Pathological neurons generate ripples at the UP-DOWN transition disrupting information transfer.

OBJECTIVE: To confirm and investigate why pathological high-frequency oscillations (pHFOs), including ripples (80-200 Hz) and fast ripples (200-600 Hz), are generated during the UP-DOWN transition of the slow wave and if information transmission mediated by ripple temporal coupling is disrupted in the seizure-onset zone (SOZ). METHODS: We isolated 217 total units from 175.95 intracranial electroencephalography (iEEG) contact-hours of synchronized macro- and microelectrode recordings from 6 patients. Sleep slow oscillation (.1-2 Hz) epochs were identified in the iEEG recording. iEEG HFOs that occurred superimposed on the slow wave were transformed to phasors and adjusted by the phase of maximum firing in nearby units (i.e., maximum UP). We tested whether, in the SOZ, HFOs and associated action potentials (APs) occur more often at the UP-DOWN transition. We also examined ripple temporal correlations using cross-correlograms. RESULTS: At the group level in the SOZ, HFO and HFO-associated AP probability was highest during the UP-DOWN transition of slow wave excitability (p < < .001). In the non-SOZ, HFO and HFO-associated AP was highest during the DOWN-UP transition (p < < .001). At the unit level in the SOZ, 15.6% and 20% of units exhibited more robust firing during ripples (Cohen's d = .11-.83) and fast ripples (d = .36-.90) at the UP-DOWN transition (p < .05 f.d.r. corrected), respectively. By comparison, also in the SOZ, 6.6% (d = .14-.30) and 8.5% (d = .33-.41) of units had significantly less firing during ripples and fast ripples at the UP-DOWN transition, respectively. Additional data shows that ripple and fast ripple temporal correlations, involving global slow waves, between the hippocampus, entorhinal cortex, and parahippocampal gyrus were reduced by >50% in the SOZ compared to the non-SOZ (N = 3). SIGNIFICANCE: The UP-DOWN transition of slow wave excitability facilitates the activation of pathological neurons to generate pHFOs. Ripple temporal correlations across brain regions may be important in memory consolidation and are disrupted in the SOZ, perhaps by pHFO generation.

Fast ripples reflect increased excitability that primes epileptiform spikes.

The neuronal circuit disturbances that drive inter-ictal and ictal epileptiform discharges remain elusive. Using a combination of extra-operative macro-electrode and micro-electrode inter-ictal recordings in six pre-surgical patients during non-rapid eye movement sleep, we found that, exclusively in the seizure onset zone, fast ripples (200-600 Hz), but not ripples (80-200 Hz), frequently occur <300 ms before an inter-ictal intra-cranial EEG spike with a probability exceeding chance (bootstrapping, P < 1e-5). Such fast ripple events are associated with higher spectral power (P < 1e-10) and correlated with more vigorous neuronal firing than solitary fast ripple (generalized linear mixed-effects model, P < 1e-9). During the intra-cranial EEG spike that follows a fast ripple, action potential firing is lower than during an intra-cranial EEG spike alone (generalized linear mixed-effects model, P < 0.05), reflecting an inhibitory restraint of intra-cranial EEG spike initiation. In contrast, ripples do not appear to prime epileptiform spikes. We next investigated the clinical significance of pre-spike fast ripple in a separate cohort of 23 patients implanted with stereo EEG electrodes, who underwent resections. In non-rapid eye movement sleep recordings, sites containing a high proportion of fast ripple preceding intra-cranial EEG spikes correlate with brain areas where seizures begin more than solitary fast ripple (P < 1e-5). Despite this correlation, removal of these sites does not guarantee seizure freedom. These results are consistent with the hypothesis that fast ripple preceding EEG spikes reflect an increase in local excitability that primes EEG spike discharges preferentially in the seizure onset zone and that epileptogenic brain regions are necessary, but not sufficient, for initiating inter-ictal epileptiform discharges.

Pathological neurons generate ripples at the UP-DOWN transition disrupting information transfer.

Objective: To confirm and investigate why pathological HFOs (pHFOs), including Ripples [80-200 Hz] and fast ripples [200-600 Hz], are generated during the UP-DOWN transition of the slow wave and if pHFOs interfere with information transmission. Methods: We isolated 217 total units from 175.95 iEEG contact-hours of synchronized macro- and microelectrode recordings from 6 patients. Sleep slow oscillation (0.1-2 Hz) epochs were identified in the iEEG recording. iEEG HFOs that occurred superimposed on the slow wave were transformed to phasors and adjusted by the phase of maximum firing in nearby units (i.e., maximum UP). We tested whether, in the seizure onset zone (SOZ), HFOs and associated action potentials (AP) occur more often at the UP-DOWN transition. We also examined ripple temporal correlations using cross correlograms. Results: At the group level in the SOZ, HFO and HFO-associated AP probability was highest during the UP-DOWN transition of slow wave excitability (p<<0.001). In the non-SOZ, HFO and HFO-associated AP was highest during the DOWN-UP transition (p<<0.001). At the unit level in the SOZ, 15.6% and 20% of units exhibited more robust firing during ripples (Cohen's d=0.11-0.83) and fast ripples (d=0.36-0.90) at the UP-DOWN transition (p<0.05 f.d.r corrected), respectively. By comparison, also in the SOZ, 6.6% (d=0.14-0.30) and 8.5% (d=0.33-0.41) of units had significantly less firing during ripples and fast ripples at the UP-DOWN transition, respectively. Additional data shows ripple temporal correlations, involving global slow waves, between the hippocampus, entorhinal cortex, and parahippocampal gyrus were reduced by ~50-80% in the SOZ compared to the non-SOZ (N=3). Significance: The UP-DOWN transition of slow wave excitability facilitates the activation of pathological neurons to generate pHFOs. The pathological neurons and pHFOs disrupt ripple temporal correlations across brain regions that transfer information and may be important in memory consolidation.

Fast ripples reflect increased excitability that primes epileptiform spikes.

The neuronal circuit disturbances that drive interictal and ictal epileptiform discharges remains elusive. Using a combination of extraoperative macro- and micro-electrode interictal recordings in six presurgical patients during non-rapid eye movement (REM) sleep we found that, exclusively in the seizure onset zone, fast ripples (FR; 200-600Hz), but not ripples (80-200 Hz), frequently occur <300 msec before an interictal intracranial EEG (iEEG) spike with a probability exceeding chance (bootstrapping, p<1e-5). Such FR events are associated with higher spectral power (p<1e-10) and correlated with more vigorous neuronal firing than solitary FR (generalized linear mixed-effects model, GLMM, p<1e-3) irrespective of FR power. During the iEEG spike that follows a FR, action potential firing is lower than during a iEEG spike alone (GLMM, p<1e-10), reflecting an inhibitory restraint of iEEG spike initiation. In contrast, ripples do not appear to prime epileptiform spikes. We next investigated the clinical significance of pre-spike FR in a separate cohort of 23 patients implanted with stereo EEG electrodes who underwent resections. In non-REM sleep recordings, sites containing a high proportion of FR preceding iEEG spikes correlate with brain areas where seizures begin more than solitary FR (p<1e-5). Despite this correlation, removal of these sites does not guarantee seizure freedom. These results are consistent with the hypothesis that FR preceding EEG spikes reflect an increase in local excitability that primes EEG spike discharges preferentially in the seizure onset zone and that epileptogenic brain regions are necessary, but not sufficient, for initiating interictal epileptiform discharges.

Neuronal Network Excitability in Alzheimer's Disease: The Puzzle of Similar versus Divergent Roles of Amyloid ß and Tau.

Alzheimer's disease (AD) is the most frequent neurodegenerative disorder that commonly causes dementia in the elderly. Recent evidence indicates that network abnormalities, including hypersynchrony, altered oscillatory rhythmic activity, interneuron dysfunction, and synaptic depression, may be key mediators of cognitive decline in AD. In this review, we discuss characteristics of neuronal network excitability in AD, and the role of Aß and tau in the induction of network hyperexcitability. Many patients harboring genetic mutations that lead to increased Aß production suffer from seizures and epilepsy before the development of plaques. Similarly, pathologic accumulation of hyperphosphorylated tau has been associated with hyperexcitability in the hippocampus. We present common and divergent roles of tau and Aß on neuronal hyperexcitability in AD, and hypotheses that could serve as a template for future experiments.

BC RNA Mislocalization in the Fragile X Premutation.

Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5' untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor-dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.

Early-Onset Network Hyperexcitability in Presymptomatic Alzheimer's Disease Transgenic Mice Is Suppressed by Passive Immunization with Anti-Human APP/Aß Antibody and by mGluR5 Blockade.

Cortical and hippocampal network hyperexcitability appears to be an early event in Alzheimer's disease (AD) pathogenesis, and may contribute to memory impairment. It remains unclear if network hyperexcitability precedes memory impairment in mouse models of AD and what are the underlying cellular mechanisms. We thus evaluated seizure susceptibility and hippocampal network hyperexcitability at ~3 weeks of age [prior to amyloid beta (Aß) plaque deposition, neurofibrillary pathology, and cognitive impairment] in a triple transgenic mouse model of familial AD (3xTg-AD mouse) that harbors mutated human Aß precursor protein (APP), tau and presenilin 1 (PS1) genes. Audiogenic seizures were elicited in a higher proportion of 3xTg-AD mice compared with wild type (WT) controls. Seizure susceptibility in 3xTg-AD mice was attenuated either by passive immunization with anti-human APP/Aß antibody (6E10) or by blockade of metabotropic glutamate receptor 5 (mGluR5) with the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). In in vitro hippocampal slices, suppression of synaptic inhibition with the GABAA receptor antagonist, bicuculline, induced prolonged epileptiform (>1.5 s in duration) ictal-like discharges in the CA3 neuronal network in the majority of the slices from 3xTg-AD mice. In contrast, only short epileptiform (<1.5 s in duration) interictal-like discharges were observed following bicuculline application in the CA3 region of WT slices. The ictal-like activity in CA3 region of the hippocampus was significantly reduced in the 6E10-immunized compared to the saline-treated 3xTg-AD mice. MPEP acutely suppressed the ictal-like discharges in 3xTg-AD slices. Remarkably, epileptiform discharge duration positively correlated with intraneuronal human (transgenic) APP/Aß expression in the CA3 region of the hippocampus. Our data suggest that in a mouse model of familial AD, hypersynchronous network activity underlying seizure susceptibility precedes Aß plaque pathology and memory impairment. This early-onset network hyperexcitability can be suppressed by passive immunization with an anti-human APP/Aß antibody and by mGluR5 blockade in 3xTg-AD mice.

APP Causes Hyperexcitability in Fragile X Mice.

Amyloid-beta protein precursor (APP) and metabolite levels are altered in fragile X syndrome (FXS) patients and in the mouse model of the disorder, Fmr1KO mice. Normalization of APP levels in Fmr1KO mice (Fmr1KO /APPHET mice) rescues many disease phenotypes. Thus, APP is a potential biomarker as well as therapeutic target for FXS. Hyperexcitability is a key phenotype of FXS. Herein, we determine the effects of APP levels on hyperexcitability in Fmr1KO brain slices. Fmr1KO /APPHET slices exhibit complete rescue of UP states in a neocortical hyperexcitability model and reduced duration of ictal discharges in a CA3 hippocampal model. These data demonstrate that APP plays a pivotal role in maintaining an appropriate balance of excitation and inhibition (E/I) in neural circuits. A model is proposed whereby APP acts as a rheostat in a molecular circuit that modulates hyperexcitability through mGluR5 and FMRP. Both over- and under-expression of APP in the context of the Fmr1KO increases seizure propensity suggesting that an APP rheostat maintains appropriate E/I levels but is overloaded by mGluR5-mediated excitation in the absence of FMRP. These findings are discussed in relation to novel treatment approaches to restore APP homeostasis in FXS.

Extracellular glutamate exposure facilitates group I mGluR-mediated epileptogenesis in the hippocampus.

Stimulation of group I mGluRs elicits several forms of translation-dependent neuronal plasticity including epileptogenesis. The translation process underlying plasticity induction is controlled by repressors including the fragile X mental retardation protein (FMRP). In the absence of FMRP-mediated repression, a condition that occurs in a mouse model (Fmr1(-/-)) of fragile X syndrome, group I mGluR-activated translation is exaggerated causing enhanced seizure propensity. We now show that glutamate exposure (10 µm for 30 min) reduced FMRP levels in wild-type mouse hippocampal slices. Downregulation of FMRP was dependent on group I mGluR activation and was blocked by a proteasome inhibitor (MG-132). Following glutamate exposure, synaptic stimulation induced prolonged epileptiform discharges with properties similar to those observed in Fmr1(-/-) preparations. In both cases, prolonged epileptiform discharges were blocked by group I mGluR antagonists (LY367385 + MPEP) and their induction was prevented by protein synthesis inhibitor (anisomycin). The results suggest that stimulation of group I mGluRs during glutamate exposure caused proteolysis of FMRP. Reduction of FMRP led to enhanced synaptic group I mGluR-mediated translation. Elevated translation facilitated the recruitment of group I mGluR-mediated prolonged epileptiform discharges.

Interactions of noncanonical motifs with hnRNP A2 promote activity-dependent RNA transport in neurons.

A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell's activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca(2+) levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca(2+) through voltage-dependent calcium channels upon ß-adrenergic receptor activation. The combined data establish a functional correspondence between Ca(2+)-dependent RNA-protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.

Persistent receptor activity underlies group I mGluR-mediated cellular plasticity in CA3 neuron.

Plastic changes in cortical activities induced by group I metabotropic glutamate receptor (mGluR) stimulation include epileptogenesis, expressed in vitro as the conversion of normal neuronal activity to persistent, prolonged synchronized (ictal) discharges. At present, the mechanism that maintains group I mGluR-induced plasticity is not known. We examined this issue using hippocampal slices from guinea pigs and mice. Agonist [(S)-3,5-dihydroxyphenylglycine (DHPG), 30-50 µm)] stimulation of group I mGluRs induces persistent prolonged synchronized (ictal-like) discharges in CA3 that are associated with three identified excitatory cellular responses-suppression of spike afterhyperpolarizations, activation of a voltage-dependent cationic current, and increase in neuronal input resistance. Persistent prolonged synchronized discharges and the underlying excitatory cellular responses maintained following induction were reversibly blocked by mGluR1 antagonists [(S)-+-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385), 50, 100 µm; CPCCOEt (hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester, 100 µm], and to a lesser extent by the mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride, 50 µm]. Activation of persistent cellular responses to DHPG were unaffected by tetrodotoxin (0.5-1 µm) or perfusion with low Ca(2+)(0.2 mm)-Mn(2+)(0.5 mm) media-conditions that suppress endogenous glutamate release. The pharmacological profile of the blocking action of the group I mGluR antagonist MCPG [(RS)-α-methyl-4-carboxyphenylglycine, 50-500 µm] on persistent cellular responses was different from that on cellular responses directly activated by DHPG. These data indicate that transient stimulation of group I mGluRs alters receptor properties, rendering them persistently active in the absence of applied agonist or endogenous glutamate activation. Persistent receptor activities, primarily involving mGluR1, maintain excitatory cellular responses and emergent prolonged synchronized discharges.

Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome.

Many neuropsychiatric symptoms of fragile X syndrome (FXS) are believed to be a consequence of altered regulation of protein synthesis at synapses. We discovered that lovastatin, a drug that is widely prescribed for the treatment of high cholesterol, can correct excess hippocampal protein synthesis in the mouse model of FXS and can prevent one of the robust functional consequences of increased protein synthesis in FXS, epileptogenesis. These data suggest that lovastatin is potentially disease modifying and could be a viable prophylactic treatment for epileptogenesis in FXS.

Dual regulation of fragile X mental retardation protein by group I metabotropic glutamate receptors controls translation-dependent epileptogenesis in the hippocampus.

Group I metabotropic glutamate receptors (mGluRs) stimulation activates translation-dependent epileptogenesis in the hippocampus. This translation is regulated by repressors, including BC1 RNA and fragile X mental retardation protein (FMRP). Recent data indicate that group I mGluR stimulation exerts bidirectional control over FMRP level by activating translation and ubiquitin-proteasome system (UPS)-dependent proteolysis for the up- and downregulation of the protein, respectively. At present, the temporal relationship of translation and proteolysis on FMRP and their interplay for group I mGluR-mediated translation and epileptogenesis are unknown. We addressed these issues by using mouse hippocampal slices. Agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] stimulation of group I mGluRs caused a biphasic change in FMRP level. An initial decrease (within 10 min) was followed by an increase at 30 min. When slices were pretreated with translation inhibitor (anisomycin or cycloheximide), group I mGluRs elicited a sustained decrease in FMRP. This decrease was prevented by a proteasome inhibitor [Z-Leu-Leu-Leu-CHO (MG-132)]. When slices were pretreated with MG-132 alone, DHPG no longer elicited any change in FMRP. MG-132 also suppressed increase in other proteins, including postsynaptic density-95 and α-calcium/calmodulin-dependent protein kinase II, normally elicited by group I mGluR stimulation. Physiological experiments showed that proteasome inhibitor suppressed group I mGluR-induced prolonged synchronized discharges. However, proteasome inhibitor did not affect group I mGluR-induced prolonged synchronized discharges in Fmr1(-/-) preparations, where functional FMRP is absent. The results suggest that constitutive FMRP in hippocampal cells acts as a brake on group I mGluR-mediated translation and epileptogenesis. FMRP downregulation via UPS removes this brake enabling group I mGluR-mediated translation and epileptogenesis.

Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain.

BACKGROUND: BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability. METHODOLOGY/PRINCIPAL FINDINGS: We generated BC1-/- Fmr1-/- double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1-/- Fmr1-/- dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1-/- Fmr1-/- dKO animals in response to auditory stimulation. In place learning, BC1-/- Fmr1-/- dKO animals were severely impaired, in contrast to BC1-/- or Fmr1-/- sKO animals which exhibited only mild deficits. CONCLUSIONS/SIGNIFICANCE: Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.

BC1 regulation of metabotropic glutamate receptor-mediated neuronal excitability.

Regulatory RNAs have been suggested to contribute to the control of gene expression in eukaryotes. Brain cytoplasmic (BC) RNAs are regulatory RNAs that control translation initiation. We now report that neuronal BC1 RNA plays an instrumental role in the protein-synthesis-dependent implementation of neuronal excitation-repression equilibria. BC1 repression counter-regulates translational stimulation resulting from synaptic activation of group I metabotropic glutamate receptors (mGluRs). Absence of BC1 RNA precipitates plasticity dysregulation in the form of neuronal hyperexcitability, elicited by group I mGluR-stimulated translation and signaled through the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway. Dysregulation of group I mGluR function in the absence of BC1 RNA gives rise to abnormal brain function. Cortical EEG recordings from freely moving BC1(-/-) animals show that group I mGluR-mediated oscillations in the gamma frequency range are significantly elevated. When subjected to sensory stimulation, these animals display an acute group I mGluR-dependent propensity for convulsive seizures. Inadequate RNA control in neurons is thus causally linked to heightened group I mGluR-stimulated translation, neuronal hyperexcitability, heightened gamma band oscillations, and epileptogenesis. These data highlight the significance of small RNA control in neuronal plasticity.

Cellular plasticity for group I mGluR-mediated epileptogenesis.

Stimulation of group I metabotropic glutamate receptors (mGluRs) by the agonist (S)-dihydroxyphenylglycine in the hippocampus transforms normal neuronal activity into prolonged epileptiform discharges. The conversion is long lasting in that epileptiform discharges persist after washout of the inducing agonist and serves as a model of epileptogenesis. The group I mGluR model of epileptogenesis took on special significance because epilepsy associated with fragile X syndrome (FXS) may be caused by excessive group I mGluR signaling. At present, the plasticity mechanism underlying the group I mGluR-mediated epileptogenesis is unknown. I(mGluR(V)), a voltage-gated cationic current activated by group I mGluR agonists in CA3 pyramidal cells in the hippocampus, is a possible candidate. I(mGluR(V)) activation is associated with group I mGluR agonist-elicited epileptiform discharges. For I(mGluR(V)) to play a role in epileptogenesis, long-term activation of the current must occur after group I mGluR agonist exposure or synaptic stimulation. We observed that I(mGluR(V)), once induced by group I mGluR agonist stimulation in CA3 pyramidal cells, remained undiminished for hours after agonist washout. In slices prepared from FXS model mice, repeated stimulation of recurrent CA3 pyramidal cell synapses, effective in eliciting mGluR-mediated epileptiform discharges, also induced long-lasting I(mGluR(V)) in CA3 pyramidal cells. Similar to group I mGluR-mediated prolonged epileptiform discharges, persistent I(mGluR(V)) was no longer observed in preparations pretreated with inhibitors of tyrosine kinase, of extracellular signal-regulated kinase 1/2, or of mRNA protein synthesis. The results indicate that I(mGluR(V)) is an intrinsic plasticity mechanism associated with group I mGluR-mediated epileptogenesis.

Signaling mechanisms underlying group I mGluR-induced persistent AHP suppression in CA3 hippocampal neurons.

Activation of group I metabotropic glutamate receptors (mGluRs) leads to a concerted modulation of spike afterpotentials in guinea pig hippocampal neurons including a suppression of both medium and slow afterhyperpolarizations (AHPs). Suppression of AHPs may be long-lasting, in that it persists after washout of the agonist. Here, we show that persistent AHP suppression differs from short-term, transient suppression in that distinct and additional signaling processes are required to render the suppression persistent. Persistent AHP suppression followed DHPG application for 30 min, but not DHPG application for 5 min. Persistent AHP suppression was temperature dependent, occurring at 30-31 degrees C, but not at 25-26 degrees C. Preincubation of slices in inhibitors of protein synthesis (cycloheximide or anisomycin) prevented the persistent suppression of AHPs by DHPG. Similarly, preincubation of slices in an inhibitor of p38 MAP kinase (SB 203580) prevented persistent AHP suppression. In contrast, a blocker of p42/44 MAP kinase activation (PD 98059) had no effect on persistent AHP suppression. Additionally, we show that the mGluR5 antagonist MPEP, but not the mGluR1 antagonist LY 367385, prevented DHPG-induced persistent AHP suppression. Thus persistent AHP suppression by DHPG in hippocampal neurons requires activation of mGluR5. In addition, activation of p38 MAP kinase signaling and protein synthesis are required to impart persistence to the DHPG-activated AHP suppression.

Group I metabotropic glutamate receptor-dependent TRPC channel trafficking in hippocampal neurons.

The group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited two phases of synchronized neuronal (epileptiform) discharges in hippocampal slices: an initial phase of short duration discharges followed by a phase of prolonged discharges. We assessed the involvement of transient receptor potential canonical (TRPC) channels in these responses. Pre-treatment of hippocampal slices with TRPC channel blockers, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365) or 2-aminoethoxydiphenyl borate, did not affect the short epileptiform discharges but blocked the prolonged epileptiform discharges. SKF96365 suppressed ongoing DHPG-induced prolonged epileptiform discharges. Western blot analysis showed that the total TRPC4 or TRPC5 proteins in hippocampal slices were unchanged following DHPG. DHPG increased TRPC4 and TRPC5 in the cytoplasmic compartment and decreased these proteins in the plasma membrane. Translocation of TRPC4 and TRPC5 was suppressed when the epileptiform discharges were blocked by ionotropic glutamate receptor blockers. Translocation of TRPC4 and TRPC5 was also prevented in slices from phospholipase C (PLC) beta1 knockout mice, even when synchronized discharges were elicited by the convulsant 4-aminopyridine. The results suggest that TRPC channels are involved in generating DHPG-induced prolonged epileptiform discharges. This function of TRPC channels is associated with a neuronal activity- and PLCbeta1-dependent translocation of TRPC4 and TRPC5 proteins from the plasmalemma to the cytoplasmic compartment.

Pharmacology of a slowly inactivating outward current in hippocampal CA3 pyramidal neurons.

The pharmacology of a slowly inactivating outward current was examined using whole cell patch-clamp recordings in CA3 pyramidal cells of guinea pig hippocampal slices. The current had a low activation threshold (about -60 mV) and inactivated slowly (time constant of 3.4 +/- 0.5 s at -50 mV) and completely at membrane voltages depolarized to -50 mV. The slowly inactivating outward current was mainly mediated by K+ with a reversal potential close to the equilibrium potential for K+. The slowly inactivating outward current had distinct pharmacological properties: its time course was not affected by extracellular Cs+ (1 mM) or 4-AP (1-5 mM)-broad spectrum inhibitors of K+ currents and of inactivating K+ currents, respectively. The presence of extracellular Mn2+ (0.5-1 mM), which suppresses several Ca2+ -dependent K+ currents, also did not affect the slowly inactivating outward current. The current was partially suppressed by TEA (50 mM) and was blocked by intracellular Cs+ (134 mM). In addition, intracellular QX-314 (5 mM), a local anesthetic derivative, inhibited this current. The slowly inactivating outward current with its low activation threshold should be operational at the resting potential. Our results suggest that the transient outward current activated at subthreshold membrane potentials in hippocampal pyramidal cells consists of at least three components. In addition to the well-described A- and D-currents, the slowest decaying component reflects the time course of a distinct current, suppressible by QX-314.