Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

NMDA-dependent currents in granule cells of the dentate gyrus contribute to induction but not permanence of kindling

Single-electrode voltage-clamp techniques and bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) were used to study the time course of seizure-induced alterations in NMDA-dependent synaptic currents in granule cells of the dentate gyrus in hippocampal slices from kindled and normal rats. In agreement with previous studies, granule cells from kindled rats examined within 1 wk after the last of 3 or 30-35 generalized tonic-clonic (class V) seizures demonstrated an increase in the NMDA receptor-dependent component of the perforant path-evoked synaptic current. Within 1 wk of the last kindled seizure, NMDA-dependent charge transfer underlying the perforant path-evoked current was increased by 63-111% at a holding potential of -30 mV. In contrast, the NMDA-dependent component of the perforant-evoked current in granule cells examined at 2.5-3 mo after the last of 3 or 90-120 class V seizures did not differ from age-matched controls. Because the seizure-induced increases in NMDA-dependent synaptic currents declined toward control values during a time course of 2.5-3 mo, increases in NMDA-dependent synaptic transmission cannot account for the permanent susceptibility to evoked and spontaneous seizures induced by kindling. The increase in NMDA receptor-dependent transmission was associated with the induction of kindling but was not responsible for the maintenance of the kindled state. The time course of alterations in NMDA-dependent synaptic current and the dependence of the progression of kindling and kindling-induced mossy fiber sprouting on repeated NMDA receptor activation are consistent with the possibility that the NMDA receptor is part of a transmembrane signaling pathway that induces long-term cellular alterations and circuit remodeling in response to repeated seizures, but is not required for permanent seizure susceptibility in circuitry altered by kindling.     

Epileptogenesis is associated with enhanced glutamatergic transmission in the perforant path

The perforant path provides the main excitatory input into the hippocampus and has been proposed to play a critical role in the generation of temporal lobe seizures. It has been hypothesized that changes in glutamatergic transmission in this pathway promote the epileptogenic process and seizure generation. We therefore asked whether epileptogenesis is associated with enhanced glutamatergic transmission from the perforant path to dentate granule cells. We used a rat model of temporal lobe epilepsy in which spontaneous seizures occur after an episode of pilocarpine-induced status epilepticus. Whole cell patch-clamp recordings were obtained from dentate granule cells in hippocampal slices from control and epileptic animals 3 wk after pilocarpine-induced status epilepticus. The paired pulse ratio of perforant path-evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) was reduced in tissue obtained from epileptic rats. This is consistent with an increase in release probability. N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs were also prolonged. This prolongation could not be accounted for by decreased activity of glutamate transporters or by a change in NMDA receptor subunit composition in dentate granule cells, implying a change in NMDA receptor kinetics. This change in NMDA receptor kinetics was associated with the emergence of significant synaptic cross-talk, detected as a use-dependent block of receptors activated by medial perforant path synapses after lateral perforant path stimulation in MK-801. Enhanced glutamatergic transmission and the emergence of cross-talk among perforant path-dentate granule cell synapses may contribute to lowering seizure threshold.     

Intracellular responses from granule cell layer in slices of rat hippocampus: perforant path synapse.

Intracellular responses were recorded in vitro from the denate granule cell layer of hippocampal slices prepared from adult rats. Spontaneous activity of granule cells in vitro consisted of action potentials and small, graded depolarizations, presumably of synaptic origin. Granule cells could be activated by injection of depolarizing current or release of hyperpolarizing current. Individual granule cells spatially summed input from the perforant path and fired multiple action potentials in vitro following strong presynaptic volleys. Depolarization decreased and hyperpolarization increased the EPSP amplitude, which is consistent with a conductance-increase mechanism. Although we could demonstrate postexcitatory inhibition in some cells, granule cells in vitro appeared to receive less inhibitory feedback than in vivo, EPSP amplitude and spike output of granule cells showed frequency potentiation and posttetanic potentiation to perforant path stimulation. These intracellular responses in vitro complement some of the findings from field-potential analyses of the dentate gyrus in intact animals.     

Action of norepinephrine in the dentate gyrus. I. Stimulation of locus coeruleus

Summary The effect of stimulating locus coeruleus (LC) on the response of dentate gyrus granule cells to medial perforant pathway stimulation was studied in anesthetized rats. Field responses were recorded simultaneously at the mid-dendritic and granule cell levels. Two types of responses were recorded: those due to the synchronous firing of granule cell action potentials (population spikes) and those produced by excitatory synaptic activity (evoked synaptic potentials or ESPs). Stimulation of LC prior to stimulating the perforant pathway resulted in a decrease in the ESP (inward current) measured at the dendrites and, in most animals, an increase of the population spike measured at the granule cell level. Although LC stimulation decreased the ESP at the dendrites, the ESP at the granule cell body level (outward current) was not affected. The changes in granule cell responses following LC stimulation are discussed in relation to previous findings in freely moving rats.     

Commissural and perforant path interactions in the rat hippocampus

Summary The interaction of the commissural and perforant path systems was studied by recording extracellular field potentials and single unit activity in the dentate gyrus in urethane-anesthetized rats. Conditioning commissural volleys suppressed extracellular synaptic potentials, population spikes and single unit discharges evoked by perforant path stimulation. Commissural stimulation (single or repetitive) failed to induce a population spike, however strong the stimulation. About half of the cells fired monosynaptically to perforant path volleys and 20% to commissural volleys. Half of the commissurally driven units fired before or coincided with field potential onset. The antidromic mechanism of these short latency unitary spikes was shown by the collision test. Commisural activation reduced spontaneous cell firing without previous excitation in 25% of the neurons. Less than 6% of the cells responded to stimulation of both inputs, indicating little convergence between the two pathways. We contend that a simple form of recurrent inhibition fails to explain the above findings, and the possibility of feed-forward inhibition by commissural activation has been raised.     

Laminar profiles of the changes in extracellular calcium concentration induced by repetitive stimulation and excitatory amino acids in the rat dentate gyrus

Changes in extracellular free calcium concentration ([Ca2+]o) were measured with ion-selective microelectrodes in the dentate gyrus of the rat hippocampal slice preparation. Repetitive stimulation (20 Hz/10 s) of the perforant path or mossy fibers, or alternatively, iontophoresis of the excitatory amino acids N-methyl-D-aspartate (NMDA) or quisqualate (Quis) elicited decreases in [Ca2+]o (delta Ca2+) which were unequally distributed along the axis of the granule cells. Laminar profiles of the [Ca2+]o changes revealed marked differences between stimulus- and amino acid-induced responses. The delta Ca2+ induced by either anti- or orthodromic stimulation were relatively small (less than 0.15 mM) and were found to be maximal at the cell body layer (stratum granulosum). In contrast, the excitatory amino acids NMDA and Quis evoked large delta Ca2+ (greater than 1.2 mM) which were maximal at dendritic sites, 100 microns away from the cell body layer in the inner stratum moleculare. The effect of NMDA was reversibly blocked by 2-amino-5-phosphonovaleric acid without antagonism of the synaptic responses or the stimulus-induced changes in [Ca2+]o. Therefore, under normal conditions, NMDA receptors appear not to participate in synaptically induced delta Ca2+ in the dentate gyrus.     

Evoked responses of the dentate gyrus during seizures in developing gerbils with inherited epilepsy

When they are 1-2 mo old, domesticated Mongolian gerbils begin having initially mild seizures which become more severe with age. To evaluate the development of this increasing seizure severity, we obtained field potential responses of the dentate gyrus to paired-pulse stimulation of the perforant path during seizures. In 18 gerbils that were 1.5-8.0 mo old, 73 seizures were analyzed. We measured population spike amplitude, the slope of the field excitatory postsynaptic potential (fEPSP), and the population spike amplitude ratio (2nd/1st) to evaluate excitatory and inhibitory synaptic processes. In gerbils <2 mo old, exposure to a novel environment was followed by an increase in population spike amplitude and then by seizure onset, but population spike amplitude ratio and fEPSP slope remained at baseline levels, and multiple population spikes were never evoked. As previously reported for chronically epileptic gerbils, these findings provide little evidence of a disinhibitory seizure-initiating mechanism in the dentate gyrus when young gerbils begin having seizures. In young gerbils evoked responses changed little during the behaviorally mild seizures. In contrast, most seizures in older gerbils included generalized convulsions, postictal depression, and evoked responses that changed dramatically. In older gerbils, shortly after seizure onset the dentate gyrus became hyperexcitable. Population spike amplitude and fEPSP slope peaked, and multiple population spikes were evoked, suggesting that mechanisms for seizure amplification and spread are more developed in older gerbils. Next, dentate gyrus excitability decreased precipitously, and population spike amplitude and fEPSP slope diminished. This period of hypoexcitability began before the end of the seizure, suggesting it may contribute to seizure termination. After the convulsive phase of the seizure, older gerbils remained motionless during a period of postictal depression, and population spike amplitude remained suppressed until the abrupt switch to normal exploratory activity. These findings suggest that the mechanisms of postictal depression may suppress granule cell excitability. The population spike amplitude ratio peaked after the convulsive phase and then gradually returned to the baseline level an average of 12 min after seizure onset, suggesting that granule cell inhibition recovers within minutes after a spontaneous seizure. Although it is unclear whether the seizure-related changes in evoked responses are a cause or an effect of increased seizure severity in older gerbils, their analysis provides clues about developmental changes in the mechanisms of seizure spread and termination.     

Electrophysiology of dentate gyrus granule cells

The orthodromic synaptic responses, membrane properties, and responses of dentate gyrus granule cells (DGCs) to several convulsant agents were studied in the in vitro hippocampal slice preparation. Orthodromic stimulation via the perforant pathway (PP) evoked excitatory-inhibitory postsynaptic potentials (EPSP-IPSP) sequences in 27 of 34 DGCs studied. In the majority, only one action potential could be evoked by supramaximal orthodromic stimulation. In recordings from DGC somata, overshooting spikes could be evoked either orthodromically or by current injections. Small-amplitude, fast transients were seen in 5 of 34 DGCs. The current/voltage (I-V) characteristic of most DGCs was linear throughout a range of membrane potentials between 15 and 20 mV negative and 5 and 15 mV positive to the resting potential. At the extremes of this range nonohmic behavior was noted. Exposure of slices to agents that block IPSPs, such as penicillin, bicuculline, picrotoxin, and media containing low Cl- concentrations, eliminated PP-evoked hyperpolarizations in DGCs and prolonged the repolarizing phase of the PP EPSP. In contrast to findings in hippocampal pyramidal cells and neocortical neurons, blockade of IPSPs did not lead to the development of orthodromically evoked slow depolarizations and burst discharges. After slices were exposed to 5 mM tetraethylammonium, current pulses evoked slow spikes, which were resistant to tetrodotoxin and presumably mediated by Ca2+. Spontaneous burst discharges or bursts evoked by brief depolarizing pulses did not occur under these conditions. Substitution of Ba2+ for Ca2+ in the perfusion solution resulted in development of spontaneous slow membrane depolarizations and burst discharges in DGCs. Burst discharges could be directly evoked and spikes were prolonged and resistant to tetrodotoxin (TTX). After hyperpolarizations lasting 200-1,000 ms, associated with a conductance increase and presumably due to a Ca2+-activated K+ conductance, followed directly evoked spike trains in 5 of 20 DGCs. These data suggest that Ca2+ conductances may be evoked in DGCs under certain circumstances but are not prominent during activation of DGCs under standard in vitro recording conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of excitation reveals inhibition of dentate spiny hilar neurons recorded in rat hippocampal slices.

1. Extracellular and intracellular recordings in rat hippocampal slices were used to compare the synaptic responses to perforant path stimulation of granule cells of the dentate gyrus, spiny "mossy" cells of the hilus, and area CA3c pyramidal cells of hippocampus. Specifically, we asked whether aspects of the local circuitry could explain the relative vulnerability of spiny hilar neurons to various insults to the hippocampus. 2. Spiny hilar cells demonstrated a surprising lack of inhibition after perforant path activation, despite robust paired-pulse inhibition and inhibitory postsynaptic potentials (IPSPs) in adjacent granule cells and area CA3c pyramidal cells in response to the same stimulus in the same slice. However, when the slice was perfused with excitatory amino acid antagonists [6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), or CNQX with 2-amino-5-phosphonovaleric acid (APV)], IPSPs could be observed in spiny hilar cells in response to perforant path stimulation. 3. The IPSPs evoked in spiny hilar cells in the presence of CNQX were similar in their reversal potentials and bicuculline sensitivity to IPSPs recorded in dentate granule cells or hippocampal pyramidal cells in the absence of CNQX. 4. These results demonstrate that, at least in slices, perforant path stimulation of spiny hilar cells is primarily excitatory and, when excitation is blocked, underlying inhibition can be revealed. This contrasts to the situation for dentate and hippocampal principal cells, which are ordinarily dominated by inhibition, and only when inhibition is compromised can the full extent of excitation be appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enkephalin enhances responsiveness to perforant path input while decreasing spontaneous activity in the dentate gyrus

The opioid peptide [D-Ala2-Met5]-enkephalin-amide (DAMEA) was electrophoretically applied to the dentate gyrus in vivo to investigate opioid effects on evoked responsiveness to perforant path input and on spontaneous granule cell activity. DAMEA increased the amplitude of population spikes evoked by stimulation of the perforant path, and decreased sequential (recurrent or feed-forward) inhibition as determined by a paired-pulse paradigm. However, DAMEA concurrently inhibited spontaneous activity of individual granule cells. The effects of DAMEA on population and unit responses were antagonized by intravenous naloxone. These results confirm previous findings of opioid enhanced responsiveness to perforant path input and decreased sequential inhibition, but demonstrate a paradoxical inhibition of spontaneous unit activity by opioids in the granule cell layer.     

Morphine augments excitatory synaptic transmission in the dentate gyrus through GABAergic disinhibition

The present study investigated the effect of morphine on synaptic transmission and long-term potentiation (LTP) in the dentate gyrus using rat hippocampal slice preparations. Field excitatory postsynaptic potential (fEPSP) and population spike (PS), evoked by stimulation of the perforant path, were recorded from the dentate molecular layer and the stratum granulosum, respectively. Following application of 10 microM morphine, PS amplitude increased gradually in 10 min and was eventually potentiated by approximately 50%. The phenomenon showed a concentration-dependent manner and was completely canceled by naloxone, a mu opioid receptor antagonist. Furthermore, morphine-induced PS augmentation was not detected in disinhibited hippocampal slices, which suggests that the inhibitory input to the dentate granule cells was required for the facilitatory effect of morphine. Neither fEPSP nor tetanus-induced LTP of PS was altered by morphine application. The data support the hypothesis that mu opioid receptor activity modulates inhibitory recurrent circuits in the dentate gyrus and thereby, indirectly plays a regulatory role for hippocampal excitatory neurotransmission.     

Effect of adrenalectomy on membrane properties and synaptic potentials in rat dentate granule cells

Adrenalectomy is known to accelerate both neurogenesis and cell death of granule cells located in the suprapyramidal blade of the rat dentate gyrus. Three days after adrenalectomy, some granule cells have already died by apoptosis while newly formed cells are not yet incorporated in the cell layer, resulting in a temporary loss of granule cells. Concomitantly, the field response to stimulation of perforant path afferents is reduced. While the temporary cell loss is likely to attenuate synaptic field responses, adrenalectomy-induced changes in properties of the surviving cells may also contribute to the reduction in field response amplitude. To address this possibility, we here investigated the membrane properties and synaptic responses of dentate granule cells, 3 days after adrenalectomy. We found that passive and most of the active membrane properties of granule cells in adrenalectomized rats were not significantly different from the cell properties in sham-operated controls. However, intracellularly recorded synaptic responses from surviving granule cells were markedly reduced after adrenalectomy. The N-methyl-D-aspartate (NMDA)- and the non-NMDA receptor-mediated components were reduced to a similar extent, suggesting that the attenuation of synaptic transmission after adrenalectomy could be partly of presynaptic origin. The data indicate that the earlier observed attenuation of synaptic field responses after adrenalectomy may be partly due to a diminished glutamatergic input to the dentate gyrus and not exclusively to a loss of granule cells participating in the synaptic circuit.     

Functional consequences of the lack of amyloid precursor protein in the mouse dentate gyrus in vivo

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. Here, we studied whether the lack of APP affects the synaptic properties in the dentate gyrus by measuring granule cell field potentials evoked by perforant path stimulation in anesthetized 9-11-month-old APP-deficient mice in vivo. We found decreased paired-pulse facilitation, indicating altered presynaptic short-term plasticity in the APP-deficient dentate gyrus. In contrast, excitatory synaptic strength and granule cell firing were unchanged in APP knockout mice. Likewise, long-term potentiation (LTP) induced by a theta-burst stimulation protocol was not impaired in the absence of APP. These findings suggest that the deletion of APP may affect presynaptic plasticity of synaptic transmission at the perforant path-granule cell synapse but leaves synaptic efficacy intact and LTP preserved, possibly due to functional redundancy within the APP gene family.     

Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures

Granule cells in the dentate gyrus are born throughout life, and various stimuli can affect their development in the adult brain. Following seizures, for instance, neurogenesis increases greatly, and some new cells migrate to abnormal (ectopic) locations, such as the hilus. Previous electrophysiological studies of this population have shown that they have intrinsic properties that are similar to normal granule cells, but differ in other characteristics, consistent with abnormal integration into host circuitry. To characterize the response of ectopic hilar granule cells to perforant path stimulation, intracellular recordings were made in hippocampal slices from rats that had pilocarpine-induced status epilepticus and subsequent spontaneous recurrent seizures. Comparisons were made with granule cells located in the granule cell layer of both pilocarpine- and saline-treated animals. In addition, a few ectopic hilar granule cells were sampled from saline-treated rats. Remarkably, hilar granule cells displayed robust responses, even when their dendrites were not present within the molecular layer, where perforant path axons normally terminate. The evoked responses of hilar granule cells were similar in several ways to those of normally positioned granule cells, but there were some differences. For example, there was an unusually long latency to onset of responses evoked in many hilar granule cells, especially those without molecular layer dendrites. Presumably this is due to polysynaptic activation by the perforant path. These results indicate that synaptic reorganization after seizures can lead to robust activation of newly born hilar granule cells by the perforant path, even when their dendrites are not in the terminal field of the perforant path. Additionally, the fact that these cells can be found in normal tissue and develop similar synaptic responses, suggests that seizures, while not necessary for their formation, strongly promote their generation and the development of associated circuits, potentially contributing to a lowered seizure threshold.     

Activation of N-methyl-D-aspartate receptors contributes to the EPSP at perforant path synapses in the rat dentate gyrus in vitro

The excitatory postsynaptic potential (EPSP) evoked in the granule cells of the rat dentate gyrus following low frequency stimulation of the perforant path has been investigated using intracellular recording. The EPSP was reduced by low microM concentrations of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). A small CNQX-resistant component of the EPSP remained. This could be blocked by the NMDA receptor antagonist (+/-)-2-amino-5-phosphonovalerate, was enhanced in Mg2+-free medium and showed a potential-dependency characteristic of the activation of NMDA ionophores. These results demonstrate that NMDA receptors contribute to the EPSP in the granule cell.     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Somatostatin depresses long-term potentiation and Ca2+ signaling in mouse dentate gyrus

The selective loss of somatostatin (SST)-containing interneurons from the hilus of the dentate gyrus is a hallmark of epileptic hippocampus. The functional consequence of this loss, including its contribution to postseizure hyperexcitability, remains unclear. We address this issue by characterizing the actions of SST in mouse dentate gyrus using electrophysiological techniques. Although the majority of dentate SST receptors are located in the outer molecular layer adjacent to lateral perforant path (LPP) synapses, we found no consistent action of SST on standard synaptic responses generated at these synapses. However, when SST was present during application of high-frequency trains that normally generate long-term potentiation (LTP), the induction of LTP was impaired. SST did not alter the maintenance of LTP when applied after its induction. To examine the mechanism by which SST inhibits LTP, we recorded from dentate granule cells and examined the actions of this neuropeptide on synaptic transmission and postsynaptic currents. Unlike findings in the CA1 hippocampus, we observed no postsynaptic actions on K(+) currents. Instead, SST inhibited Ca(2+)/Ba(2+) spikes evoked by depolarization. This inhibition was dependent on N-type Ca(2+)currents. Blocking these currents also blocked LTP, suggesting a mechanism through which SST may inhibit LTP. Our results indicate that SST reduction of dendritic Ca(2+) through N-type Ca(2+) channels may contribute to modulation of synaptic plasticity at LPP synapses. Therefore the loss of SST function postseizure could result in abnormal synaptic potentiation that contributes to epileptogenesis.     

Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus.We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the axon loses parvalbumin or immunoreactivity for parvalbumin. The hyperinnervation of axon initial segments might be a compensatory change in the inhibitory network, but at the same time may lead to a more effective synchronization of granule cell firing that could contribute to the generation or amplification of epileptic seizures.