EphB3 signaling propagates synaptic dysfunction in the traumatic injured brain.

Traumatic brain injury (TBI), ranging from mild concussion to severe penetrating wounds, can involve brain regions that contain damaged or lost synapses in the absence of neuronal death. These affected regions significantly contribute to sensory, motor and/or cognitive deficits. Thus, studying the mechanisms responsible for synaptic instability and dysfunction is important for protecting the nervous system from the consequences of progressive TBI. Our controlled cortical impact (CCI) injury produces ~20% loss of synapses and mild changes in synaptic protein levels in the CA3-CA1 hippocampus without neuronal losses. These synaptic changes are associated with functional deficits, indicated by >50% loss in synaptic plasticity and impaired learning behavior. We show that the receptor tyrosine kinase EphB3 participates in CCI injury-induced synaptic damage, where EphB3(-/-) mice show preserved long-term potentiation and hippocampal-dependent learning behavior as compared with wild type (WT) injured mice. Improved synaptic function in the absence of EphB3 results from attenuation in CCI injury-induced synaptic losses and reduced d-serine levels compared with WT injured mice. Together, these findings suggest that EphB3 signaling plays a deleterious role in synaptic stability and plasticity after TBI.

Phosphodiesterase inhibition rescues chronic cognitive deficits induced by traumatic brain injury.

Traumatic brain injury (TBI) modulates several cell signaling pathways in the hippocampus critical for memory formation. Previous studies have found that the cAMP-protein kinase A signaling pathway is downregulated after TBI and that treatment with a phosphodiesterase (PDE) 4 inhibitor rolipram rescues the decrease in cAMP. In the present study, we examined the effect of rolipram on TBI-induced cognitive impairments. At 2 weeks after moderate fluid-percussion brain injury or sham surgery, adult male Sprague Dawley rats received vehicle or rolipram (0.03 mg/kg) 30 min before water maze acquisition or cue and contextual fear conditioning. TBI animals treated with rolipram showed a significant improvement in water maze acquisition and retention of both cue and contextual fear conditioning compared with vehicle-treated TBI animals. Cue and contextual fear conditioning significantly increased phosphorylated CREB levels in the hippocampus of sham animals, but not in TBI animals. This deficit in CREB activation during learning was rescued in TBI animals treated with rolipram. Hippocampal long-term potentiation was reduced in TBI animals, and this was also rescued with rolipram treatment. These results indicate that the PDE4 inhibitor rolipram rescues cognitive impairments after TBI, and this may be mediated through increased CREB activation during learning.

EphrinBs regulate D-serine synthesis and release in astrocytes.

There is growing evidence that astrocytes play critical roles in neuron-glial interactions at the synapse. Astrocytes are believed to regulate presynaptic and postsynaptic structures and functions, in part, by the release of gliotransmitters such as glutamate, ATP, and d-serine; however, little is known of how neurons and astrocytes communicate to regulate these processes. Here, we investigated a family of transmembrane proteins called ephrinBs and Eph receptors that are expressed in the synapse and are known to regulate synaptic transmission and plasticity. In addition to their presence on CA1 hippocampal neurons, we determined that ephrins and Eph receptors are also expressed on hippocampal astrocytes. Stimulation of hippocampal astrocytes with soluble ephrinB3, known to be expressed on CA1 postsynaptic dendrites, enhanced d-serine synthesis and release in culture. Conversely, ephrinB3 had no effect on d-serine release from astrocytes deficient in EphB3 and EphA4, which are the primary receptors for ephrinB3. Eph receptors mediate this response through interactions with PICK1 (protein interacting with C-kinase) and by dephosphorylating protein kinase C α to activate the conversion of l-serine to d-serine by serine racemase. These findings are supported in vivo, where reduced d-serine levels and synaptic transmissions are observed in the absence of EphB3 and EphA4. These data support a role for ephrins and Eph receptors in regulating astrocyte gliotransmitters, which may have important implications on synaptic transmission and plasticity.

An Exploratory Report on Electrographic Changes in the Cerebral Cortex Following Mild Traumatic Brain Injury with Hyperthermia in the Rat.

Traumatic brain injury (TBI) has the potential to perturb perception by disrupting electrical propagation within and between the thalamus and cerebral cortex. Moderate and severe TBI may result in posttraumatic epilepsy, a condition characterized by convulsive tonic-clonic seizures. Spike/wave discharges (SWDs) of generalized nonconvulsive seizures, also called absence seizures, may also occur as a consequence of brain trauma. As mild hyperthermia has been reported to exacerbate histopathological and behavioral outcomes, we used an unbiased algorithm to detect periodic increases in power across different frequency bands following single or double closed head injury (CHI) under normothermia and hyperthermia conditions. We demonstrated that mild TBI did not significantly alter the occurrence of events containing increases in power between the delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), and beta1 (12-20 Hz) frequency bands in the Sprague Dawley rat 12 weeks after injury. However, when hyperthermia (39°C) was induced before and after CHI, electrographic events containing a similar waveform and harmonic frequency to SWDs were observed in a subset of animals. Further experiments utilizing chronic recordings will need to be performed to determine if these trends lead to absence seizures.

Post-traumatic seizure susceptibility is attenuated by hypothermia therapy.

Traumatic brain injury (TBI) is a major risk factor for the subsequent development of epilepsy. Currently, chronic seizures after brain injury are often poorly controlled by available antiepileptic drugs. Hypothermia treatment, a modest reduction in brain temperature, reduces inflammation, activates pro-survival signaling pathways, and improves cognitive outcome after TBI. Given the well-known effect of therapeutic hypothermia to ameliorate pathological changes in the brain after TBI, we hypothesized that hypothermia therapy may attenuate the development of post-traumatic epilepsy and some of the pathomechanisms that underlie seizure formation. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury, and were then maintained at normothermic or moderate hypothermic temperatures for 4 h. At 12 weeks after recovery, seizure susceptibility was assessed by challenging the animals with pentylenetetrazole, a GABA(A) receptor antagonist. Pentylenetetrazole elicited a significant increase in seizure frequency in TBI normothermic animals as compared with sham surgery animals and this was significantly reduced in TBI hypothermic animals. Early hypothermia treatment did not rescue chronic dentate hilar neuronal loss nor did it improve loss of doublecortin-labeled cells in the dentate gyrus post-seizures. However, mossy fiber sprouting was significantly attenuated by hypothermia therapy. These findings demonstrate that reductions in seizure susceptibility after TBI are improved with post-traumatic hypothermia and provide a new therapeutic avenue for the treatment of post-traumatic epilepsy.

Neurotherapeutic capacity of P7C3 agents for the treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a significant public health problem around the world. A promising area of research is the characterization of small, drug-like molecules that have potent clinical properties. One pharmacotherapeutic agent in particular, an aminopropyl carbazole called P7C3, was discovered using an in vivo screen to identify new agents that augmented the net magnitude of adult hippocampal neurogenesis. P7C3 greatly enhanced neurogenesis by virtue of increasing survival rates of immature neurons. The potent neuroprotective efficacy of P7C3 is likely due to enhanced nicotinamide phosphoribosyltransferase (NAMPT) activity, which supports critical cellular processes. The scaffold of P7C3 was found to have favorable pharmacokinetic properties, good bioavailability, and was nontoxic. Preclinical studies have shown that administration of the P7C3-series of neuroprotective compounds after TBI can rescue and reverse detrimental cellular events leading to improved functional recovery. In several TBI models and across multiple species, P7C3 and its analogues have produced significant neuroprotection, axonal preservation, robust increases in the net magnitude of adult neurogenesis, protection from injury-induced LTP deficits, and improvement in neurological functioning. This review will elucidate the exciting and diverse therapeutic findings of P7C3 administration in the presence of a complex and multifactorial set of cellular and molecular challenges brought forth by experimental TBI. The clinical potential and broad therapeutic applicability of P7C3 warrants much needed investigation into whether these remedial effects can be replicated in the clinic. P7C3 may serve as an important step forward in the design, understanding, and implementation of pharmacotherapies for treating patients with TBI. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

Increased Expression of Epileptiform Spike/Wave Discharges One Year after Mild, Moderate, or Severe Fluid Percussion Brain Injury in Rats.

In this study, we describe increased expression of cortical epileptiform spike/wave discharges (SWD) in rats one year after mild, moderate, or severe fluid percussion traumatic brain injury (fpTBI). Groups of rats consisted of animals that had received mild, moderate, or severe fpTBI, or sham operation one year earlier than electrocorticography (ECoG) recordings. In addition, we included a group of age-matched naïve animals. ECoG was recorded from awake animals using epidural electrodes implanted on the injured hemisphere (right), sham-operated hemisphere (right), or right hemisphere in naïve animals. The SWDs were detected automatically using Fast Fourier Transformation and a novel algorithm for comparing changes in spectral power to control (nonepileptical) ECoG. The fpTBI resulted in increased expression of SWDs one year after injury compared with sham-operated or naïve animals. The number of SWD-containing ECoG epochs recorded in a 1 h recording session were: naïve 12.9 ± 10.3, n = 8, sham 23.6 ± 8.2, n = 10, mild TBI 78.9 ± 23.9, n = 10, moderate TBI 61.3 ± 32.5, n = 12, severe TBI 72.5 ± 28.3, n = 11 (mean ± standard error of the mean). Increased expression of SWDs was not related to injury severity. SWDs were observed to a lesser extent even in sham-operated and naïve animals. The data indicate that fpTBI exacerbates expression of SWDs in the rat and that this increase may be observed at least one year after injury. As others have discussed, the spontaneous occurrence of these epileptiform events in rodents limits the use of this model for investigations of acquired epilepsy, at least of the nonconvulsive type, after TBI.

Automated approach to detecting behavioral states using EEG-DABS.

Electrocorticographic (ECoG) signals represent cortical electrical dipoles generated by synchronous local field potentials that result from simultaneous firing of neurons at distinct frequencies (brain waves). Since different brain waves correlate to different behavioral states, ECoG signals presents a novel strategy to detect complex behaviors. We developed a program, EEG Detection Analysis for Behavioral States (EEG-DABS) that advances Fast Fourier Transforms through ECoG signals time series, separating it into (user defined) frequency bands and normalizes them to reduce variability. EEG-DABS determines events if segments of an experimental ECoG record have significantly different power bands than a selected control pattern of EEG. Events are identified at every epoch and frequency band and then are displayed as output graphs by the program. Certain patterns of events correspond to specific behaviors. Once a predetermined pattern was selected for a behavioral state, EEG-DABS correctly identified the desired behavioral event. The selection of frequency band combinations for detection of the behavior affects accuracy of the method. All instances of certain behaviors, such as freezing, were correctly identified from the event patterns generated with EEG-DABS. Detecting behaviors is typically achieved by visually discerning unique animal phenotypes, a process that is time consuming, unreliable, and subjective. EEG-DABS removes variability by using defined parameters of EEG/ECoG for a desired behavior over chronic recordings. EEG-DABS presents a simple and automated approach to quantify different behavioral states from ECoG signals.

Enhanced astrocytic d-serine underlies synaptic damage after traumatic brain injury.

After traumatic brain injury (TBI), glial cells have both beneficial and deleterious roles in injury progression and recovery. However, few studies have examined the influence of reactive astrocytes in the tripartite synapse following TBI. Here, we have demonstrated that hippocampal synaptic damage caused by controlled cortical impact (CCI) injury in mice results in a switch from neuronal to astrocytic d-serine release. Under nonpathological conditions, d-serine functions as a neurotransmitter and coagonist for NMDA receptors and is involved in mediating synaptic plasticity. The phasic release of neuronal d-serine is important in maintaining synaptic function, and deficiencies lead to reductions in synaptic function and plasticity. Following CCI injury, hippocampal neurons downregulated d-serine levels, while astrocytes enhanced production and release of d-serine. We further determined that this switch in the cellular source of d-serine, together with the release of basal levels of glutamate, contributes to synaptic damage and dysfunction. Astrocyte-specific elimination of the astrocytic d-serine-synthesizing enzyme serine racemase after CCI injury improved synaptic plasticity, brain oscillations, and learning behavior. We conclude that the enhanced tonic release of d-serine from astrocytes after TBI underlies much of the synaptic damage associated with brain injury.

Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice.

Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

Protein kinase C delta cleavage initiates an aberrant signal transduction pathway after cardiac arrest and oxygen glucose deprivation.

Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

Quantitative analysis of brain NADH in the presence of hemoglobin using microfiber spectrofluorometry: a pre-calibration approach.

Dysfunction of mitochondria links a variety of central nervous system disorders and other neurodegenerative diseases. The primary respiratory chain substrate reduced-form nicotinamide adenine dinucleotide (NADH) is an important regulator of respiratory chain function in mitochondria and, because of its fluorescent properties, has been used to assess mitochondrial pathophysiology in cells and tissues. However, assessment of changes in tissue NADH has been limited to qualitative analysis primarily because hemoglobin (Hb) interferes with NADH fluorescence measurements by absorbing both excitation and emission light. This report presents a computer-assisted approach to estimate tissue NADH and Hb concentrations quantitatively at the same time. The method is based on a two-dimensionally interpolated database model that is calibrated by fluorescence emission spectra with known-value standard chemical solutions. Quantitative concentrations for NADH and Hb can be determined by the corresponding known-value spectral data that have the minimum error to the sample spectrum obtained from an experiment. Repeatability and reliability tests are also presented in this report. Results demonstrate that this method can feasibly quantify the NADH content regardless of the Hb background in living hippocampal cells during hypoxia, suggesting that it has the potential to be applied to in vivo experiments in the future.

Mitochondria consume energy and compromise cellular membrane potential by reversing ATP synthetase activity during focal ischemia in rats.

The direction of the chemical reaction of ATP synthetase is reversible. The present study was designed to determine whether mitochondria produce or consume ATP during ischemia. For this purpose, changes in mitochondrial membrane potential were measured in vivo at the site of a direct current (DC) electrode using a potentiometric dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and a rat model of focal ischemia. Two microL of dye (control group) or dye with oligomycin, an ATP synthetase inhibitor (oligomycin group), was injected into the parietotemporal cortex through the DC electrode. With the initiation of ischemia, a decrease in mitochondrial potential was observed within 20 seconds in the oligomycin group (earlier than the onset of DC deflection, P = 0.02). In contrast, in the control group, mitochondrial potential was maintained at 91 +/- 5% of the preischemia level for 118 +/- 38 seconds before showing full depolarization simultaneously with DC deflection. During the period of ischemia, the mitochondrial potential was higher in the control group (66 +/- 9%) than in the oligomycin group (46 +/- 8%, P = 0.0002), whereas DC potential was lower in the control group (-18 +/- 3) than in the oligomycin group (-15 +/- 2 mV, P = 0.04). These observations suggest that mitochondria consume ATP during ischemia by reversing ATP synthetase activity, which compromises cellular membrane potential by consuming ATP.

Epsilon protein kinase C mediated ischemic tolerance requires activation of the extracellular regulated kinase pathway in the organotypic hippocampal slice.

Ischemic preconditioning (IPC) promotes brain tolerance against subsequent ischemic insults. Using the organotypic hippocampal slice culture, we conducted the present study to investigate (1) the role of adenosine A1 receptor (A1AR) activation in IPC induction, (2) whether epsilon protein kinase C (epsilonPKC) activation after IPC is mediated by the phosphoinositol pathway, and (3) whether epsilonPKC protection is mediated by the extracellular signal-regulated kinase (ERK) pathway. Our results demonstrate that activation of A1AR emulated IPC, whereas blockade of the A1AR during IPC diminished neuroprotection. The neuroprotection promoted by the A1AR was also reduced by the epsilonPKC antagonist. To determine whether epsilonPKC activation in IPC and A1AR preconditioning is mediated by activation of the phosphoinositol pathway, we incubated slices undergoing IPC or adenosine treatment with a phosphoinositol phospholipase C inhibitor. In both cases, preconditioning neuroprotection was significantly attenuated. To further characterize the subsequent signal transduction pathway that ensues after epsilonPKC activation, mitogen-activated protein kinase kinase was blocked during IPC and pharmacologic preconditioning (PPC) (with epsilonPKC, NMDA, or A1AR agonists). This treatment significantly attenuated IPC- and PPC-induced neuroprotection. In conclusion, we demonstrate that epsilonPKC activation after IPC/PPC is essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures and that the ERK pathway is downstream to epsilonPKC.

Changes in trkB-ERK1/2-CREB/Elk-1 pathways in hippocampal mossy fiber organization after traumatic brain injury.

Traumatic brain injury (TBI) leads to mossy fiber reorganization, which is considered to be a causative factor in the development of temporal lobe epilepsy. However, the underlying mechanism is not fully understood. Emerging evidence suggests that TrkB-ERK1/2-CREB/Elk-1 pathways are highly related to synaptic plasticity. This study used the rat fluid-percussion injury model to investigate activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways after TBI. Rats were subjected to 2.0-atm parasagittal TBI followed by 30 minutes, 4 hours, 24 hours, and 72 hours of recovery. After TBI, striking activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways in mossy fiber organization were observed with confocal microscopy and Western blot analysis. ERK1/2 was highly phosphorylated predominantly in hippocampal mossy fibers, whereas TrkB was phosphorylated both in the mossy fibers and the dentate gyrus region at 30 minutes and 4 hours of recovery after TBI. CREB was also activated at 30 minutes, peaked at 24 hours of recovery, and returned to the control level at 72 hours of recovery in dentate gyrus granule cells. Elk-1 phosphorylation was seen in CA3 neurons at 4 hours after TBI. The results suggest that the signaling pathways of TrkB-ERK1/2-CREB/Elk-1 are highly activated in mossy fiber organization, which may contribute to mossy fiber reorganization seen after TBI.

Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia.

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1h following IPC, and the other one that occurs 1-3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min 'test' ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N,N,N',N'-tetramethyl-p-phenylenediamine ( P< 0.01). The activities of complex I-III decreased in ischemic group by 16, 23 (P < 0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the 'first window'.

Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures.

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.

Mild cardiopulmonary arrest promotes synaptic dysfunction in rat hippocampus.

Cardiac arrest (CA) patients exhibit learning and memory disabilities. These deficits suggest that synaptic dysfunction may underlie such disabilities. The hypothesis of the present study was that synaptic dysfunction occurs following CA and that this precedes cell death. To test this hypothesis, we used histopathological and electrophysiological markers in the hippocampus of rats subjected to CA. Evoked potentials (EP) were determined in the CA1 region of hippocampal slices harvested from animals subjected to CA or sham-operated rats by stimulating the Schaffer collaterals and recording in the CA1 pyramidal region. EP amplitudes were significantly attenuated by approximately 60% in hippocampal slices harvested from animals subjected to CA. Hippocampal slices harvested from sham rats exhibited normal long-term potentiation (LTP). In contrast, hippocampal slices harvested 24 h after CA exhibited no LTP response, even when no histopathological abnormalities were observed. These data suggest that synaptic dysfunction occurs before and without overt histopathology. We suggest that the synaptic dysfunction precedes and may be an early marker for delayed neuronal cell death in the hippocampus after CA.

Mitochondrial complex I inhibition produces selective damage to hippocampal subfield CA1 in organotypic slice cultures.

The effects of mitochondrial respiratory chain inhibitors and the excitotoxin N-methyl-D-aspartate (NMDA) on cell death in hippocampal subfields CA1 and CA3 were examined in hippocampal organotypic slice cultures. Slice cultures, 2-3 week old, were exposed for 1 h to either the Complex I inhibitors, rotenone or 1-methyl-4-phenylpyridium (MPP+), the Complex II inhibitor 3-nitropropionic acid (3-NP), or the excitotoxin NMDA. Cell death was examined 24 and 48 h following treatment, by measuring propidium iodide (PI) fluorescence. Treatment with 1 micro M Rotenone caused greater cell death in hippocampal subfield CA1 than CA3. Exposure of hippocampal slice cultures to 10 micro M rotenone, to MPP+ or to NMDA resulted in damage to both CA1 and CA3 subfields. 3-NP produced little damage in either subfield. The data suggest that mitochondrial Complex I inhibition can produce selective cell damage in hippocampus and in this regard is similar to that observed following hypoxia/ischemia.