that exposing organotypic cultures of fetal rat cerebral cortex to ethanol results in changes similar to those described in vivo. Organotypic cultures of brains from 16-day-old fetuses were exposed to ethanol (0, 200, 400 or 800 mg/dl) for 72 h. Stereological methods were used to assess the frequency of viable and dying cells. Dying cells were identified as having DNA with polyadenylated tails or as having condensed chromatin. A small amount of cell death was evident in the marginal zone (MZ) and cortical plate (CP) of control cultures. The MZ, normally a cell body-poor layer, was enriched with somata following exposure to 400 mg/dl ethanol. Ethanol-induced cell death in the MZ; the amount of cell death was doubled following exposure to 800 mg/dl ethanol. The CP was more sensitive than the MZ; cell death increased following treatment with 400 mg/dl ethanol. Thus, organotypic cultures show that ethanol disrupts neuronal migration and increases cell death in the developing cerebral cortex. The effects of ethanol were site-specific and concentration-dependent. These changes are similar to those described in vivo.     

Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.     

Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxicological studies, including further studies of neurotoxic mechanisms of TMT. Four-week-old cultures, derived from 7-day-old donor rats and grown in serum-free medium, were exposed to TMT (0.5–100 μM) for 24 h followed by 24 h in normal medium. TMT-induced neurodegeneration was then monitored by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed by densitometric measurements of PI uptake displayed a dose and time-dependent increase, with the following ranking of vulnerability of the hippocampal subfields: FD>CA4≥CA3c>CA1>CA3ab. This differential neuronal vulnerability observed by PI uptake was confirmed by MAP-2 immunostaining and corresponded to in vivo cell stain observations of rats acutely exposed to TMT. The mean PI uptake of the cultures and the LDH efflux into the medium were highly correlated. The combined results obtained by the different markers indicate that the hippocampal slice culture method is a feasible model for further studies of TMT neurotoxicity.     

Nestin expression persists in astrocytes of organotypic slice cultures from rat cortex

Nestin is an intermediate filament protein typical for neural precursor cells that is down-regulated in the post-natal rodent brain. Re-expression of nestin has been observed in reactive astrocytes after injury. In this study, organotypic slice cultures from rat cortex were examined for expression of nestin and glial fibrillary acidic protein between 2 and 8 weeks in culture. Immunoreactivity for nestin and glial fibrillary acidic protein was seen in astrocytes which persisted throughout the observation period. Immunofluorescence double labeling showed widespread co-localization of nestin and glial fibrillary acidic protein. Image analysis revealed that levels of nestin-immunoreactivity plateaued after 5 weeks in culture. By comparison nestin immunoreactivity was absent from glial cells of the cortex in mature rats. These immunohistochemical findings of a persistent expression of nestin in glial cells of organotypic slice culture of the rat cortex indicate a different time course of glial maturation in vitro. This difference could be related to the altered trophic stimulation in vitro; differences in neuronal maturation, activity or survival; slow degeneration of the vasculature; or intrinsic properties of astrocytes.     

Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic cultures

Exposure to high levels of bilirubin in hyperbilirubinemia patients and animal models can result in sensorineural deafness. However, the mechanisms underlying bilirubin-induced damage to the inner ear, including the cochlear and vestibular organs, remain unknown. The present analyses of cochlear and vestibular organotypic cultures obtained from postnatal day 3 rats exposed to bilirubin at varying concentrations (0, 10, 50, 100, or 250 μM) for 24 h revealed that auditory nerve fibers (ANFs) and vestibular nerve endings were destroyed even at low doses (10 and 50 μM). Additionally, as the bilirubin dose increased, spiral ganglion neurons (SGNs) and vestibular ganglion neurons (VGNs) exhibited gradual shrinkage in conjunction with nuclei condensation or fragmentation in a dose-dependent manner. The loss of cochlear and vestibular hair cells (HCs) was only evident in explants treated with the highest concentration of bilirubin (250 μM), and bilirubin-induced major apoptosis most likely occurred via the extrinsic apoptotic pathway. Thus, the present results indicate that inner ear neurons and fibers were more sensitive to, and exhibited more severe damage following, bilirubin-induced neurotoxicity than sensory HCs, which illustrates the underlying causes of auditory neuropathy and vestibulopathy in hyperbilirubinemia patients.     

Human fetal myelinated organotypic cultures

We have previously reported the establishment of organotypic cultures derived from human fetal brain tissue. Although these cultures permit the testing of multiple hypotheses about normal human neurodevelopment and neuropathologic conditions, they have the limitation of not being myelinated and therefore preclude the study of questions related to myelinogenesis and diseases of myelin. In the current communication, we describe recent developments that allow us to overcome this limitation and permit the establishment of a myelinated organotypic culture model. Sections of dorsal column dissected from the lumbar spinal cord of human fetuses ranging in age 21-23 weeks of gestation were placed in culture. The explants were maintained for up to 12 weeks during which time they were characterized and shown to express a number of CNS cell-type-specific markers including glial fibrillary acidic protein (astrocytes), nerve growth factor receptor and neurofilament protein (neurons), CD68 (microglia), and myelin basic protein, HNK-1 and galactocerebroside (oligodendrocytes). In addition, lectin histochemistry using Ricinus communis agglutinin-1 detected microglia and endothelial cells. Upon explantation, abundant myelin was seen by electron microscopy in the cultures. Although during the culture period there was degradation of myelin, there was also evidence of maintenance of intact myelin sheaths around small caliber axons and de novo myelin synthesis. This model system may permit the further use of human organotypic cultures to investigate issues related to neurodevelopment and to pathologic conditions including those relevant to dysmyelination and demyelination.     

NGF increases neuritic complexity of cholinergic interneurons in organotypic cultures of neonatal rat striatum

The influence of NGF on cholinergic interneurons in organotypic roller tube cultures of 4 day postnatal rat striatum was examined after 13 to 16 days in vitro. Cultures were divided into four groups. The medium of the NGF treated group was supplemented with 5 ng/ml NGF, whereas control groups were cultured either without NGF, by adding 20 ng/ml neutralising anti-NGF antibody, or by adding both NGF and anti-NGF antibody to the medium. Two different cell populations were identified by an image analysis system which measured acetylcholinesterase staining intensity. It was demonstrated that NGF promotes survival of the large, intensely stained population. Eighty computer-assisted reconstructions of intensely stained cells, 20 for each treatment group, were performed in a random order by means of a neuron tracing system. Axons and dendrites were analysed separately. © Wiley-Liss, Inc. NGF enhanced complexity of neuritic, predominantly axonal trees by increasing the number of axonal segments by 91% to 100% (P < 0.01), the number of dendritic segments by 33% to 63% (P = 0.09 to P < 0.01), maximal axonal branch order by 37% to 50% (P < 0.05), and maximal dendritic branch order by 22% to 37% (P < 0.05). Further evidence of more complex neuritic trees was given by Sholl concentric sphere analysis. Anti-NGF antibody could block all these effects. General rules of branching architecture were not affected by NGF treatment as shown by analysing mean segment length in relation to the branch order, branch point exit angles, total tortuosity, Rall's ratio, and tapering of neuritic trees. © Wiley-Liss, Inc.     

The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 microM [3H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (125I seeds and a clinical 60Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with 125I, 5 microM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous 125I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a 60Co source; and (4) 5 microM hemicholinium significantly depressed the accumulation of tritium in both the control and the 125I-irradiated groups, and there was no longer a difference between 125I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed.     

The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus

Abstract

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 µ M [³H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (¹²⁵I seeds and a clinical ⁶⁰Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with ¹²⁵I, 5 µM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous ¹²⁵I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a ⁶⁰Co source; and (4) 5 µM hemicholinium significantly depressed the accumulation of tritium in both the control and the ¹²⁵I-irradiated groups, and there was no longer a difference between ¹²⁵I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed. [Neurol Res 2001; 23: 669-675]     

Electrophysiological characteristic of corticoaccumbens synapses in rat mesolimbic system reconstructed using organotypic slice cultures

The nucleus accumbens (NAc) is a component of the mesolimbic system involved in drug dependence. Activity of nucleus accumbens neurons is modulated by glutamatergic afferents from the prefrontal cortex and by dopaminergic afferents from the ventral tegmental area (VTA). In the present study, we reconstructed the mesolimbic system using organotypic slice cultures and examined the effects of dopaminergic agents on synaptic activity in the prefrontal cortex-nucleus accumbens synapses. A slice of each of the prefrontal cortex, nucleus accumbens and ventral tegmental area in newborn rat, was arranged on a multi-electrode dish (MED) filled with culture medium so that they contacted each other, termed a 'triple culture'. Extracellular recording using microelectrodes on the multi-electrode dish showed that a single electrical stimulation of the prefrontal cortex slice evoked field excitatory postsynaptic potential, and that population spikes occurred spontaneously in the nucleus accumbens area of the triple culture. The amplitude of evoked field excitatory postsynaptic potentials and the frequency of spontaneous population spikes were decreased by glutamatergic antagonists, D(-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The D1-like receptor agonist SKF38393, but not the D2-like receptor agonist quinpirole, reduced both the amplitude of field excitatory postsynaptic potential and frequency of spontaneous population spikes. Cocaine depressed field excitatory postsynaptic potential and this depression was reversed by D1-like receptor antagonist SCH23390, but not by D2-like receptor antagonist sulpiride. These results suggest that evoked field excitatory postsynaptic potentials and spontaneous population spikes were driven by glutamatergic neurons and were subject to exogenous and endogenous dopaminergic modulation in the triple culture that was similar to that shown in in vivo.     

Calbindin-D 28K in hippocampal organotypic cultures

Slices of hippocampus from 6-day-old rats were cultured for 2-4 weeks using the roller-tube technique. The organization of these explants was studied by immunocytochemical labeling of calbindin-D 28K (CaBP 28K). The development of the CaBP 28K staining was very close to that of the rat hippocampus in vivo with only 3 subpopulations of labeled cells: granule cells and their mossy fibers, pyramidal cells in the subiculum-CA1 zone and interneurons scattered in strata oriens and radiatum.     

Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device

Our aim is the biolistic transfection of organotypic cultures of rat visual cortex with plasmids coding for neurotrophic factors, which then become expressed for limited periods of time during postnatal ontogenesis. Out of two commercially available devices, we adopted the handheld 'Helios Gene Gun' instead of the stationary PDS-1000He (both Biorad, Munich, Germany). This device allows multiple transfections of single targets and the transfection of a distinct part of a co-culture when utilising an aperture. Unfortunately, the most detailed protocols are limited to the stationary device and not compatible with the hand-held device. We report here the construction of a support for the gene gun including an aperture and the establishment of a protocol to efficiently transfect rat cortical slice cultures. We achieve a high degree of co-expression of independent plasmids coated on the same particles. The expression of the neurotrophin plasmids is demonstrated on mRNA and protein level.