Rat Organotypic Hippocampal Cultures Research Articles

Calmodulin in ischemic neurotoxicity of rat hippocampus in vitro.

To determine whether calmodulin plays a role in neurodegeneration after ischemia, effects of the selective calmodulin inhibitors calmidazolium and W7 were studied in organotypic cultures of rat hippocampus. Protection of pyramidal cells in the CA1 region of the hippocampus by calmidazolium and W7 against hypoxia/hypoglycemia suggests that activation of intracellular calmodulin plays a significant role in ischemic neuronal injury. Both ryanodine and TMB-8, inhibitors of intracellular Ca2+ release, failed to prevent ischemic neuronal injury. These results indicate that calmodulin, a major intracellular Ca2+ binding protein, plays a significant role in experimental ischemia-induced hippocampal neuronal injury in vitro.

Production of reactive oxygen species and glutamate neurotoxicity in a rat organotypic hippocampal culture

Production of reactive oxygen species and glutamate neurotoxicity in a rat organotypic hippocampal culture

Glutamate induces rapid nuclear changes and a delayed cell death selectively in CA1 neurons of a rat organotypic hippocampal culture

Glutamate induces rapid nuclear changes and a delayed cell death selectively in CA1 neurons of a rat organotypic hippocampal culture

1207 Production of reactive oxygen species and glutamate neurotoxicity in a rat organotypic hippocampal culture

1207 Production of reactive oxygen species and glutamate neurotoxicity in a rat organotypic hippocampal culture

1206 Glutamate induces rapid nuclear changes and a delayed cell death selectively in CA1 neurons of a rat organotypic hippocampal culture

1206 Glutamate induces rapid nuclear changes and a delayed cell death selectively in CA1 neurons of a rat organotypic hippocampal culture

Correlation between anti-ubiquitin immunoreactivity and region-specific neuronal death in N-methyl- d-aspartate-treated rat hippocampal organotypic cultures

Neuronal degeneration appears to be associated with changes in anti-ubiquitin immunoreactivity (UIR). To elucidate the relationship between the two events, we examined the time course of changes in UIR in pyramidal neurons of hippocampal organotypic cultures following exposure to an excitotoxin, N-methyl- d-aspartate (NMDA). In nontreated cultures, weak UIR was confined to the nucleus. Exposure to 100 μM NMDA for 15 min induced degeneration of pyramidal neurons, within 24 h, in the CA1 and CA3c regions. In these neurons, the nuclear UIR was reduced, and instead, UIR developed in the cytoplasm. In response to the same procedure, CA3a,b pyramidal neurons showed slight shrinkage but otherwise virtually normal morphological features. Little perikaryal (cytoplasmic) UIR developed in CA3a,b neurons. Both degeneration and perikaryal UIR were observed in CA3a,b neurons, however, when the culture was exposed to 300 μM NMDA. Immunoblot analysis showed that changes in the amount of a ubiquitin protein conjugate (24 kDa), presumably ubiquitinated histone, are similar to those of nuclear UIR in the same time course. We propose that the changes in the expression of nuclear and perikaryal ubiquitinated proteins represent some process closely related to neuronal death.

Ca 2+- and Cl −-dependent, NMDA receptor-mediated neuronal death induced by depolarization in rat hippocampal organotypic cultures

The neurotoxicity induced by depolarization with high-K + was investigated in rat hippocampal organotypic slice cultures. The exposure of cultures to 90 mM K + solution for 30 min caused a severe neuronal injury in CA1 region while less damage was observed in CA3 and dentate gyrus over the following day. This neurotoxicity was prevented in a concentration dependent manner by NMDA antagonist MK-801 or CPP. Non-NMDA antagonist, DNQX, had no protective effect. Omission of Ca 2+ from the exposure solution prevented the neurotoxicity. Voltage-dependent Ca 2+ channel blockers, nifedipine and flunarizine, failed to prevent the neurotoxicity. These results suggest that the Ca 2+ influx through the NMDA receptor is predominantly involved in this neurotoxicity. Apparent tissue swelling was observed immediately after the depolarization. This swelling was completely inhibited by omission of Cl − from the exposure solution, accompanied with complete protection against neurotoxicity. This suggests that Cl −-dependent tissue swelling also largely contributes to the neurotoxicity. Depolarization with application of MK-801 (10 μM) or omission of Ca 2+ from the solution still caused apparent swelling, despite these treatment protected neuronal death. We hypothesize that Cl −-dependent tissue swelling may be involved in the release of the excitatory amino acid, which activates the NMDA receptor.

Sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures

The understanding of the mechanisms of a functional synaptic plasticity in the hippocampus has been expanded greatly by the use of in vitro slice preparations. The question addressed in the present study was whether morphological plasticity observed in vivo can also be reproduced in hippocampal slices. In vivo, hippocampal commissural and association fibers are known to sprout and occupy synaptic sites vacated by deafferentation of the dentate gyrus (DG). In hippocampal slice preparations, the major input to the DG is eliminated, so that the DG is deafferented. Might intrinsic neurons sprout to the DG if the slice preparation is maintained for weeks? In this study hippocampal slices obtained from 6-day-old rats were cultured. Stimulation of the dentate stratum moleculare produced antidromic field potentials in the CA3 of the slices cultivated for more than 1 week. The antidromic response was not observed in CA1 pyramidal neurons. The CA3 to DG projection response was also observed in a CA3 mini-slice placed near a co-cultured whole hippocampal slice, when the DG in the latter was stimulated. Moreover, stimulation of the CA3 mini-slice induced synaptic responses in the DG of the whole-slice. The conclusion drawn is that deafferentation could induce axonal sprouting in a neuron-specific manner in hippocampal organotypic culture. This preparation would be potentially useful for the screening of chemical factors that influence sprouting of central neurons.

Selective degeneration of CA1 pyramidal cells by chronic application of bismuth

The heavy metal bismuth induces a new type of selective neuronal degeneration that shares some common aspects with that seen following hypoxia and ischemia. Continuous application of 3 microns bismuth to organotypic cultures of rat hippocampus resulted after 2-3 weeks in selective degeneration of CA1 pyramidal cells, while CA3 pyramidal cells, dentate granule cells, and subicular neurons were resistant. With 10 microns bismuth, the majority of hippocampal neurons degenerated. The addition of 20 microns MK-801, a noncompetitive NMDA-antagonist, during the entire culturing period failed to prevent neuronal degeneration induced by 3 microns bismuth. GABA-immunoreactive interneurons were also affected by bismuth, but were generally less sensitive than CA1 pyramidal cells. Acute application of up to 100 microns bismuth did not change the electrophysiological properties of CA1 pyramidal cells.

Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischemia.

The hippocampus demonstrates a regional pattern of vulnerability to ischemic injury that depends on its characteristic differentiation and intrinsic connections. We now describe a model of ischemic injury using organotypic hippocampal culture, which preserves the anatomic differentiation of the hippocampus in long-term tissue culture. Ischemic conditions were modeled by metabolic inhibition. Cultures were briefly exposed to potassium cyanide to block oxidative phosphorylation and 2-deoxyglucose to block glycolysis. The fluorescent dye propidium iodide was used to observe membrane damage in living cultures during recovery. 2-Deoxyglucose/potassium cyanide incubation resulted in dose-dependent, regionally selective neuronal injury in CA1 and the dentate hilus, which began slowly after 2 to 6 hours of recovery. Subsequent histological examination of cultures after 1 to 7 days of recovery demonstrated neuronal pyknosis that was correlated with the early, direct observation of membrane damage with propidium. Both propidium staining and histological degeneration were prevented by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 when administered 30 minutes after the end of the exposure to 2-deoxyglucose and potassium cyanide. Tetrodotoxin, which blocks voltage-dependent sodium channels, had protective effects that were greatest during the period of 2-deoxyglucose and potassium cyanide incubation but also produced protection against the mildest conditions of metabolic inhibition when administered after 30 minutes of recovery. This in vitro model reproduced elements of the time course, regional vulnerability, and pharmacologic sensitivities of in vivo ischemic hippocampal injury. Inhibition of metabolism in organotypic culture provides a rapid, easily controlled injury and reproduces the in vitro pattern of hippocampal regional vulnerability to ischemia. It is the first in vitro model of ischemia to exhibit complete protection by delayed administration of an NMDA receptor antagonist during recovery from a brief insult. The protective effects of tetrodotoxin suggest that an early period of sodium entry into cells during and after ATP depletion may be responsible for the more prolonged period of toxic NMDA receptor activation.

P-51 - Involvement of NMDA receptors in AMPA-induced degeneration of CA1 pyramidal neurons in rat hippocampal organotypic cultures.

P-51 - Involvement of NMDA receptors in AMPA-induced degeneration of CA1 pyramidal neurons in rat hippocampal organotypic cultures.

Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures

By sectioning, using a razor blade, one- and three-week-old rat hippocampal organotypic cultures, we have tested the possibility that neurite outgrowth and reactive synaptogenesis would take place even after several weeks in culture in this in vitro model. At the light-microscopic level, recovery from the section and formation of a thin scar were observed within six days following the lesion. Immunostainings using neurofilament antibodies showed the presence of numerous degenerative and regenerative images one day after the cut and many fibres crossing the section six days after the lesion. Electrophysiological recordings of synaptic responses elicited across the section indicated the formation of new functional synaptic contacts and complete recovery of transmission within three to six days. Interestingly, functional recovery in three-week-old cultures was found to be significantly slower than in one-week-old tissue. These findings were confirmed at the electron-microscopic level. Evidence was obtained for an effective cleaning of the lesion site by macrophages and astroglial cells, the existence of many degenerative and regenerative images one day after the cut and the presence of new dendrites, axonal fibres and synapses in the area of the section six days after the lesion. All these changes were slower in three- than in one-week-old cultures. These results indicate that organotypic cultures can be used as an interesting model for studies of reactive synaptogenesis.

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

We have compared rapid freezing followed by freeze-substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 microns without visible ice crystals. Freeze-substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze-substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques.

1324 Axonal sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures

1324 Axonal sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures

Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures

Using morphological techniques, we characterized the developmental reorganization that takes place during the first weeks after explantation in area CA1 of organotypic hippocampal cultures maintained at the interface between medium and a CO 2-enriched atmosphere. Pyramidal neurones redistributed from a vertical into an horizontal cell layer in the middle of a three-dimensional culture, with apical dendrites running above the pyramidal layer. Glial cells redistributed into a thin layer at the bottom of the culture, forming an interface between tissue and culture medium. Astrocytes were identified as the most numerous non neuronal cells. No sign of glial proliferation could be observed, except for a transient increase during the first days after explantation. The density of synaptic contacts in the stratum radiatum decreased immediately after explantation and then increased by about 20-fold to reach values in the proximal part of the apical layer after 4 weeks in culture which were only slightly smaller than those measured in 1-month-old rats. The synaptic density in the most distal part of the dendritic layer which receives connections extrinsic to the hippocampus remained significantly lower than in vivo. The ratio of spine to shaft contacts was comparable to that found in vivo. These results indicate that interface type of organotypic cultures can be used as an interesting model for studies of synaptic development in vitro.

Differentiation of dentate granule cells in slice cultures of rat hippocampus: a Golgi/electron microscopic study

The differentiation of granule cells in organotypic cultures of rat hippocampus was studied by means of the Golgi/electron microscope (EM) technique. Like in vivo, the granule cells have a small round or ovoid cell body which gives rise to apical dendrites densely covered with spines. However, the apical dendrites of the cultured granule cells are more horizontally oriented than in the normal fascia dentata where they form a cone-shaped dendritic arbor. Granule cells in slice cultures occasionally have basal dendrites invading the hilar region. Electron microscopic examination revealed many synaptic contacts on identified apical and basal dendrites of the gold-toned granule cells in culture. This suggests that a considerable synaptic reorganization takes place since all extrinsic afferents normally innervating the granule cells are lost. Several granule cells displayed deep infoldings of their nuclei which are known from in vivo studies to be a characteristic feature of non-granule cells in this region, i.e. basket cells. The presence of basal dendrites and nuclear infoldings indicates an increased variability of this cell type which in situ displays a rather stereotyped morphology.

The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus

Intracellular recordings were made from CA1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N- methyl- d-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist d-2-amino-5-phosphonovalerate ( d-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.