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P L Carlen

Publications and source records attributed to P L Carlen.

At least 19 recordsLinked to original sources
PubMed2006

Bidirectional multisite seizure propagation in the intact isolated hippocampus: the multifocality of the seizure "focus".

Localizing the seizure focus is difficult and frequently, multiple sites are found. This reflects our poor understanding of the fundamental mechanisms of seizure generation and propagation. We used multisite electrophysiological recordings in two seizure models and voltage-sensitive dye imaging, to spatiotemporally characterize the initiation and propagation of seizures in an intact epileptogenic brain region, the isolated hippocampus. In low-magnesium perfusate, seizures always originated in the temporal region, and propagated along the septotemporal axis to the septal region. After the seizure spread across the hippocampus, the bursts within a seizure became bidirectional, with different propagation patterns at different frequencies. When the intact hippocampus was separated along the septotemporal axis, independent bidirectional activity was observed in the two halves, and region-specific cuts to the tissue reveal that the CA3 region is critical for seizure generation and propagation. In a second seizure model, using focal tetanic stimulation of the septal and temporal CA3 region, seizures always originated at the stimulated site with bidirectionality later developing at different frequencies, as noted in the low magnesium model, behavior compatible with coupled neuronal network oscillators. These data provide novel insights into the dynamic multifocality of seizure onset and propagation, revealing that the current concept of a single seizure "focus" is complex.

Animals↗
PubMed2005

Postnatal development of intrinsic GABAergic rhythms in mouse hippocampus.

The local circuitry of the mammalian limbic cortices, including the hippocampus, is capable of generating spontaneous rhythmic activities of 0.5-4 Hz when isolated in vitro. These rhythmic activities are mediated by synchronous inhibitory postsynaptic potentials in pyramidal neurons as the result of repeated discharges of inhibitory interneurons. As such, they are thought to represent an intrinsic inhibitory rhythm. It is unknown at present whether such a rhythm occurs in the immature rodent hippocampus and, if so, the postnatal time window in which it develops. We explored these issues using our recently developed whole mouse hippocampal isolate preparation in vitro. We found that spontaneous rhythmic field potentials started to emerge in mouse hippocampal isolates around postnatal day 10, stabilized after postnatal day 15 and persisted into adulthood. In postnatal days 11-14 mouse hippocampi, the properties of these rhythmic potentials were in keeping with a CA3-driven, IPSP-based intrinsic network activity. The lack of spontaneous field rhythm in neonatal (postnatal days 2-7) hippocampi cannot be attributed to the excitatory activities mediated by gamma-aminobutyric acid type A (GABA-A) receptors, as chloride-dependent hyperpolarizing inhibitory postsynaptic potentials were detectable in neonatal pyramidal neurons at voltages near resting potentials and pharmacological antagonisms of GABA-A receptors produced robust epileptiform discharges in neonatal hippocampi. High frequency afferent stimulation or applications of 4-aminopyridine at low micromolar concentrations failed to induce persistent field rhythm in neonatal hippocampi, suggesting that an overall weak glutamatergic drive is not the sole causing factor. We suggest that the inhibitory postsynaptic potential-based spontaneous rhythmic field potentials develop in a discrete time window during the second postnatal week in the mouse hippocampus due to a fine-tuning in the structure and function of CA3 recurrent circuitry and associated GABAergic inhibitory interneurons.

2-Amino-5-phosphonovalerate↗
PubMed2004

Model of frequent, recurrent, and spontaneous seizures in the intact mouse hippocampus.

This study presents a model of chronic, recurrent, spontaneous seizures in the intact isolated hippocampal preparation from mice aged P8-P25. Field activity from the CA1 pyramidal cell layer was recorded and recurrent, spontaneous seizure-like events (SLEs) were observed in the presence of low Mg2+ (0.25 mM) artificial cerebrospinal fluid (ACSF). Hippocampi also showed interictal epileptiform discharges (IEDs) of 0.9-4.2 Hz occurring between seizures. No age-specific differences were found in SLE occurrence (2 SLEs per 10 min, on average), duration, and corresponding frequencies. After long exposure to low Mg2+ ACSF (>3 h), SLEs were completely reversible within minutes with the application of normal (2 mM Mg2+) ACSF. The AMPA antagonist, CNQX, blocked all epileptiform activity, whereas the NMDA antagonist, APV, did not. The gamma-aminobutyric acid (GABA)A antagonist, bicuculline, attenuated and fragmented SLEs, implicating interneurons in SLE generation. The L-type Ca2+ blocker, nifedipine, enhanced epileptiform activity. Analysis of dual site recordings along the septotemporal hippocampus demonstrated that epileptiform activity began first in the temporal pole of the hippocampus, as illustrated by disconnection experiments. Once an SLE had been established, however, the septal hippocampus was sometimes seen to lead the epileptiform activity. The whole hippocampus with intact local circuitry, treated with low Mg2+, provides a realistic model of recurrent spontaneous seizures, which may be used, in normal and genetically modified mice, to study the dynamics of seizures and seizure evolution, as well as the mechanisms of action of anti-epileptic drugs and other therapeutic modalities.

2-Amino-5-phosphonovalerate↗
PubMed2001

Dynamics of intracellular calcium and free radical production during ischemia in pyramidal neurons.

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction. We investigated the contribution of mitochondrial dysfunction to free radical (FR) overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced FR generation was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydroRhod-2, revealed that FR production during and after the anoxic episode correlates with the accumulation of mitochondrial calcium. However, the mitochondrial calcium uptake inhibitor Ru360 did not prevent FR generation during ischemia and attenuated it to some degree during reoxygenation. On the other hand, the mitochondrial permeability transition blocker cyclosporinA (CsA) completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented deterioration of neuronal intrinsic membrane properties. CsA had no effect on the accumulation of intracellular calcium during ischemia-reperfusion. Nicotinamide, a blocker of NAD+ hydrolysis, reproduced the CsA effects on FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases. These observations suggest that a major determinant of ischemic FR generation in pyramidal neurons is the uncoupling of the mitochondrial respiratory chain, which may be associated with the mitochondrial permeability transition.

Aniline Compounds↗
PubMed2001

Artificial electrotonic coupling affects neuronal firing patterns depending upon cellular characteristics.

While there have been numerous theoretical studies indicating that electrotonic coupling via gap junctions interacts with the intrinsic characteristics of the coupled neurons to modify their electrical behaviour, little experimental evidence has been provided in coupled mammalian neurons. Using an artificial electrotonic junction, two distant uncoupled neurons were coupled through the computer, and the coupling conductance was varied. Tonically firing CA1 hippocampal pyramidal neurons reduced their spike firing frequency when coupled to thalamic or pyramidal cells, showing that the electrical coupling can be considered as a low-pass filter. The strength of coupling needed to entrain spike bursts of pyramidal neurons was considerably lower than the coupling needed to synchronize two neurons with different cellular characteristics (thalamic and pyramidal cells). Coupling promoted burst firing in a non-bursting cell if it was coupled to a spontaneously bursting neuron. These results support modelling studies that indicate a role for gap-junctional coupling in the synchronization of neuronal firing and the expression of low-frequency bursting.

Action Potentials↗
PubMed2001

Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus.

Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.

Action Potentials↗
PubMed2000

Antiepileptic efficacy of topiramate: assessment in two in vitro seizure models.

The antiepileptic efficacy of topiramate (TPM) has been demonstrated in both whole animal seizure models and clinical trials; however, there is no consensus concerning its mechanism of action. We determined first whether the antiepileptic effect of TPM generalized to in vitro seizure models. Epileptiform discharges, recorded extracellularly, were evoked by repeated tetanic stimulation of Schaffer collaterals and layer III association fibers in entorhinal cortex/hippocampus and piriform cortex slices, respectively. TPM was applied at concentrations of 20 or 100 microM. Whole cell recordings were made from CA1 pyramidal neurons and the effect of TPM was assessed on a variety of intrinsic membrane properties including resting membrane potential, input resistance and postspike potentials. TPM (20 microM) was without effect in entorhinal cortex/hippocampus (N=6); however, 100 microM TPM decreased significantly the Coastline Burst Index from 358.3+/-65.8 to 225. 5+/-77.1 (N=4), the frequency of spontaneous epileptiform discharges to 44.6+/-21.8 (N=5) and the duration of primary afterdischarge (PAD) to 65.9+/-10.1 (N=10) percent of control. In contrast, phenytoin (50 microM, N=7; 100 microM, N=8) reduced PAD to 96.9+/-14. 8 and 86.5+/-17.3 percent of control, respectively. TPM (100 microM) did not reduce significantly the frequency of spontaneous discharges in piriform cortex (85.4+/-12.3 percent of control; N=5). TPM (100 microM) was without significant effect on intrinsic membrane properties in CA1 pyramidal neurons. Likely candidate mechanisms underlying the antiepileptic effect produced by TPM include enhancement of chloride-mediated GABA(A) currents and reduction of kainate and L-type calcium currents.

Action Potentials↗
PubMed2000

The role of gap junctions in seizures.

Electrotonic synaptic communication between neurons via gap junctions (gjs) is increasingly recognized as an important synchronizing mechanism in the brain. At the same time, the biology of central nervous system (CNS) gjs is being unravelled. The pathogenesis of the abnormal neuronal synchrony underlying seizures, formerly thought to be based mainly on chemical synaptic transmission, now includes a role for gap junctional communication. This concept has been strengthened by evidence from several in vitro seizure models, in which pharmacological manipulations of gap junctional communication predictably affect the generation of seizures: blockers diminishing seizures and enhancers increasing the seizures. Evidence for interneurons, coupled in part by gjs, generating synchronous neural network activity including seizures, is presented. Also neuromodelling studies, which have enhanced our ability to understand the functional role that gap junctional communication plays in the generation and maintenance of neural synchrony and seizures, are presented. Gap junctional communication appears to be a promising target for the development of future anticonvulsant therapy.

Animals↗
PubMed2000

Gap junctions, synchrony and seizures.

The old concept that the direct intercellular cytoplasmic connections between neurones participate in the coordination of neuronal activity has gained new relevance, owing to recent theoretical and experimental evidence, particularly with regard to neuronal synchronization and epileptogenesis. Computer simulations demonstrating that neurones synchronize and alter their firing patterns depending on gap-junctional communication, have provided insights into the interactions between electrotonic coupling and cellular and synaptic characteristics. Experimental manipulations of gap-junctional communication support its role in the generation and maintenance of synchronized neuronal firing and seizures. Hence, in addition to chemical transmission, direct electrotonic coupling might contribute to normal and abnormal physiological brain rhythms.

Action Potentials↗
PubMed2000

Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy.

Reactive oxygen species have been implicated in the development of seizures under pathological conditions and linked to seizure-induced neurodegeneration. There has been little direct evidence, however, of free radical production resulting from seizures. Using amygdala-kindled rats, we have examined the generation of reactive oxygen species following seizures, and their possible contribution to seizure development and seizure-induced neuronal loss. The concentrations of two products of free radical-induced lipid peroxidation, malonaldehyde and 4-hydroxy-2(E)-nonenal, were measured using colorimetric assays. Lipid peroxidation was increased in both hemispheres of kindled rats as compared to sham-operated controls. Cell death was also significantly increased in all hippocampal areas. Antioxidants (vitamin E and glutathione) prevented the rise in lipid peroxides and hippocampal neuronal death during kindling, but did not arrest the development of seizures.Thus, epileptiform activity can result in free radical production which may be one of the factors leading to cell death.

Animals↗
PubMed2000

Progestin receptors mediate progesterone suppression of epileptiform activity in tetanized hippocampal slices in vitro.

Clinical and laboratory studies suggest that progesterone reduces epileptic seizure activity. The mechanisms underlying this effect are not known. The present study determined the effects of progesterone on extracellular evoked responses recorded in the CA1 field of hippocampal slices, as well as epileptiform responses recorded from tetanized slices. Slices were prepared from ovariectomized rats, with or without estrogen replacement. Hippocampal slices were superfused in vitro with one of the following treatments: progesterone with or without RU486 (a progesterone receptor antagonist); allopregnanolone (a progesterone metabolite that potentiates GABA action at GABA(A) receptors); RU5020 (a high-affinity progesterone receptor agonist); or cholesterol (control). In non-tetanized slices, a twofold increase in the excitatory postsynaptic field potential and population spike amplitude occurred during both cholesterol and progesterone superfusion. In contrast, under the same conditions, exposure to allopreganolone caused a 25% reduction in both field potential and population spike amplitude of evoked responses within 30min of treatment. In tetanized slices, progesterone and RU5020, but not allopregnanolone or cholesterol, caused significant reductions in the field potential and population spike amplitude of evoked responses. Progesterone and RU5020 also significantly reduced the duration of tetanic stimulus-induced afterdischarges and the frequency of spontaneous interictal discharges. The effects of allopregnanolone were restricted to a reduction in the primary afterdischarge duration. Estrogen replacement slightly attenuated progesterone's suppression of spontaneous discharges and depression of evoked responses. All responses to progesterone were blocked by prior or concurrent exposure to RU486. These data indicate that allopregnanolone suppresses evoked potentials in non-tetanized hippocampal slices, consistent with previous reports that this neurosteroid has marked anxiolytic and anticonvulsant effects. After tetanization, however, progesterone receptor-mediated responses become quantitatively more important as a mechanism for suppressing hippocampal electrical activity.

Animals↗
PubMed2000

Mitochondrial porin required for ischemia-induced mitochondrial dysfunction and neuronal damage.

The precise molecular events of mitochondrial dysfunction, one of the last steps that irreversibly determines cellular degeneration and death, remain unknown. We introduce a novel strategy to isolate and assess the molecular mechanisms underlying mitochondrial dysfunction. Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Then, mitochondria were isolated from brain slices and mitochondrial proteins were purified on a cyclosporin-A affinity column. Cyclosporin-A is the most potent inhibitor of mitochondrial dysfunction, in particular the mitochondrial permeability transition, and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote this event. Mitochondrial porin was reproducibly eluted from the affinity column using proteins from ischemic brain mitochondria, or from mitochondria exposed to oxidative stress that were used as a positive control. Anti-porin antibodies prevented mitochondrial depolarization and electrophysiological deterioration of hippocampal neurons during hypoxia-reperfusion, as measured by simultaneous fluorescence imaging and whole-cell recordings. These observations provide biochemical and functional evidence that porin is directly involved in mitochondrial dysfunction and neuronal impairment during ischemia-reperfusion, and indicate that porin could be a novel therapeutic target to prevent cellular degeneration.

Animals↗
PubMed2000

Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.

Animals↗
PubMed2000

Free radical production correlates with cell death in an in vitro model of epilepsy.

Free radical (FR) production, a major step in calcium-dependent neurodegeneration, has been linked to the generation of epileptiform activity and seizure-induced cell death. However, direct evidence of FR production in neurons during seizures has never been presented. Using hippocampal cultured slices we demonstrate that FRs are produced in CA3 but not CA1 pyramidal neurons during the rhythmic synchronous activity induced by the GABAA receptor antagonist bicuculline. The production of FRs (measured as changes in the fluorescence emission of dihydrorhodamine 123) was correlated with an increase in the baseline levels of intracellular calcium ([Ca2+]i) estimated by fluo-3 injected into individual neurons via a patch pipette. [Ca2+]i increased during spike bursting and returned to baseline levels after the burst termination in CA1, but not in CA3, pyramidal neurons where 'interburst' calcium concentrations progressively increased. Measurement of cell death, performed with propidium iodide 48 h after a 30-min exposure to bicuculline, revealed most prominent degeneration of pyramidal neurons in the CA3 pyramidal layer. The FR scavengers vitamin E and glutathione significantly reduced the seizure-induced neurodegeneration without supressing spontaneous epileptiform activity. These observations indicate that FR overproduction is related to seizure-induced neuronal death.

Action Potentials↗
PubMed2000

Modulation of high-voltage-activated calcium channels in dentate granule cells by topiramate.

PURPOSE: In this study, we assessed the effects of topiramate (TPM) on high-voltage-activated calcium channel (HVACC) currents in vitro. METHODS: HVACC currents were recorded from rat dentate gyrus granule cells by using whole-cell patch-clamp techniques. The biophysical properties of HVACCs were used to separate voltage-activated Ca2+ currents into different subtypes. Three concentrations of TPM were tested: 1, 10, and 50 microM. RESULTS: TPM inhibited L-type currents and was more effective at 10 microM than at 50 microM, suggesting that there may be an optimal concentration at which TPM decreases L-type currents. Non-L-type currents were transiently increased by TPM at a high concentration (50 microM). CONCLUSIONS: Because the location of L-type calcium channels on soma and proximal dendrites gives these channels a crucial role in controlling dendritic excitability and in providing calcium for intracellular effectors, the decrease in the L-type HVA Ca2+ currents may be an important anticonvulsant mechanism of TPM.

Action Potentials↗
PubMed2000

Analysis of single K(ATP) channels in mammalian dentate gyrus granule cells.

ATP-sensitive potassium (K(ATP)) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K(+) channel and the ATP-binding cassette protein superfamilies. K(ATP) channels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic beta cells, where their activation inhibits insulin release, K(ATP) channels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of K(ATP) channels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single K(ATP) channels in DG granule cells. We recorded single K(ATP) channels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an E(K) close to 0 mV (symmetrical K(+)) and were organized in bursts with a duration of 19.3 +/- 1.6 (SE) ms and a frequency of 3.5 +/- 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening (P(open), 0.04 +/- 0.01). Open and closed dwell-time histograms were fitted best with one (tau(open) = 1.3 +/- 0.2 ms) and the sum of two (tau(closed,fast) = 2.6 +/- 0.9 ms, tau(closed,slow) = 302.7 +/- 67. 7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The P(open) was reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 microM,11/14 patches) and tolbutamide (20 microM, 4/6; 100 microM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 +/- 2.6% of control I(open channel)). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 microM) increased significantly P(open) (0.12 +/- 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 +/- 2.0 Hz). Diazoxide was without effect on tau(open) and tau(closed,fast) but decreased significantly tau(closed,slow) (24.4 +/- 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly P(open) (0.09 +/- 0.03) and the frequency of bursts (7.1 +/- 1.7 Hz) and decreased significantly tau(closed,slow) (29.5 +/- 1.8 ms). We have presented convergent evidence consistent with single K(ATP) channel activity in DG granule cells. The subunit composition of K(ATP) channels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.

Animals↗
PubMed1999

A submersion method to induce hypoxic damage in organotypic hippocampal cultures.

Organotypic brain slices cultured on semi-porous membranes is an increasingly popular in vitro preparation for studying mechanisms of ischemic brain damage. To model in vivo hypoxia, cultured brain slices are exposed to anaerobic atmosphere by placing them into a special incubator. This requirement limits the use of in vitro ischemic models to highly specialized laboratories. Here, we describe a simple method that reproduces hypoxic injury, where cultured hippocampal slices are submerged into glucose-free deoxygenated medium for 1 h. The extent and distribution of hippocampal neuronal loss obtained with this treatment resembled that caused by hypoxia in living tissue in situ, i.e. CA1 pyramidal cell layer was most vulnerable and dentate granular cell layer was least susceptible to hypoxia as measured with fluorescence of the viability marker propidium iodide (PI). Electrophysiologic functional impairment determined by field recordings of CA1 pyramidal neurones temporally coincided with the extent of neuronal death. In addition, known neuroprotective treatments, such as hypothermia and phenytoin application ameliorated neuronal damage in a pattern similar to previously published reports. Therefore, the present in vitro model of ischemia is simple, reliable and of low cost. It is well suited for short and long-term studies of the mechanisms of hypoxic brain damage.

Animals↗