Ethanol induces MAP2 changes in organotypic hippocampal slice cultures

Microtubule-associated protein 2 (MAP2) and neuron-specific protein (NeuN) immunostains were used to demonstrate neurotoxic effects in mature hippocampal slice cultures exposed to ethanol (50, 100, 200 mM) for 4 weeks. At the low dose the density of MAP2 immunostaining in the dentate molecular layer was 118% of the control cultures, with no detectable changes in CA1 and CA3. At 100 mM no changes were detected, while 200 mM ethanol significantly reduced the MAP2 density in both dentate (19%) and hippocampal dendritic fields (CA3, 52%; CA1, 55%). At this dose NeuN staining showed considerable loss of CA3 pyramidal cells and moderate loss of dentate granule cells, as seen in vivo. The results indicate that brain slice cultures combined with immunostaining for cytoskeleton and neuronal markers can be used for studies of ethanol and organic solvent neurotoxicity.     

Receptor-oriented intercellular calcium waves evoked by vasopressin in rat hepatocytes

Agonist-induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2-loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D-myo-inositol 1,4, 5-trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4-. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin-binding sites, vasopressin-induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+-dependent processes.     

Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures

The aim of this study was to analyse microglial reactions to excitotoxic N-methyl-D-aspartic acid (NMDA)-induced degeneration of rat dentate and hippocampal neurons in vitro. We used a migration model combining the techniques of microglial single cell culture and organotypic hippocampal slice culture (OHSC). Site-specific oxidative damage in OHSCs was induced by pretreatment with 50 microM NMDA. Neuronal injury determined by propidium iodide (PI) uptake included the hippocampal cell layers of the dentate gyrus (DG) and the cornu ammonis (CA). Fluorescence-prelabelled microglial cells with ameboid morphology were transferred onto the OHSC and migrated predominantly to the prelesioned cell layers of DG and CA when compared with unlesioned areas of the OHSC. In NMDA pretreated slices, microglial cells clustered around degenerating granule cells in the DG and pyramidal cells in the CA. This effect was significantly inhibited in unlesioned slice cultures and in NMDA-exposed cultures that were pretreated with the NMDA-antagonist MK-801. Our observations suggest that microglia -- attracted by the presence of stimuli provided by NMDA-induced neuronal death -- migrate specifically towards these lesioned neurons.     

Properties of spontaneous miniature GABAA receptor mediated synaptic currents in area CA3 of rat hippocampal slice cultures

Miniature, gamma-aminobutyric acid A receptor mediated inhibitory postsynaptic currents (mIPSCs) were recorded from CA3 pyramidal cells in hippocampal slice cultures using whole-cell techniques in the presence of tetrodotoxin. The kinetics and amplitudes of the mIPSCs were analyzed with the aim of determining whether subclasses of events arising from distinct populations of presynaptic interneurons could be distinguished. Histograms of mIPSC amplitude, rise time constant, and decay time constant were all positively skewed, but discrete subsets of events could not be distinguished. The positive skew did not appear to result from electrotonic filtering of distal synaptic currents because there was no correlation among mIPSC amplitudes and the kinetic parameters. Analysis of the intervals between mIPSCs indicated that each event occurred independently. The analysis of spontaneous mIPSCs does not provide evidence of the innervation of pyramidal cells by heterogeneous interneurons.     

Markers for neuronal degeneration in organotypic slice cultures

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5-100 microM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5-25 microM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6-24 microM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.     

Temporal characterization of microglia, IL-1 beta-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures

These studies demonstrate that murine hippocampal slice cultures possess neural-immune elements that show responses parallel to comparable in vivo models of neural-immune activation. Using immunocytochemical techniques, this study characterized the phenotypes of specific glial elements and the expression of the cytokine, interleukin-1 (IL-1 beta), in the hippocampal dentate gyrus over a period of 10 days in vitro (DIV). Preparation of organotypic slice cultures of neonatal mouse hippocampus produced cellular damage including axotomy of afferent fibers within the molecular layer of the dentate gyrus. This form of lesion-induced injury caused activation of neural-immune elements in the slice cultures. Staining with the microglial specific biotinylated Griffonia simplicifolia B4-isolectin revealed reactive microglia were most prevalent at 2 DIV and decreased in number from 4 to 10 DIV, whereas the initial population of resting microglia at 2 DIV increased approximately four-fold from 4 to 10 DIV. The presence of a round IL-1 beta-like immunophenotype closely paralleled the temporal and spatial distribution of the reactive form of microglia observed in the dentate gyrus. In addition, between 4 and 10 DIV, some IL-1 beta-like immunoreactive cells exhibited a stellate-like morphology with numerous branching processes, similar to resting microglia. At 2 DIV astrocytes showed minimal labeling with antibodies directed against glial fibrillary acidic protein (GFAP), while from 4 to 10 DIV, a dramatic hypertrophic astrocytic response occurred, resulting in a gliotic scar forming over the entire dentate gyrus. We conclude that neural-immune activation in the hippocampal organotypic slice culture preparation closely parallels similar responses observed in vivo and thus slice cultures represent an excellent model for further studies of neural-immune interactions resulting from lesion-induced injury in the central nervous system.     

Brain-derived neurotrophic factor promotes the differentiation of various hippocampal nonpyramidal neurons, including Cajal-Retzius cells, in organotypic slice cultures

Brain-derived neurotrophic factor (BDNF) is widely expressed in the central nervous system, where its function is poorly understood. The aim of this study was to investigate the effects of BDNF on the differentiation of hippocampal nonpyramidal neurons using organotypic slice cultures prepared from postnatal rats. The application of BDNF induced an increase in immunostaining for the microtubule-associated protein (MAP)-2 in non-pyramidal neurons of the stratum oriens. BDNF promotes the elongation of the dendrites of these neurons, as demonstrated by analysis after biocytin labeling. Calbindin-D- and calretinin-containing subgroups of nonpyramidal cells in the stratum oriens were responsive to BDNF but not to nerve growth factor, as shown by an increase in the number of neurons immunostained for these proteins. BDNF also induced an increase in neuropeptide Y immunostaining of stratum oriens neurons. In contrast, BDNF had no effect on parvalbumin immunostaining, despite the fact that these cells express the BDNF receptor trkB. In addition, BDNF increased calretinin immunoreactivity in Cajal-Retzius cells situated around the hippocampal fissure. The Cajal-Retzius neurons persisted in slices beyond the time at which they degenerate in vivo. However, BDNF is not required for the survival of these cells, because they also persisted in slices from BDNF knock-out mice. The present results indicate that BDNF exerts an effect on the morphology of stratum oriens nonpyramidal cells and their calcium-binding protein levels. BDNF also regulates the calretinin content of Cajal-Retzius cells but is not necessary for their survival.     

Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells

Mechanical stimulation was used to initiate Ca2+ waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca2+ to adjacent cells and to assess alternative Ca2+ signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca2+ wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca2+ wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18beta-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca2+ wave. In contrast, treatment with suramin, a P2-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca2+ wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca2+ wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.     

Suppression of synaptogenesis by epileptiform discharges in hippocampal slice culture

Epidemiological and laboratory studies provide preliminary evidence that a compound may prevent certain types of clinical cancer. The final proof for practical application demands two controlled trials with similar, decisive results. Controlled chemoprevention trials on clinical cancer are large, time-consuming and expensive, whereas studies on cancer surrogates are smaller but less reliable. Rational trial design often lacks sufficient information about the sensitive period and the time from that point to clinically detectable cancer. The correct dose of chemopreventive agent and an expected preventive fraction of cancer are also often based on informed guesswork. Long trials call for special arrangements to guarantee the staying will of the participants and key research personnel. Although large chemoprevention trials are currently being carried out without any certainty of successful outcome, the situation is not so different from the early days of chemoprevention trials for cardiovascular diseases. Cancer trials will be conducted based on the 'learning-by-doing' approach, and in the more distant future based on research designed to provide information for trial needs.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Spiral waves in heart (theoretical analysis)

Sensitivity of two kinds of spiral wave sources of excitation of fast inward current (INa) inhibition was compared. These spiral wave sources were: circulation in a ring around an obstacle--re-entry and circulation in tissue without an obstacle--reverbarator (leading circle). It was shown that moderate inhibition of INa ceased the circulation of the reverbarator but was ineffective against the circulation in the ring. It is known that auricular flutter and fibrillation are based on wave circulation in the ring. So a conclusion was made that all antiarrhythmic drugs inhibiting mainly the sodium current should be ineffective against auricular flutter and fibrillation.     

Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18-39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP-containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA-immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker.     

Aging-related increase in hippocampal calcium channels

This paper briefly reviews more than 10 years of our studies on brain aging and voltage-activated calcium (Ca) currents in rat hippocampal CA1 neurons. Initial studies in the hippocampal slice preparations found that synaptic plasticity was impaired with aging, apparently due to excess Ca influx. In subsequent analyses it was found that the Ca-dependent afterhyperpolarization, the Ca action potential and voltage-activated Ca currents were all increased in aged CA1 neurons. This was not due to impaired inactivation processes. Multiple types of Ca channels appear to be affected by aging. A long Ca tail current was also found in these studies, which seems to represent an unrecognized and significant Ca entry pathway at resting potential. In primary cell cultures, Ca currents and single Ca channels increase steadily over the life cycle of the cultured neurons and are correlated with cell death. Single L-type Ca channels were also studied in brain neurons of an aged mammal (rat), using the partially dissociated ("zipper") hippocampal slice preparation. A substantial increase in the density of functionally available Ca channels was present in CA1 neurons of aged rats, similar to the increase seen in cultured neurons. Thus, a gradual increase in the density of Ca channels appears to be a consistent property of hippocampal neuronal aging and might well be a factor in the vulnerability of aged neurons to Alzheimer's disease and other neurodegenerative/traumatic conditions.     

Mechanisms and function of intercellular calcium signaling

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.     

The effects of extracellular pH and calcium manipulation on protein synthesis and response to anoxia/aglycemia in the rat hippocampal slice

Extracellular pH modulates the function of the N-methyl-D-aspartate (NMDA) receptor, which may influence pathophysiological responses to glutamate. While damage due to oxygen and glucose deprivation or glutamate exposure is attenuated by acidification of the incubating medium of cultured neurons, neuron damage is enhanced in vivo following ischemia in hyperglycemic animals. A persistent inhibition of protein synthesis (to less than 5% of normoxic levels) is a reliable index of damage to neurons both in vivo and in the rat hippocampal slice. We explored the influence of extracellular pH and calcium manipulation on protein synthesis inhibition and energy failure due to anoxia/aglycemia or exposure to N-methyl-D-aspartate in the rat hippocampal slice. Moderate acidification of the medium during anoxia/aglycemia did not reduce the damage to protein synthesis in hippocampal neurons (9% of normoxic levels) and did not alter basal ATP levels or the rate of ATP depletion during anoxia/aglycemia. However, when calcium levels were lowered during the acidification and following the anoxia/aglycemia, protein synthesis was almost completely protected (84% of normoxic levels). Calcium reduction itself also attenuated the protein synthesis inhibition due to anoxia/aglycemia (to 55.6% of normoxic controls), but the protection was not as complete. In contrast, moderate acidification of the medium significantly reduced the damage to protein synthesis due to a brief exposure to NMDA (37% of control with NMDA, 78.9% of control with acidification during NMDA), even in the presence of extracellular calcium. Alkalinization of the medium exacerbated the protein synthesis inhibition following anoxia/aglycemia, and significantly reduced basal ATP levels (to 52% of normoxic control levels). Thus, pHo changes influence neuronal metabolism and response to anoxia/aglycemia. In addition, while acidification can reduce the excitotoxic damage caused by direct exposure to NMDA, it cannot reduce damage due to anoxia/aglycemia unless calcium is lowered concomitantly. Thus, both NMDA receptor activation and calcium are involved in the damage due to oxygen and glucose deprivation in the slice.     

The hamster hippocampal slice: I. Physiological properties.

Describes the physiological properties of the in vitro hippocampal slice of the golden hamster. Hamster hippocampal tissue displays features similar to those seen in other species in terms of postsynaptic response characteristics to stimulation of monosynaptic afferents in the 3 primary subfields of the hippocampus. This pattern of physiological similarity supports the contention of the uniformity of hippocampal function across species, an important consideration with regard to the role of the hippocampus in brain and behavioral function. (14 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The hamster hippocampal slice: II. Neuroendocrine modulation.

Studied effects of exposing golden hamster hippocampal slices to the gonadal steroids estradiol and testosterone and to delta-9-tetra-hydrocannabinol (THC). At 10–20M, estradiol and testosterone acted as facilitatory neuromodulators on hippocampal slices obtained from male and diestrous female hamsters, respectively. At 10–21M, THC facilitated responses in hippocampal slices from males. Implications for the use of the hamster in investigations of the biobehavioral role of steroid modulation of brain excitability are discussed. (14 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra- or extracellular recording from the mouse hippocampal slice preparation. Bath-applied substance P (2-4 microM) or the selective NK1 receptor agonist substance P methylester (SPME, 10 nM-5 microM) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK1 receptors since it was completely blocked by the selective NK1 antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or 10 microM N-methyl-D-aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

Changes in mitochondrial function resulting from synaptic activity in the rat hippocampal slice

Digital imaging microfluorimetry was used to visualize changes in mitochondrial potential and intracellular Ca2+ concentration, [Ca2+]i, in thick slices of rat hippocampus. Electrical activity, especially stimulus train-induced bursting (STIB) activity, produced slow, prolonged changes in mitochondrial potential within hippocampal slices as revealed by fluorescence measurements with rhodamine dyes. Changes in mitochondrial potential showed both temporal and spatial correlations with the intensity of the electrical activity. Patterned changes in mitochondrial potential were observed to last from tens of seconds to minutes as the consequence of epileptiform discharges. STIB-associated elevations in [Ca2+]i were also prolonged and exhibited a spatial pattern similar to that of the mitochondrial depolarization. The mitochondrial depolarization was sensitive to TTX and glutamate receptor blockers ([Mg2+]o and CNQX or DNQX plus D-AP-5) and to the inhibition of glutamate release by activation of presynaptic NPY receptors. The monitoring of mitochondrial potential in slice preparations provides a new tool for mapping synaptic activity in the brain and for determining the roles of mitochondria in regulation of brain synaptic activity.