Faculty Bibliography

Although it is now established that neurogenesis of dentate gyrus granule cells increases after experimental seizures, little is currently known about the function of the new granule cells. One question is whether they become integrated into the network around them. Recent experiments that focused on the newly born granule cells in the hilus showed that indeed the new cells appear to become synchronized with host hippocampal neurons [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. To address this issue further, we asked whether the new hilar granule cells were active during spontaneous limbic seizures that follow status epilepticus induced by pilocarpine injection. Thus, we perfused rats after spontaneous seizures and stained sections using antibodies to c-fos, a marker of neural activity, and calbindin, a marker of the newly born hilar granule cells [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. We asked whether calbindin-immunoreactive hilar neurons were also c-fos-immunoreactive.C-fos was highly expressed in calbindin-immunoreactive hilar neurons. Approximately 23% of hilar cells that expressed c-fos were double-labeled for calbindin. In addition, other types of hilar neurons, i.e. those expressing parvalbumin or neuropeptide Y, also expressed c-fos. Yet other hippocampal neurons, including granule cells and pyramidal cells, had weak expression of c-fos at the latency after the seizure that hilar neuron expression occurred. In controls, there was very little c-fos or calbindin expression in the hilus.These results indicate that calbindin-immunoreactive hilar cells are activated by spontaneous seizures. Based on the evidence that many of these cells are likely to be newly born, the data indicate that new cells can become functionally integrated into limbic circuits involved in recurrent seizure generation. Furthermore, they appear to do so in a manner similar to many neighboring hilar neurons, apparently assimilating into the local environment. Finally, the results show that a number of hilar cell types are activated during chronic recurrent seizures in the pilocarpine model, a surprising result given that many hilar neurons are thought to be damaged soon after pilocarpine-induced status epilepticus

Various studies have shown that brain-derived neurotrophic factor (BDNF) increases neuronal excitability and is localized and upregulated in areas implicated in epileptogenesis. Seizure activity increases the expression of BDNF mRNA and protein, and recent studies have shown that interfering with BDNF signal transduction inhibits the development of the epileptic state in vivo. These results suggest that BDNF contributes to epileptogenesis. Further analysis of the cellular and molecular mechanisms by which BDNF influences excitability and connectivity in adult brain could provide novel concepts and targets for anticonvulsant or anti-epileptogenic therapy

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures ('epileptic' rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus.Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first.From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these 'granule-like' neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry

Limbic seizures have often been attributed to pathology in the hippocampus, such as the well described condition termed Ammon's Horn sclerosis, in which many of the hippocampal principal cells have degenerated. However, several studies in both the clinical and basic literature indicate that the parahippocampal region may also play an important role. This region sustains a characteristic pattern of damage in most animal models of epilepsy that is similar to that identified in humans with intractable temporal lobe epilepsy. Perhaps the most striking aspect of parahippocampal pathology is the marked loss of neurons in layer III of the entorhinal cortex. The similarity of cell loss in layer III and cell loss in the hilus of the dentate gyrus is compared, as is the characteristic resistance of layer II neurons and dentate granule cells. Cellular electrophysiological results are used as a basis for the hypothesis that synaptic inhibition plays a role in the relative vulnerability of these neurons. Studies of neurogenesis in both areas is also discussed. It is proposed that this may be an additional factor that influences vulnerability in these areas

Competing enzymatic mechanisms degrade the tryptophan metabolite L-kynurenine to kynurenate, an inhibitory and neuroprotective compound, and to the neurotoxins 3-hydroxykynurenine and quinolinate. Kynurenine 3-hydroxylase inhibitors such as PNU 156561 shift metabolism towards enhanced kynurenate production, and this effect may underlie the recently discovered anticonvulsant and neuroprotective efficacy of these drugs. Using electrophysiological and neurotoxicological endpoints, we now used PNU 156561 as a tool to examine the functional interplay of kynurenate, 3-hydroxykynurenine and quinolinate in the rat hippocampus in vivo. First, population spike amplitude in area CA1 and the extent of quinolinate-induced excitotoxic neurodegeneration were studied in animals receiving acute or prolonged intravenous infusions of L-kynurenine, PNU 156561, (L-kynurenine+PNU 156561) or kynurenate. Only the latter two treatments, but not L-kynurenine or PNU 156561 alone, caused substantial inhibition of evoked responses in area CA1, and only prolonged (3h) infusion of (L-kynurenine+PNU 156561) or kynurenate was neuroprotective. Biochemical analyses in separate animals revealed that the levels of kynurenate attained in both blood and brain (hippocampus) were essentially identical in rats receiving extended infusions of L-kynurenine alone or (L-kynurenine+PNU 156561) (4 and 7microM, respectively, after an infusion of 90 or 180min). However, addition of the kynurenine 3-hydroxylase inhibitor resulted in a significant decrement in the formation of 3-hydroxykynurenine and quinolinate in both blood and brain.These data suggest that the ratio between kynurenate and 3-hydroxykynurenine and/or quinolinate in the brain is a critical determinant of neuronal excitability and viability. The anticonvulsant and neuroprotective potency of kynurenine 3-hydroxylase inhibitors may therefore be due to the drugs' dual action on both branches of the kynurenine pathway of tryptophan degradation

In this review, recent studies on the electrophysiological effects of de novo synthesized ('endogenous') kynurenic acid (KYNA) are discussed. Endogenous KYNA is normally formed as a byproduct of tryptophan metabolism. Evidence for a physiological role in neuronal excitability has not been strong, in part because brain levels are much lower than the KD of KYNA at the glycine site of the NMDA receptor, where KYNA is thought to exert its most potent effect. The results suggest that, unexpectedly, even low concentrations of endogenous KYNA have physiological consequences. These levels of KYNA reduced the number of hippocampal slices with spontaneous epileptiform discharges after exposure to buffer lacking magnesium. However, effects on evoked responses to single afferent stimuli were not detected. Taken together, the data argue for a potentially important role of endogenous KYNA in suppression of seizure-like activity, and suggest a novel approach to anticonvulsant drug development that could have few side effects

Kynurenic acid (KYNA) is an antagonist of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors and it blocks the glycine site of the NMDA receptor preferentially (IC50 = 7.9 microM). KYNA is produced endogenously by transamination of its precursor L-kynurenine (L-KYN). We tested the hypothesis that effects of endogenous, de novo produced KYNA, following bath-application of L-KYN to slices, would be different than effects of commercially-synthesized (exogenous) KYNA. The ability to block spontaneous epileptiform activity, induced by lowering extracellular magnesium, was examined in area CA3 of hippocampus and the entorhinal cortex. At a concentration of 200 microM L-KYN, which produced 0.89 +/- 0.20 microM KYNA, there were fewer slices with spontaneous epileptiform activity than slices exposed to 2 microM exogenous KYNA. The results indicate a more potent neuromodulatory action of endogenous KYNA than has been previously realized

This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell 'mossy fiber' axons. This reorganization, referred to as 'sprouting,' involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy