NMDA receptor activation during epileptiform responses in the dentate gyrus of epileptic patients.

We previously showed that a low frequency (1 Hz) train of perforant path stimulation evokes burst discharges in the dentate gyrus of hippocampal slices obtained from patients surgically treated for intractable temporal lobe epilepsy. We report here that multiple population spikes that characterize the burst discharge are blocked reversibly by the specific NMDA receptor antagonist, D-(-)-2-amino-5-phosphonovaleric acid (D-APV). The epileptiform discharge evoked in human dentate gyrus by stimulation trains of 1 Hz could be reproduced in the rat dentate gyrus in vitro by the same stimulation protocol but required the presence of low concentrations (0.2-0.6 mM) of extracellular magnesium. We suggest that low frequency orthodromic stimulation of dentate granule cells through the perforant path progressively evokes an increase in the activation of NMDA receptors resulting in burst discharges in tissue from epileptic patients.     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

Effects of pentetrazol on neuronal activity and on extracellular calcium concentration in rat hippocampal slices

Effects of pentetrazol (PTZ) were studied on neuronal responses in dentate granule cells and area CA1 hippocampal pyramidal cells with intra- and extracellular recording techniques. PTZ induced spontaneous epileptiform field potential transients in areas CA3 and CA1, but not in the dentate gyrus. The concentration optimum for induction of spontaneous epileptiform activity was 2 mM. The epileptiform activity compared in many respects to that induced by GABA antagonists such as picrotoxin, bicuculline and penicillin. Paired pulse stimulus induced responses were affected by concentrations of 0.5 mM. In the concentration range 0.5-2 mM mostly disinhibitory effects were noted. Stimulus induced Ca2+ concentration changes were found to be maximally augmented at concentrations of 2-5 mM. In this range, intracellular studies revealed a block of frequency habituation and an increase in input resistance. The convulsant action of PTZ decreased at concentrations above 5 mM, probably due to a decrease of inward currents. We suggest that the action of PTZ in screening studies for anticonvulsants is mostly due to a decrease of GABAA-receptor mediated IPSPs.     

Spread of Excitation in Chronically Lesioned Mouse Hippocampus Determined by Laser Scanning Microscopy

Fast optical recordings by means of laser scanning microscopy in conjunction with a voltage-sensitive dye (RH 414) were performed to monitor the spatio-temporal spread of neuronal activity in CA3/CA4-lesioned C57BL6 mouse hippocampal slices prepared approximately 3 months after intracerebroventricular kainic acid (KA) injection. The aim of our study was to assess the effects of a circumscribed neuronal loss on the propagation of electrical activity along the trisynaptic hippocampal circuit. Both in physiological bathing solution and in bicuculline (10 μM), hilar stimulation failed to activate the downstream pathway, so that, under these conditions, the chronically disinhibited CA1 region appeared to be effectively isolated from burst activity arising upstream; however, epileptiform discharges evoked in zero Mg²⁺solution were reliably transmitted from the dentate gyrus to the CA1 region. That these bursts were indeed spreading across the lesion, and not along newly formed connections (e.g., between dentate gyrus and CA1), was confirmed by acute transection experiments of the Schaffer collateral/commissural pathway, which completely abolished translesional burst propagation. The fact that the surviving CA3–CA1 connections are unable to trigger epileptiform bursts after suppression of GABAergic inhibition suggests that the lesioned region might serve as a filter that shields hyperexcitable CA1 neurons from epileptic activity arising upstream, in particular from chronically disinhibited granule cells of the dentate gyrus. An impaired GABAergic inhibition will thus only have minor facilitating effects on seizure propagation in the hippocampus of CA3-lesioned animals.     

Activity-dependent induction and maintenance of epileptiform activity produced by group I metabotropic glutamate receptors in the rat hippocampal slice

Activation of group I metabotropic glutamate receptors (mGluRs) produces a long-lasting change in hippocampal excitability that persists in the absence of an agonist. Exposure to the group I mGluR agonist dihydroxyphenylglycine (DHPG) results in the induction of spontaneously occurring epileptiform activity in the CA3 region of rat hippocampal slices that includes both brief interictal discharges and longer synchronous activity that resembles seizure or ictal activity (>2s duration oscillating at a frequency greater than 2Hz). We evaluated activity-dependent mechanisms for the induction and maintenance of epileptiform activity. Both the induction and maintenance of epileptiform activity was blocked by inhibiting action potential generation with tetrodotoxin or substitution of sodium with choline or by blocking AMPA/KA ionotropic glutamate receptors. The ictal epileptiform activity induced by DHPG was composed of synchronous synaptic activity. Antagonists of group I mGluRs, either mGluR1 or mGluR5, suppressed the induction of ictal activity but had minimal effects on the maintenance of epileptiform activity. Group I mGluRs activate phospholipase C and inhibition of phospholipase C suppressed the induction but not the maintenance of epileptiform activity. Taken together, these results point to a use dependent change in CA3 neuronal network function produced by group I mGluR activation. Furthermore, activation of both mGluR1 and 5 is required to induce ictal discharges. The induction of epileptiform activity by DHPG is an in vitro model of epileptogenesis, and the development of epileptiform activity in this model depends on neuronal activity and synaptic transmission.     

Regulation of synaptic currents by mGluR2 at reciprocal synapses in the mouse accessory olfactory bulb

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23‐ to 36‐day‐old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from –70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin‐perforated or conventional whole‐cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG‐IV [(2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)‐α‐amino‐α‐[(1S,2S)‐2‐carboxycyclopropyl]‐9H‐xanthine‐9‐propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG‐IV and LY341495 on dendrodendritic inhibition. DCG‐IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG‐IV inhibited high‐voltage‐activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.     

Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus

Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. To assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous γ-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units.     

Long-term maintenance of mature hippocampal slices in vitro

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin, a GABAergic Cl(-) channel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CA1 region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity, and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function.     

Glutamate receptor involvement in dentate granule cell epileptiform activity evoked by mossy fiber stimulation

In many persons with temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network. This recurrent mossy fiber circuit mediates reverberating excitation that may facilitate seizure propagation by synchronizing granule cell discharge. The involvement of specific glutamate receptors in granule cell epileptiform activity evoked by stimulating the mossy fibers was investigated with use of rat hippocampal slices superfused with bicuculline, with or without increasing [K+](o) to 6 mM. The occurrence of short-latency mossy fiber-evoked granule cell epileptiform activity in slices from pilocarpine-treated rats correlated with the presence and extent of recurrent mossy fiber growth. Blockade of AMPA receptors nearly abolished the orthodromic component of the response; subsequent antagonism of kainate receptors as well appeared to have no further action. Antagonism of NMDA receptors reduced the duration of epileptiform discharge, but increased the amplitude of population spikes within the evoked burst. Thus AMPA and NMDA, but perhaps not kainate, receptors play an important role in this type of epileptiform activity. Activation of type II metabotropic glutamate receptors, which inhibits the release of glutamate from mossy fiber boutons, reduced the magnitude of epileptiform discharge. This action was reversed by a partial agonist of these receptors. However, neither an agonist nor an antagonist of type III metabotropic glutamate receptors significantly altered the response. Considering the importance of synchronous granule cell discharge for seizure propagation from the entorhinal cortex to the hippocampus, agonists of type II metabotropic glutamate receptors may be useful in suppressing such discharge both experimentally and clinically.     

Interneuron subtype specific activation of mGluR1/5 during epileptiform activity in hippocampus

Purpose: Specific inhibitory interneurons in area CA1 of the hippocampus, notably those located in stratum oriens–alveus (O/A‐INs), are selectively vulnerable in patients and animal models of temporal lobe epilepsy (TLE). The excitotoxic mechanisms underlying the selective vulnerability of interneurons have not been identified but could involve group I metabotropic glutamate receptor subtypes (mGluR1/5), which have generally proconvulsive actions and activate prominent cationic currents and calcium responses specifically in O/A‐INs. Methods: In this study, we examine the role of mGluR1/5 in interneurons during epileptiform activity using whole‐cell recordings from CA1 O/A‐INs and selective antagonists of mGluR1α (LY367385) and mGluR5 (MPEP) in a disinhibited rat hippocampal slice model of epileptiform activity. Results: Our data indicate more prominent epileptiform burst discharges and paroxysmal depolarizations (PDs) in O/A‐INs than in interneurons located at the border of strata radiatum and lacunosum/moleculare (R/LM‐INs). In addition, mGluR1 and mGluR5 significantly contributed to epileptiform responses in O/A‐INs but not in R/LM‐INs. Epileptiform burst discharges in O/A‐INs were partly dependent on mGluR5. PDs and associated postsynaptic currents were dependent on both mGluR1α and mGluR5. These receptors contributed differently to postsynaptic currents underlying PDs, with mGluR5 contributing to the fast and slow components and mGluR1α to the slow component. Discussion: These findings support interneuron subtype‐specific activation and differential contributions of mGluR1α and mGluR5 to epileptiform activity in O/A‐INs, which could be important for their selective vulnerability in TLE.     

Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures

Generation of dentate granule cells and its modulation by glutamate receptor antagonists, growth factors and pilocarpine-induced seizure-like activity was investigated in rat hippocampal slice cultures derived from 1-week-old rats and grown for 2 weeks. Focussing on the dentate granule cell layer facing CA1 and the immediate subgranular zone, exposure for 3 days to the NMDA receptor blocking agents MK-801 (10 microM) or APV (25 microM) in the culture medium, increased the number of TOAD-64/Ulip/CRMP-4 (TUC-4)-positive cells as counted in the slice cultures at the end of the 3-day treatment period. Exposure to IGF-I (200 ng/ml) and EGF (20 ng/ml) also increased the number of TUC-4-positive cells. Combining APV with IGF-I/EGF had an additive effect. Similar results were obtained by 3 days treatment with the AMPA receptor antagonist CNQX (25 microM). Surprisingly, addition of 5 mM pilocarpine reduced the number of TUC-4-positive cells, just as combining pilocarpine with the neurogenesis-stimulating compounds, prevented or reduced the increase of TUC-4-positive cells. None of the treatments were found to induce dentate granule cell death within the observed period. Labeling of dividing cells by adding 5-bromo-2-deoxyuridine (BrdU) to the culture medium did not result in cells double-labeled with BrdU and TUC-4. The induced increase in TUC-4-positive cells therefore represent neuronal differentiation of existing neural precursor cells when investigated at the 3-day time point. We conclude that 3 days treatment of 2-week-old hippocampal slice cultures with IGF-I and EGF and NMDA and AMPA glutamate receptor antagonists increase granule cell neurogenesis from preexisting neural precursors.     

Group I metabotropic glutamate receptor activation produces prolonged epileptiform neuronal synchronization and alters evoked population responses in the hippocampus

Glutamate activates a class of receptors coupled to G-proteins that initiate second messenger cascades, change ion channel function, cause release of calcium from intracellular stores, and produce long-term changes in synaptic strength. We used the CA3 region of the adult rat hippocampal slice to evaluate group I metabotropic glutamate receptor (mGluR) activation on epileptiform activity and the population response recorded extracellularly evoked by stratum radiatum stimulation. The selective group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) accelerated the rate of bicuculline-induced interictal discharges at concentrations of 10 and 30 microM. At a concentration of 100 microM, DHPG produced prolonged recurrent discharges that last more than 2s and consisted of an oscillation of the field potential at 2-20 Hz that resembled electrographic seizure activity (ictal). DHPG (100 microM) when bath-applied alone for 30-120 min produced both ictal and interictal discharges that persisted following removal of DHPG from the bathing solution. DHPG (100 microM) reduced the amplitude of the first population spike evoked by stratum radiatum stimulation and changed the relationship of paired evoked population spikes from suppression of the second response relative to the first to facilitation of the second response at interpulse intervals of 15 and 25 ms. To test the possibility that a reduction of the first evoked population spike and loss of inhibition of a second evoked population spike generated prolonged ictal discharges, we used 4-aminopyridine (4-AP 50 microM) to enhance synaptic transmission. 4-AP converted ictal discharges produced by DHPG to an interictal pattern of synchronous activity, reversed the DHPG-induced reduction in the first evoked population spike, and changed paired-pulse facilitation to inhibition. Reversing the changes of evoked population neuronal activity produced by group I mGluR activation favored interictal patterns of epileptiform activity. These results confirm that group I mGluR activation promotes epileptiform activity in the hippocampus and support the hypothesis that a lower efficacy of synaptic transmission favors the generation of prolonged synchronization of neurons that underlies seizures.     

Hippocampal granule cell activity and c-Fos expression during spontaneous seizures in awake, chronically epileptic, pilocarpine-treated rats: implications for hippocampal epileptogenesis

The process of postinjury hippocampal epileptogenesis may involve gradually developing dentate granule cell hyperexcitability caused by neuron loss and synaptic reorganization. We tested this hypothesis by repeatedly assessing granule cell excitability after pilocarpine-induced status epilepticus (SE) and monitoring granule cell behavior during 235 spontaneous seizures in awake, chronically implanted rats. During the first week post-SE, granule cells exhibited diminished paired-pulse suppression and decreased seizure discharge thresholds in response to afferent stimulation. Spontaneous seizures often began during the first week after SE, recruited granule cell discharges that followed behavioral seizure onsets, and evoked c-Fos expression in all hippocampal neurons. Paired-pulse suppression and epileptiform discharge thresholds increased gradually after SE, eventually becoming abnormally elevated. In the chronic epileptic state, interictal granule cell hyperinhibition extended to the ictal state; granule cells did not discharge synchronously before any of 191 chronic seizures. Instead, granule cells generated only low-frequency voltage fluctuations (presumed "field excitatory postsynaptic potentials") during 89% of chronic seizures. Granule cell epileptiform discharges were recruited during 11% of spontaneous seizures, but these occurred only at the end of each behavioral seizure. Hippocampal c-Fos after chronic seizures was expressed primarily by inhibitory interneurons. Thus, granule cells became progressively less excitable, rather than hyperexcitable, as mossy fiber sprouting progressed and did not initiate the spontaneous behavioral seizures. These findings raise doubts about dentate granule cells as a source of spontaneous seizures in rats subjected to prolonged SE and suggest that dentate gyrus neuron loss and mossy fiber sprouting are not primary epileptogenic mechanisms in this animal model.     

Differential effects of the group II mGluR agonist, DCG-IV, on depolarization-induced suppression of inhibition in hippocampal CA1 and CA3 neurons

We investigated the role of metabotropic glutamate receptors in the mediation of depolarization-induced suppression of inhibition (DSI), using whole-cell electrophysiological techniques in rat hippocampal slice preparation. In a previous work, we showed that a retrograde signal travels from CA1 pyramidal cells to GABA interneurons and prevents them from releasing GABA for tens of seconds at 30 degrees C. The resulting suppression of inhibition is DSI. The retrograde signal appeared to be glutamate, or a glutamate analog, which acted on group I metabotropic receptors on the interneurons. It is not known if DSI occurs in hippocampal subregions besides CA1. If DSI does occur in other regions, it will be important to know if the role of metabotropic glutamate receptors (mGluRs) in mediating DSI is the same everywhere. The distribution of mGluR subtypes varies among hippocampal subregions. In the CA3 region, unlike CA1, group II mGluRs are prevalent. It was possible, therefore, that in CA3, the group II mGluRs would mediate DSI. We have begun to investigate these issues. We now report that: 1) DSI does occur in CA3. 2) Carbachol induces IPSC activity that can be recorded in CA1 and CA3a. This carbachol-induced activity can be reduced by the selective group II mGluR agonist, DCG-IV, and by DSI. 3) Evoked IPSCs in CA3a, but not in CA1, can be reduced by DCG-IV; hence the interneurons activated by carbachol may reside in CA3a. 4) Despite the group II mGluR agonist sensitivity of CA3a interneurons, DSI in this region is not affected by a group II mGluR antagonist, CPPG, and therefore does not appear to be mediated by group II mGluRs.     

Exposure of hippocampal slices to magnesium-free medium produces epileptiform activity and simultaneously decreases calcium and calmodulin-dependent protein kinase II activity.

The effect of magnesium-free medium on electrical and CaM kinase II activity in the rat hippocampal slice was examined. Experimental slices were incubated in 2 mM Mg, then exposed to magnesium-free medium for 1 h. Control slices were concurrently run in 2 mM Mg. Slices were then frozen and CaM kinase II activity was measured in homogenates. Exposure of hippocampal slices to magnesium-free medium resulted in spontaneous epileptiform activity and a concurrent 38 +/- 5.47% decrease in CaM kinase II activity (range 38.8-75.4% of control; n = 7, P less than 0.001, paired Student's t test). The decrease in CaM kinase II activity was not reversible by treatment with protein phosphatases 1 and 2A (58.8 +/- 4.77% of control activity; range 28.6-69.7, P less than 0.01, paired Student's t-test), indicating that the decrease in CaM kinase II activity cannot be accounted for exclusively by autophosphorylation. The results demonstrate that magnesium-free medium treatment can induce spontaneous epileptiform activity and simultaneous changes in CaM kinase II activity.     

Adenosine-mediated synaptic inhibition: partial blockade by barium does not prevent anti-epileptiform activity.

Adenosine-induced inhibition of evoked postsynaptic potentials (PSPs) and epileptiform burst firing in the CA1 subfield of rat hippocampal slices was studied with intracellular recordings in vitro. Adenosine (50 microM) caused a membrane hyperpolarization which was abolished during superfusion with 2 mM Ba2+. The adenosine-induced inhibition of the PSPs was still evident, although the magnitude of the effect was significantly reduced. Adenosine also reduced Ba(2+)-induced burst firing, but less effectively than it did bursts evoked by TEA (5 mM). The results suggest that adenosine inhibits synaptic transmission and epileptiform activity by at least 2 mechanisms: a postsynaptic barium-sensitive increase in gK and a presynaptic effect independent of this adenosine-evoked outward potassium conductance.     

Modulation of calcium channels by group I and group II metabotropic glutamate receptors in dentate gyrus neurons from patients with temporal lobe epilepsy

PURPOSE: Metabotropic glutamate receptors (mGluRs) might be promising new drug targets for the treatment of epilepsy because the expression of certain mGluRs is regulated in epilepsy and because activation of mGluRs results in distinctive anti- and proconvulsant effects. Therefore, we examined how mGluR activation modulates high-voltage-activated (HVA) Ca2+ channels. METHODS: Whole-cell patch-clamp recordings were obtained from granule cells and interneuron-like cells acutely isolated from the dentate gyrus of patients with pharmacoresistent temporal lobe epilepsy. RESULTS: Agonists selective for either group I or group II mGluRs rapidly and reversibly reduced HVA currents in most dentate gyrus cells. These modulatory effects were inhibited by the respective group I and group II mGluR antagonists. The specific Ca2+ channel antagonists nifedipine and omega-conotoxin GVIA potently occluded the effects of group I and II mGluR agonists, respectively, indicating that group I mGluRs acted on L-type channels and group II mGluRs affected N-type channels. About two thirds of the responsive neurons were sensitive either to group I or group II mGluRs, whereas a minority of cells showed effects to agonists of both groups, indicating a variable mGluR expression pattern. CONCLUSIONS: Group I and group II mGluRs are expressed in human dentate gyrus neurons and modulate L- and N-type HVA channels, respectively. The data shed light on the possible cellular sequelae of the mGluR1 upregulation observed in human epileptic dentate gyrus as well as on possible mGluR-mediated anticonvulsant mechanisms.     

Impact of protein kinase C activation on epileptiform activity in the hippocampal slice

There is evidence suggesting that protein kinase C (PKC) activation can prevent the enhanced network excitability associated with status epilepticus and group I metabotropic glutamate receptor (mGluR)-induced epileptogenesis. However, we observed no suppression of mGluR-induced burst prolongation in the guinea pig hippocampal slice when applied in the presence of the PKC activator phorbol-12,13-dibutyrate (PDBu). Furthermore, PDBu alone converted picrotoxin-induced interictal bursts into ictal-length discharges ranging from 2 to 6s in length. This effect could not be elicited by the inactive analog 4-alpha-PDBu and was suppressed with the PKC inhibitor chelerythrine, indicating PKC dependence. PKC activation can enhance neurotransmitter release, and both glutamate and acetylcholine are capable of eliciting similar prolonged synchronized discharges. However, neither mGluR1 nor NMDA receptor antagonist suppressed PDBu-driven burst prolongation, suggesting that increased glutamate release alone is unlikely to account for the PKC-induced expression of ictaform discharges. Similarly, atropine, a broad-spectrum muscarinic receptor antagonist, had no effect on PKC-induced burst prolongation. By contrast, AMPA/kainate receptor antagonist abolished PKC-induced burst prolongation, and mGluR5 antagonist significantly blunted the maximum burst length induced by PKC. These data suggest that PKC-induced prolongation of epileptiform bursts is dependent on changes specific to mGluR5 and AMPA/kainate receptors and not mediated simply by a generalized increase in transmitter release.     

Attenuation of epileptiform bursting by baclofen: Reduced potency in elevated potassium

Baclofen is an analog of the inhibitory neurotransmitter, GABA, which is used clinically to control spasticity. Recent evidence has accumulated showing this compound to have profound inhibitory effects upon hippocampal neural activity at both the cellular and circuit levels, and to attenuate epileptiform bursting in the hippocampal slice. However, it does not appear as an anticonvulsant on most traditional drug screens. Baclofen can produce inhibition by increasing potassium conductance, and therefore may fail to appear efficacious in typical anticonvulsant screens due to techniques that cause rapid and massive increases in interstitial potassium. We tested the hypothesis that baclofen is less effective at attenuating epileptiform bursting in the hippocampal slice under conditions of elevated extracellular potassium. Male Sprague-Dawley rats were decapitated and hippocampal slices were prepared. Epileptiform bursting was induced by bathing the slices in an artificial cerebrospinal fluid solution which contained either 7.0 mM K+ or 30 microM bicuculline methiodide, or by stimulus train-induced bursting. In each of these media, baclofen was applied in a random presentation of concentration format. Baclofen attenuated epileptiform bursting in both bicuculline and elevated K+, although considerably higher concentrations were necessary to attenuate bursting in high K+ than in bicuculline or after stimulus train-induced bursting. These results further support the antiepileptic actions of baclofen and provide evidence that this drug may be of value for attenuating epileptiform activity when there is not a tonic elevation of interstitial brain potassium.