Mechanisms of increased hippocampal excitability in the Mashl+/- mouse model of Na+ /K+ -ATPase dysfunction.

OBJECTIVE: Na+ /K+ -ATPase dysfunction, primary (mutation) or secondary (energy crisis, neurodegenerative disease) increases neuronal excitability in the brain. To evaluate the mechanisms underlying such increased excitability we studied mice carrying the D801N mutation, the most common mutation causing human disease, specifically alternating hemiplegia of childhood (AHC) including epilepsy. Because the gene is expressed in all neurons, particularly γ-aminobutyric acid (GABA)ergic interneurons, we hypothesized that the pathophysiology would involve both pyramidal cells and interneurons and that fast-spiking interneurons, which have increased firing rates, would be most vulnerable. METHODS: We performed extracellular recordings, as well as whole-cell patch clamp recordings from pyramidal cells and interneurons, in the CA1 region on hippocampal slices. We also performed immunohistochemistry from hippocampal sections to count CA1 pyramidal cells as well as parvalbumin-positive interneurons. In addition, we performed video-electroencephalography (EEG) recordings from the dorsal hippocampal CA1 region. RESULTS: We observed that juvenile knock-in mice carrying the above mutation reproduce the human phenotype of AHC. We then demonstrated in the CA1 region of these mice the following findings as compared to wild type: (1) Increased number of spikes evoked by electrical stimulation of Schaffer collaterals; (2) equalization by bicuculline of the number of spikes induced by Schaffer collateral stimulation; (3) reduced miniature, spontaneous, and evoked inhibitory postsynaptic currents, but no change in excitatory postsynaptic currents; (4) robust action potential frequency adaptation in response to depolarizing current injection in CA1 fast-spiking interneurons; and (5) no change in the number of pyramidal cells, but reduced number of parvalbumin positive interneurons. SIGNIFICANCE: Our data indicate that, in our genetic model of Atp1α3 mutation, there is increased excitability and marked dysfunction in GABAergic inhibition. This supports the performance of further investigations to determine if selective expression of the mutation in GABAergic and or glutamatergic neurons is necessary and sufficient to result in the behavioral phenotype.

Transcriptional profile of hippocampal dentate granule cells in four rat epilepsy models.

Global expression profiling of neurologic or psychiatric disorders has been confounded by variability among laboratories, animal models, tissues sampled, and experimental platforms, with the result being that few genes demonstrate consistent expression changes. We attempted to minimize these confounds by pooling dentate granule cell transcriptional profiles from 164 rats in seven laboratories, using three status epilepticus (SE) epilepsy models (pilocarpine, kainate, self-sustained SE), plus amygdala kindling. In each epilepsy model, RNA was harvested from laser-captured dentate granule cells from six rats at four time points early in the process of developing epilepsy, and data were collected from two independent laboratories in each rodent model except SSSE. Hierarchical clustering of differentially-expressed transcripts in the three SE models revealed complete separation between controls and SE rats isolated 1 day after SE. However, concordance of gene expression changes in the SE models was only 26-38% between laboratories, and 4.5% among models, validating the consortium approach. Transcripts with unusually highly variable control expression across laboratories provide a 'red herring' list for low-powered studies.

Persistent Hyperactivity of Hippocampal Dentate Interneurons After a Silent Period in the Rat Pilocarpine Model of Epilepsy.

Profile of GABAergic interneuron activity after pilocarpine-induced status epilepticus (SE) was examined in the rat hippocampal dentate gyrus by analyzing immediate early gene expression and recording spontaneous firing at near resting membrane potential (REM). SE for exact 2 h or more than 2 h was induced in the male Sprague-Dawley rats by an intraperitoneal injection of pilocarpine. Expression of immediate early genes (IEGs) was examined at 1 h, 1 week, 2 weeks or more than 10 weeks after SE. For animals to be examined at 1 h after SE, SE lasted for exact 2 h was terminated by an intraperitoneal injection of diazepam. Spontaneous firing at near the REM was recorded in interneurons located along the border between the granule cell layer and the hilus more than 10 weeks after SE. Results showed that both c-fos and activity-regulated cytoskeleton associated protein (Arc) in hilar GABAergic interneurons were up-regulated after SE in a biphasic manner; they were increased at 1 h and more than 2 weeks, but not at 1 week after SE. Ten weeks after SE, nearly 60% of hilar GABAergic cells expressed c-fos. With the exception of calretinin (CR)-positive cells, percentages of hilar neuronal nitric oxide synthase (nNOS)-, neuropeptide Y (NPY)-, parvalbumin (PV)-, and somatostatin (SOM)-positive cells with c-fos expression are significantly higher than those of controls more than 10 weeks after SE. Without the REM to be more depolarizing and changed threshold potential level in SE-induced rats, cell-attached recording revealed that nearly 90% of hilar interneurons fired spontaneously at near the REM while only 22% of the same cell population did so in the controls. In conclusion, pilocarpine-induced SE eventually leads to a state in which surviving dentate GABAergic interneurons become hyperactive with a subtype-dependent manner; this implies that a fragile balance between excitation and inhibition exists in the dentate gyrus and in addition, the activity-dependent up-regulation of IEGs may underlie plastic changes seen in some types of GABAergic cells in the pilocarpine model of epilepsy.

Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat.

To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells.

What is a seizure focus?

The seizure focus is the site in the brain from which the seizure originated and is most likely equivalent to the epileptogenic zone, defined as the area of cerebral cortex indispensable for the generation of clinical seizures. The boundaries of this region cannot be defined at present by any diagnostic test. Imaging and EEG recording can define regions of functional deficit during the interictal period, regions that generate interictal spikes, regions responsible for the ictal symptoms, regions from which the seizure is triggered, and regions of structural damage. However, these regions define the epileptogenic zone only when they are spatially concordant. The frequent discrepancies suggest the essential involvement of synaptically connected regions, that is a distributive focus, in the origination of most seizures. Here we review supporting evidence from animal studies and studies of persons undergoing surgical resection for medically-intractable epilepsy. We conclude that very few of the common seizures are truly local, but rather depend on nodal interactions that permit spontaneous network excitability and behavioral expression. Recognition of the distributive focus underlying most seizures has motivated many surgical programs to upgrade their intracranial studies to capture activity in as much of the network as possible.

Morphologic integration of hilar ectopic granule cells into dentate gyrus circuitry in the pilocarpine model of temporal lobe epilepsy.

After pilocarpine-induced status epilepticus, many granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) are hyperexcitable and may therefore increase circuit excitability. This study determined the distribution of their axons and dendrites. HEGCs and normotopic granule cells were filled with biocytin during whole-cell patch clamp recording in hippocampal slices from pilocarpine-treated rats. The apical dendrite of 86% of the biocytin-labeled HEGCs extended to the outer edge of the dentate molecular layer. The total length and branching of HEGC apical dendrites that penetrated the molecular layer were significantly reduced compared with apical dendrites of normotopic granule cells. HEGCs were much more likely to have a hilar basal dendrite than normotopic granule cells. They were about as likely as normotopic granule cells to project to CA3 pyramidal cells within the slice, but were much more likely to send at least one recurrent mossy fiber into the molecular layer. HEGCs with burst capability had less well-branched apical dendrites than nonbursting HEGCs, their dendrites were more likely to be confined to the hilus, and some exhibited dendritic features similar to those of immature granule cells. HEGCs thus have many paths along which to receive synchronized activity from normotopic granule cells and to transmit their own hyperactivity to both normotopic granule cells and CA3 pyramidal cells. They may therefore contribute to the highly interconnected granule cell hubs that have been proposed as crucial to development of a hyperexcitable, potentially seizure-prone circuit.

Aspartate release and signalling in the hippocampus.

The Ca(2+)-dependent release of aspartate from hippocampal preparations was first reported 35 years ago, but the functional significance of this process remains uncertain. Aspartate satisfies all the criteria normally required for identification of a CNS transmitter. It is synthesized in nerve terminals, is accumulated and stored in synaptic vesicles, is released by exocytosis upon nerve terminal depolarization, and activates postsynaptic NMDA receptors. Aspartate may be employed as a neuropeptide-like co-transmitter by pathways that release either glutamate or GABA as their principal transmitter. Aspartate mechanisms include vesicular transport by sialin, vesicular content sensitive to glucose concentration, release mainly outside the presynaptic active zones, and selective activation of extrasynaptic NR1-NR2B NMDA receptors. Possible neurobiological functions of aspartate in immature neurons include activation of cAMP-dependent gene transcription and in mature neurons inhibition of CREB function, reduced BDNF expression, and induction of excitotoxic neuronal death. Recent findings suggest new experimental approaches toward resolving the functional significance of aspartate release.

High ratio of synaptic excitation to synaptic inhibition in hilar ectopic granule cells of pilocarpine-treated rats.

After experimental status epilepticus, many dentate granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) have also been found in persons with epilepsy. These cells exhibit a high rate of spontaneous activity, which may enhance seizure propagation. Electron microscopic studies indicated that HEGCs receive more recurrent mossy fiber innervation than normotopic granule cells in the same animals but receive much less inhibitory innervation. This study used hippocampal slices prepared from rats that had experienced pilocarpine-induced status epilepticus to test the hypothesis that an imbalance of synaptic excitation and inhibition contributes to the hyperexcitability of HEGCs. Mossy fiber stimulation evoked a much smaller GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in HEGCs than in normotopic granule cells from either control rats or rats that had experienced status epilepticus. However, recurrent mossy fiber-evoked excitatory postsynaptic currents (EPSCs) of similar size were recorded from HEGCs and normotopic granule cells in status epilepticus-experienced rats. HEGCs exhibited the highest frequency of miniature excitatory postsynaptic currents (mEPSCs) and the lowest frequency of miniature inhibitory postsynaptic currents (mIPSCs) of any granule cell group. On average, both mEPSCs and mIPSCs were of higher amplitude, transferred more charge per event, and exhibited slower kinetics in HEGCs than in granule cells from control rats. Charge transfer per unit time in HEGCs was greater for mEPSCs and much less for mIPSCs than in the normotopic granule cell groups. A high ratio of excitatory to inhibitory synaptic function probably accounts, in part, for the hyperexcitability of HEGCs.

Postsynaptic response to stimulation of the Schaffer collaterals with properties similar to those of synaptosomal aspartate release.

Aspartate satisfies all the criteria normally required for identification of a CNS neurotransmitter. Nevertheless, little electrophysiological evidence supports the existence of aspartate transmission. In studies with rat hippocampal synaptosomes, chemically evoked aspartate release differed from glutamate release in its relative sensitivity to increased Ca(2+) concentration outside the presynaptic active zones, inefficient coupling to P/Q-type Ca(2+) channels, sensitivity to KB-R7943, and resistance to native Clostridial toxins. We took advantage of these differences to search for a potential aspartate-mediated response at Schaffer collateral synapses in organotypic hippocampal slice cultures. The slice cultures were pretreated with botulinum neurotoxin C (BoNT/C) to eliminate most of the glutamate release so that an expectedly smaller aspartate-like component of the compound EPSC could be detected by whole cell patch clamp recording. In control cultures, NMDA receptor activation accounted for only 18% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by only 20%. Block of P/Q-type Ca(2+) channels essentially eliminated the response and 0.1 muM KB-R7943 had no significant effect. In BoNT/C-pretreated cultures, however, NMDA receptor activation accounted for 77% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by 57%. Block of P/Q-type Ca(2+) channels reduced the response by only 28%, but 0.1 muM KB-R7943 reduced it by 45%. These results suggest that part of the Schaffer collateral synaptic response has pharmacological properties similar to those of synaptosomal aspartate release and may therefore be mediated at least partly by released aspartate.

Enhanced tonic GABA current in normotopic and hilar ectopic dentate granule cells after pilocarpine-induced status epilepticus.

In temporal lobe epilepsy, loss of inhibitory neurons and circuit changes in the dentate gyrus promote hyperexcitability. This hyperexcitability is compensated to the point that dentate granule cells exhibit normal or even subnormal excitability under some conditions. This study explored the possibility that compensation involves enhanced tonic GABA inhibition. Whole cell patch-clamp recordings were made from normotopic granule cells in hippocampal slices from control rats and from both normotopic and hilar ectopic granule cells in slices from rats subjected to pilocarpine-induced status epilepticus. After status epilepticus, tonic GABA current was an order of magnitude greater than control in normotopic granule cells and was significantly greater in hilar ectopic than in normotopic granule cells. These differences could be observed whether or not the extracellular GABA concentration was increased by adding GABA to the superfusion medium or blocking plasma membrane transport. The enhanced tonic GABA current had both action potential-dependent and action potential-independent components. Pharmacological studies suggested that the small tonic GABA current of granule cells in control rats was mediated largely by high-affinity alpha(4)beta(x)delta GABA(A) receptors but that the much larger current recorded after status epilepticus was mediated largely by the lower-affinity alpha(5)beta(x)gamma(2) GABA(A) receptors. A large alpha(5)beta(x)gamma(2)-mediated tonic current could be recorded from controls only when the extracellular GABA concentration was increased. Status epilepticus seemed not to impair the control of extracellular GABA concentration by plasma membrane transport substantially. Upregulated tonic GABA inhibition may account for the unexpectedly modest excitability of the dentate gyrus in epileptic brain.

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: correlation of cell loss with mossy fiber sprouting.

Mossy fiber sprouting and the genesis of ectopic granule cells contribute to reverberating excitation in the dentate gyrus of epileptic brain. This study determined whether the extent of sprouting after status epilepticus in rats correlates with the seizure-induced degeneration of GluR2-immunoreactive (GluR2+) hilar neurons (presumptive mossy cells) and also quantitated granule cell-like GluR2-immunoreactive hilar neurons. Stereological cell counting indicated that GluR2+ neurons account for 57% of the total hilar neuron population. Prolonged pilocarpine-induced status epilepticus killed 95% of these cells. A smaller percentage of GluR2+ neurons (74%) was killed when status epilepticus was interrupted after 1-3.5 h with a single injection of phenobarbital, and the number of residual GluR2+ neurons varied among animals by a factor of 6.2. GluR2+ neurons were not necessarily more vulnerable than other hilar neurons. In rats administered phenobarbital, the extent of recurrent mossy fiber growth varied inversely and linearly with the number of GluR2+ hilar neurons that remained intact (P=0.0001). Thus the loss of each GluR2+ neuron was associated with roughly the same amount of sprouting. These findings support the hypothesis that mossy fiber sprouting is driven largely by the degeneration of and/or loss of innervation from mossy cells. Granule cell-like GluR2-immunoreactive neurons were rarely encountered in the hilus of control rats, but increased 6- to 140-fold after status epilepticus. Their number did not correlate with the extent of hilar cell death or mossy fiber sprouting in the same animal. The morphology, number, and distribution of these neurons suggested that they were hilar ectopic granule cells.

Neuropeptide Y in the recurrent mossy fiber pathway.

In the epileptic brain, hippocampal dentate granule cells become synaptically interconnected through the sprouting of mossy fibers. This new circuitry is expected to facilitate epileptiform discharge. Prolonged seizures induce the long-lasting neoexpression of neuropeptide Y (NPY) in mossy fibers. NPY is released spontaneously from recurrent mossy fiber terminals, reduces glutamate release from those terminals by activating presynaptic Y2 receptors, and depresses granule cell epileptiform activity dependent on the recurrent pathway. These effects are much greater in rats than in C57BL/6 mice, despite apparently equivalent mossy fiber sprouting and neoexpression of NPY. This species difference can be explained by contrasting changes in the expression of mossy fiber Y2 receptors; seizures upregulate Y2 receptors in rats but downregulate them in mice. The recurrent mossy fiber pathway may synchronize granule cell discharge more effectively in humans and mice than in rats, due to its lower expression of either NPY (humans) or Y2 receptors (mice).

Impaired firing and sodium channel function in CA1 hippocampal interneurons after transient cerebral ischemia.

Although interneurons in area CA1 of the hippocampus are less vulnerable to cerebral ischemia than CA1 pyramidal cells, it is not clear whether their relatively intact cellular morphology implies preservation of normal function. As maintenance of cellular excitability and firing properties is essential for interneurons to regulate neural networks, we investigated these aspects of interneuronal function after transient cerebral ischemia in rats. Cerebral ischemia in rats was induced for 8 mins by a combination of bilateral common carotid artery occlusion and hypovolemic hypotension, and whole cell patch clamp recordings were made in hippocampal slices prepared 24 h after reperfusion. Interneurons located within stratum pyramidale of area CA1 exhibited normal membrane properties and action potentials under these conditions. However, their excitability had declined, as evidenced by an increased action potential threshold and a rightward shift in the relationship between injected depolarizing current and firing rate. Voltage-clamp experiments revealed that transient cerebral ischemia reduced the peak Na(+) current and shifted Na(+) channel activation to more depolarized values, but did not alter steady-state inactivation of the channel. Double immunofluorescence cytochemistry showed that transient cerebral ischemia also reduced Na(v)1.1 subunit immunoreactivity in interneurons that coexpressed parvalbumin. We conclude that transient cerebral ischemia renders CA1 interneurons less excitable, that depressed excitability involves impaired Na(+) channel activation and that Na(+) channel dysfunction is explained, at least in part, by reduced expression of the Na(v)1.1 subunit. These changes may promote interneuron survival, but might also contribute to pyramidal cell death.

Reduced aspartate release from rat hippocampal synaptosomes loaded with Clostridial toxin light chain by electroporation: evidence for an exocytotic mechanism.

Aspartate can be released from certain hippocampal pathways along with glutamate or GABA. Although aspartate immunoreactivity has been localized to synaptic vesicles and aspartate release is Ca(2+)-dependent, there has been no clear evidence favoring an exocytotic mechanism. In particular, pretreatment with Clostridial toxins has not consistently inhibited aspartate release, even when release of glutamate from the same tissue samples was markedly inhibited. To address this issue directly, rat hippocampal synaptosomes were permeabilized transiently by electroporation in the presence of active or inactivated Clostridial toxin light chains. Loading rat hippocampal synaptosomes with the active light chain of tetanus toxin or of botulinum neurotoxins A, B or C reduced the K(+)-evoked release of aspartate at least as much as that of glutamate. These results confirm that aspartate is released by exocytosis in rat hippocampus.

Facilitation of granule cell epileptiform activity by mossy fiber-released zinc in the pilocarpine model of temporal lobe epilepsy.

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.

Depressed responses to applied and synaptically-released GABA in CA1 pyramidal cells, but not in CA1 interneurons, after transient forebrain ischemia.

Transient cerebral ischemia kills CA1 pyramidal cells of the hippocampus, whereas most CA1 interneurons survive. It has been proposed that calcium-binding proteins, neurotrophins, and/or inhibitory neuropeptides protect interneurons from ischemia. However, different synaptic responses early after reperfusion could also underlie the relative vulnerabilities to ischemia of pyramidal cells and interneurons. In this study, we used gramicidin perforated patch recording in ex vivo slices to investigate gamma-aminobutyric acid (GABA) synaptic function in CA1 pyramidal cells and interneurons 4 h after a bilateral carotid occlusion accompanied by hypovolemic hypotension. At this survival time, the amplitudes of both miniature inhibitory postsynaptic currents (mIPSCs) and GABA-evoked currents were reduced in CA1 pyramidal cells, but not in CA1 interneurons. In addition, the mean rise time of mIPSCs was reduced in pyramidal cells. The reversal potential for the GABA current (E(GABA)) did not shift toward depolarizing values in either cell type, indicating that the driving force for chloride was unchanged at this survival time. We conclude that early during reperfusion GABAergic neurotransmission is attenuated exclusively in pyramidal neurons. This is likely explained by reduced GABAA receptor sensitivity or clustering and possibly also reduced GABA release, rather than by an elevation of intracellular chloride. Impaired GABA function may contribute to ischemic neuronal death by enhancing the excitability of CA1 pyramidal cells and facilitating N-methyl-D-aspartic acid channel opening. Therefore, normalizing GABAergic function might be a useful pharmacological approach to counter excessive, and potentially excitotoxic, glutamatergic activity during the postischemic period.

Spontaneous release of neuropeptide Y tonically inhibits recurrent mossy fiber synaptic transmission in epileptic brain.

In the pilocarpine model of temporal lobe epilepsy, mossy fibers coexpress the inhibitory transmitter neuropeptide Y (NPY) with glutamate. The effects of endogenous and applied NPY on recurrent mossy fiber synaptic transmission were investigated with the use of whole-cell voltage-clamp and field recordings in rat hippocampal slices. Applied NPY reversibly inhibited synaptic transmission at recurrent mossy fiber synapses on dentate granule cells but not at perforant path or associational-commissural synapses. It also reduced the frequency of miniature EPSCs (mEPSCs) in granule cells from epileptic, but not control, rats and depressed granule cell epileptiform activity dependent on the recurrent mossy fiber pathway. These actions of NPY were mediated by activation of presynaptic Y2 receptors. The Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]argininamide (BIIE0246) not only blocked the effects of NPY but also enhanced recurrent mossy fiber synaptic transmission, the frequency of mEPSCs, and the magnitude of mossy fiber-evoked granule cell epileptiform activity when applied by itself. Several observations supported the selectivity of BIIE0246. These results suggest that even the spontaneous release of NPY (or an active metabolite) from recurrent mossy fibers is sufficient to depress glutamate release from this pathway. Tonic release of NPY accounts at least partially for the low probability of glutamate release from recurrent mossy fiber terminals, impedes the ability of these fibers to synchronize granule cell discharge, and may protect the hippocampus from seizures that involve the entorhinal cortex. This pathway may synchronize granule cell discharge more effectively in human brain than in rat because of its lower expression of NPY.

The recurrent mossy fiber pathway of the epileptic brain.

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K+]o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1-2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies < 1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.

Short-term frequency-dependent plasticity at recurrent mossy fiber synapses of the epileptic brain.

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in human temporal lobe epilepsy and in animal models of this disorder, creating monosynaptic connections among granule cells. This novel granule cell network can support reverberating excitation but is difficult to activate with low-frequency stimulation. This study used hippocampal slices from pilocarpine-treated rats to explore the dependence of synaptic transmission in this pathway on stimulus frequency. Minimal electrically evoked EPSCs exhibited a high failure rate ( approximately 60%). Stimulus trains delivered at a frequency of <1 Hz depressed synaptic transmission, as evidenced by an increase in response failures. Conversely, stimulus trains delivered at higher frequencies reduced the percentage of response failures and increased the amplitude of compound EPSCs, including pharmacologically isolated NMDA receptor-mediated EPSCs. Short-term frequency-dependent facilitation was of modest size compared with mossy fiber synapses on other neuronal types. Facilitation depended on the activation of kainate receptors by released glutamate and was inhibited by feedback activation of type II metabotropic glutamate receptors. These results suggest that the recurrent mossy fiber pathway may be functionally silent during baseline asynchronous granule cell activity in vivo attributable, in part, to progressive transmission failure. The pathway may synchronize granule cell firing and may promote seizure propagation most effectively during the brief periods of high-frequency granule cell firing that occur during normal behavior, during the periods of hypersynchronous fast activity characteristic of epileptic brain and, most importantly, during the period of increasing granule cell activity that precedes a spontaneous seizure.

Quantitative and morphological analysis of dentate granule cells with recurrent basal dendrites from normal and epileptic rats.

Granule cells with recurrent basal dendrites (RBDs) were previously reported in both control and epileptic rats. RBDs are dendrites that arise from the basal half of granule cell bodies and curve toward and extend into the molecular layer. They are increased in frequency in the pilocarpine model of epilepsy. The present study was undertaken to analyze the distribution and morphology of granule cells with RBDs and the synaptic connections of RBDs. Granule cells were labeled by retrograde transport of biocytin. Those with an RBD were found throughout the granule cell layer, but were most numerous at the hilar border. The morphology of these cells varied in the different depths of the granule cell layer; the angle of their cell body's long axis was mainly vertical at the hilar margin, and changed to virtually horizontal close to the molecular layer border. Quantitative data on the distribution of granule cells with RBDs and the angle of the cell body's long axis confirmed these descriptions. At the electron microscopic level, RBDs showed the typical features of dendrites and formed numerous axodendritic and axospinous synapses with labeled and unlabeled axon terminals. These results showed that RBDs of granule cells from epileptic rats are postsynaptic to axon terminals, including mossy fibers, and thus are involved in a similar synaptic circuitry as apical dendrites of granule cells from these animals.