Cells In Hippocampal Slice Cultures Research Articles

Assessment of Neuronal Damage in Brain Slice Cultures Using Machine Learning Based on Spatial Features.

Neuronal damage presents a major health issue necessitating extensive research to identify mechanisms of neuronal cell death and potential therapeutic targets. Commonly used models are slice cultures out of different brain regions extracted from mice or rats, excitotoxically, ischemic, or traumatically lesioned and subsequently treated with potential neuroprotective agents. Thereby cell death is regularly assessed by measuring the propidium iodide (PI) uptake or counting of PI-positive nuclei. The applied methods have a limited applicability, either in terms of objectivity and time consumption or regarding its applicability. Consequently, new tools for analysis are needed. Here, we present a framework to mimic manual counting using machine learning algorithms as tools for semantic segmentation of PI-positive dead cells in hippocampal slice cultures. Therefore, we trained a support vector machine (SVM) to classify images into either “high” or “low” neuronal damage and used naïve Bayes, discriminant analysis, random forest, and a multilayer perceptron (MLP) as classifiers for segmentation of dead cells. In our final models, pixel-wise accuracies of up to 0.97 were achieved using the MLP classifier. Furthermore, a SVM-based post-processing step was introduced to differentiate between false-positive and false-negative detections using morphological features. As only very few false-positive objects and thus training data remained when using the final model, this approach only mildly improved the results. A final object splitting step using Hough transformations was used to account for overlap, leading to a recall of up to 97.6% of the manually assigned PI-positive dead cells. Taken together, we present an analysis tool that can help to objectively and reproducibly analyze neuronal damage in brain-derived slice cultures, taking advantage of the morphology of pycnotic cells for segmentation, object splitting, and identification of false positives.

Inverse relationship between seizure expression and extrasynaptic NMDAR function following chronic NMDAR inhibition

We showed previously that electrographic seizures involving dentate granule cells in organotypic hippocampal slice cultures were dramatically reduced following chronic treatment with the NR2B-selective antagonist, Ro25,6981, but were increased following chronic treatment with the high-affinity competitive antagonist, D(-)-2-amino-5-phosphonopentanoic acid (D-APV). To begin to investigate the potential mechanisms underlying the differential effects of N-methyl-D-aspartate receptor (NMDAR) antagonists on seizures, electrophysiologic experiments were conducted in dentate granule cells in hippocampal slice cultures treated for the entire 17-21 day culture period with vehicle, Ro25,6981 or D-APV. Initial experiments revealed a lack of an association between miniature excitatory postsynaptic current (mEPSC) measures and seizures suggesting that shifts in mEPSC were unlikely to account for the differential effects of D-APV and Ro25,6981 on seizures. However, the amplitude of tonic NMDAR-mediated currents was reduced in cultures treated chronically with D-APV and dramatically enhanced in cultures treated chronically with Ro25,6981. Because tonic NMDAR currents are mediated primarily by extrasynaptic NMDAR, these data show an inverse relationship between changes in extrasynaptic NMDAR function and alterations in seizure expression.

Rhythmic Synaptic Activity Induced by Mechanical Injury of Rat CA3 Hippocampal Area

Mechanical injury of the CNS frequently results from accidents but also occurs in the course of neurosurgical interventions. A great variety of anatomical and physiological changes have been described to evolve after a brain trauma yet only little is known about processes that occur during a trauma. In the present study, I obtained whole-cell patch clamp recordings from pyramidal cells in hippocampal slice cultures while mechanically lesioning the CA3 area. Electrophysiological analysis revealed that traumatic injury massively increased excitatory and inhibitory synaptic activity in the entire CA3 region. Cutting the CA3 region induced highly rhythmic excitatory postsynaptic currents (EPSCs) that reached frequencies of around 70 Hz. Blocking voltage-dependent sodium channels with tetrodotoxin prevented the increase in synaptic activity and injury-induced neurotransmitter release in CA3 remote from the lesion site. With fast synaptic transmission blocked only neurons in the immediate vicinity of a lesion depolarized and fired action potentials upon mechanical damage. I hence suggest that mechanical injury damages the membrane and induces action potential firing in only a small population of neurons. This activity is then propagated throughout the undamaged CA3 network inducing highly rhythmic discharges. Thus mechanical brain injury initiates immediate functional changes that exceed the lesion site.

Neuroprotective effects of anticonvulsants in rat hippocampal slice cultures exposed to oxygen/glucose deprivation

Some anticonvulsants show neuroprotective effects, and may be of use in reducing neuronal death resulting from stroke or traumatic brain injury. Here I report that a broad range of anticonvulsants protect cells in hippocampal slice cultures from death induced by oxygen/glucose deprivation (OGD). Hippocampal slice cultures were submitted to 1 h OGD and the resulting cell death was quantified 24 h later using a novel automated fluorescent scanning method. The classical anticonvulsants phenobarbital, phenytoin, ethosuximide, chlordiazepoxide and midazolam all significantly and dose-dependently reduced cell death induced by OGD. The newer anticonvulsants carbamazepine, felbamate, lamotrigine, tiagabine, and oxcarbazepine also had significant neuroprotective effects, but gabapentin, valproic acid (10 mM), levetiracetam and retigabine were not neuroprotective at a concentration up to 300 μM. In conclusion, several classical and newer anticonvulsants have neuroprotective properties in an in vitro model that simulates cerebral ischemia.

Cytoskeleton disruption causes apoptotic degeneration of dentate granule cells in hippocampal slice cultures

Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 μM and 10 μM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca 2+-free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.

Action-potential propagation gated by an axonal I(A)-like K+ conductance in hippocampus.

Integration of membrane-potential changes is traditionally reserved for neuronal somatodendritic compartments. Axons are typically considered to transmit reliably the result of this integration, the action potential, to nerve terminals. By recording from pairs of pyramidal cells in hippocampal slice cultures, we show here that the propagation of action potentials to nerve terminals is impaired if presynaptic action potentials are preceded by brief or tonic hyperpolarization. Action-potential propagation fails only when the presynaptic action potential is triggered within the first 15-20ms of a depolarizing step from hyperpolarized potentials; action-potential propagation failures are blocked when presynaptic cells are impaled with electrodes containing 4-aminopyridine, indicating that a fast-inactivating, A-type K+ conductance is involved. Propagation failed between some, but not all, of the postsynaptic cells contacted by a single presynaptic cell, suggesting that the presynaptic action potentials failed at axonal branch points. We conclude that the physiological activation of an I(A)-like potassium conductance can locally block propagation of presynaptic action potentials in axons of the central nervous system. Thus axons do not always behave as simple electrical cables: their capacity to transmit action potentials is determined by a time-dependent integration of recent membrane-potential changes.

Lesion-induced axonal sprouting and hyperexcitability in the hippocampus in vitro: implications for the genesis of posttraumatic epilepsy.

The delayed development of recurring seizures is a common consequence of traumatic head injury; the cause of such epilepsy is unknown. We demonstrate here that transection of the mature axons of CA3 pyramidal cells in hippocampal slice cultures leads to the formation by CA3 pyramidal cells of new axon collaterals that are immunoreactive with the growth-associated protein GAP-43. Individual CA3 cell axons had an elevated number of presynaptic boutons 14 days after the lesion, and dual intracellular recordings revealed an increased probability that any two CA3 pyramidal cells were connected by an excitatory synapse. Lesioned cultures were hyperexcitable and synaptic responses often displayed unusual prolonged polysynaptic components. We thus demonstrate that recurrent axon collaterals are newly sprouted by pyramidal cells as a consequence of axonal injury and suggest that this underlies the development of posttraumatic epilepsy.

Properties of spontaneous miniature GABAAreceptor 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.

Spine loss in experimental epilepsy: quantitative light and electron microscopic analysis of intracellularly stained CA3 pyramidal cells in hippocampal slice cultures.

The sequence of neuronal alterations resulting from epileptic activity is poorly understood. In the hippocampus of some epileptic patients, there is a loss of certain neuronal types in the hilar region and in CA3. The neuronal alterations preceding this degeneration probably affect synaptic structures. Here we have estimated the number of dendritic spines, major postsynaptic elements of hippocampal neurons, in defined dendritic segments of identified (intracellularly stained) CA3 pyramidal neurons in "epileptic" slice cultures of hippocampus and in control cultures. Slice cultures were prepared from five- or six-day-old rat pups and maintained in vivo for 23 days before epileptic activity was induced by application of the convulsants bicuculline and picrotoxin for three days. Individual CA3 pyramidal neurons were then intracellularly injected with horseradish peroxidase, and the number of dendritic spines was counted in proximodistal dendritic segments by applying the Sholl method. In addition, the total dendritic length was measured and the branching index evaluated. The number of spines on CA3 pyramidal cell dendrites in the "epileptic" cultures was found to be decreased by 40%. This spine loss affected proximal and peripheral dendritic segments of the CA3 pyramidal neurons to a similar extent. No significant differences were observed between control and "epileptic" cultures in dendritic length or in the branching index. Quantitative electron microscopic analysis did not reveal differences between "epileptic" cultures and control cultures in the spine area of the labelled CA3 pyramidal cells, indicating that there was a real spine loss, not just a reduction in the size of the spines. We conclude that epileptic activity causes morphological alterations in defined postsynaptic compartments of hippocampal pyramidal cells surviving under these conditions.

Presynaptic inhibition of excitatory synaptic transmission by muscarinic and metabotropic glutamate receptor activation in the hippocampus: are Ca 2+ channels involved?

Activation of either muscarinic cholinergic or metabotropic glutamatergic presynaptic receptors inhibits evoked excitatory synaptic responses in the hippocampus. We have investigated two possible mechanisms underlying these actions using whole-cell recording from CA3 pyramidal cells in hippocampal slice cultures. Application of either methacholine (MCh, 10 μM) or trans-aminocyclopentane-1,3-dicarboxylic acid ( t-ACPD, 10 μM) was found to reduce the frequency of miniature excitatory postsynaptic currents (mEPSCs) by roughly 50%, without changing their mean amplitude. The voltage-dependent Ca 2+ channel blocker Cd 2+ (100 μM), in contrast, had no effect on the mEPSC frequency. When the extracellular [K +] was increased from 2.7 to 16 mM, the mEPSC frequency increased from 1.7 to 4.9 Hz. This increase could be completely reversed by applying Cd 2+, indicating that it was triggered by voltage-dependent Ca 2+ influx. MCh and t-ACPD each decreased the mEPSC frequency by roughly 50% under these conditions. Because the agonists were equally effective in inhibiting spontaneous release whether voltage-dependent channels were activated or not, we conclude that presynaptic cholinergic and glutamatergic inhibition is not mediated by inhibition of presynaptic Ca 2+ channels, but rather by a direct interference in the neurotransmitter release process at some point subsequent to Ca 2+ influx.

Presynaptic enhancement of inhibitory synaptic transmission by protein kinases A and C in the rat hippocampus in vitro

The protein kinase C activator phorbol 12,13-dibutyrate (0.5 microM, PDBu) and the protein kinase A activator forskolin (20 microM) each increased evoked monosynaptic inhibitory postsynaptic current (IPSC) amplitude, without affecting its reversal potential, and increased the frequency of miniature IPSCs (mIPSCs), without affecting their amplitude or kinetics, as assessed with whole-cell recording form CA3 pyramidal cells in hippocampal slice cultures. The effects of forskolin and PDBu on both evoked IPSC amplitude and mIPSC frequency were additive and were antagonized by inhibitors of protein kinases A and C, respectively. The kinase activator-induced increases in mIPSC frequency were quantitatively comparable to the increases in evoked IPSC amplitude. The increases in mIPSC frequency were not attenuated by the voltage-dependent calcium channel blocker Cd2+ (100 microM). We conclude that stimulation of protein kinases A and C potentiates hippocampal inhibitory synaptic transmission through independent presynaptic mechanisms of action. Kinase-induced potentiation of spontaneous release does not require modulation of axon terminal Ca2+ channels. This mechanism may also contribute substantially to the potentiation of evoked release.

Inhibition of a slow synaptic response by a metabotropic glutamate receptor antagonist in hippocampal CA3 pyramidal cells.

The effects of a novel antagonist of metabotropic glutamate receptors were investigated in CA3 pyramidal cells in hippocampal slice cultures of the rat. Earlier experiments showed that selective activation of metabotropic glutamate receptors with low concentrations of an agonist, 1S, 3R-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD), induced an inward current associated with a decrease in membrane conductance and inhibition of the slow calcium-dependent potassium current. These responses were strongly and reversibly reduced by the antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.5-1 mM). In the presence of antagonists of ionotropic glutamate receptors, stimulation of the afferent mossy fibres evoked postsynaptic responses in CA3 pyramidal cells which paralleled those observed with exogenously applied metabotropic glutamate receptor agonists, i.e. a slow inward current and a reduction of calcium-dependent potassium current. Both responses were greatly reduced by bath-applied MCPG (1 mM). These results show that MCPG acts as an effective antagonist at metabotropic glutamate receptors coupled to potassium conductances in the hippocampus. Furthermore, they confirm that glutamate release from presynaptic terminals can modulate postsynaptic properties by activation of metabotropic glutamate receptors.

Effects of interferons and hydrogen peroxide on CA3 pyramidal cells in rat hippocampal slice cultures

In viral diseases of the CNS, both interferon-α/β and interferon-γ are produced intrathecally. At least some of the neurological symptoms associated with these diseases may be due to the effects of these cytokines. We have studied the actions of interferons on CA3 pyramidal cells in hippocampal slice cultures. Bath application of interferon-α/β and interferon-γ produced an excitatory effect on CA3 pyramidal cells and a decrease in evoked inhibitory postsynaptic potential amplitude, eventually leading to epileptiform bursting. These effects were slow in onset (several minutes), suggesting an indirect mechanism of action. Several lines of evidence suggest that the actions of interferons on pyramidal cells may at least in part be mediated by reactive oxygen intermediates, known to be released from non-neuronal cells: the effects of interferon on CA3 pyramidal cells were blocked by the free radical scavengers catalase and superoxide dismutase. Hydrogen peroxide reduced evoked inhibitory synaptic transmission, eventually leading to epileptiform bursting, thus mimicking several of the effects of interferons on pyramidal cells.

4C3HPG (RS-4-carboxy-3-hydroxyphenylglycine), a weak agonist at metabotropic glutamate receptors, occludes the action of trans-ACPD in hippocampus

The determination of the physiological role of glutamatergic metabotropic actions has been hampered by the lack of potent and specific antagonists. It has recently been reported that 4C3HPG (RS-4-carboxy-3-hydroxyphenylglycine) can antagonize metabotropic responses in the central nervous system. The effects of 4C3HPG on mrtabotropic responses evoked by trans-ACPD were investigated in CA3 pyramidal cells in hippocampal slice cultures. Our results show that in hippocampus 4C3HPG fails to antagonize responses mediated by metabotropic glutamate receptors.

Calculation of calcium dynamics from single wavelength fura-2 fluorescence recordings

We describe techniques for measurements of cytosolic calcium dynamics in single current- or voltage-clamped nerve cells. The calculations of calcium dynamics are based on continuous recordings of fura-2 fluorescence intensity at one excitation wavelength after an initial reference measurement at two excitation wavelengths. We show that such single wavelength recordings are not only sufficient for the calculation of Ca2+ concentrations, but also lead to a superior signal-to-noise ratio at a high temporal resolution. Moreover, this strategy diminishes requirements for the experimental setup, such as a device necessary to switch quickly between excitation filters. We have applied this approach on measurements of cytosolic free Ca2+ in single-electrode voltage-clamped CA3 pyramidal cells in hippocampal slice cultures.

Muscarine affects calcium-currents in rat hippocampal pyramidal cells in vitro

Ca2+-currents were recorded from single CA, pyramidal cells in hippocampal slice cultures, voltage-clamped through a single Cs4- or K+-filled microelectrode and perfused with Hanks' balanced salt solution containing 1 μM tetrodotoxin and 10 mM tetraethylammionium. The Ca2+-current was reversibly reduced by bath-perfused muscarine (10 100 μM). This effect was inhibited by pirenzepine (10 μM) or atropine (1 μM). In K+-filled cells, inhibition was preceded by a phase of inward current enhancement; this was considered to be secondary to rapid outward current inhibition induced by muscarine since it was reduced when outward currents were previously inhibited with Ba2+. In partially clamped or unclamped cells inhibition of Ca2+-current leads to a shortening of the Ca2+-spike plateau.