Robert K. S. Wong

Excitatory synaptic responses mediated by GABAA receptors in the hippocampus

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABAA receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABAA receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABAA receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABAA receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

Prolonged Epileptiform Discharges Induced by Altered Group I Metabotropic Glutamate Receptor-Mediated Synaptic Responses in Hippocampal Slices of a Fragile X Mouse Model

Mutations in FMR1, which encodes the fragile X mental retardation protein (FMRP), are the cause of fragile X syndrome (FXS), an X-linked mental retardation disorder. Inactivation of the mouse gene Fmr1 confers a number of FXS-like phenotypes including an enhanced susceptibility to epileptogenesis during development. We find that in a FXS mouse model, in which the function of FMRP is suppressed, synaptically released glutamate induced prolonged epileptiform discharges resulting from enhanced group I metabotropic glutamate receptor (mGluR)-mediated responses in hippocampal slices. The induction of the group I mGluR-mediated, prolonged epileptiform discharges was inhibited in preparations that were pretreated with inhibitors of ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation or of mRNA translation, and their maintenance was suppressed by group I mGluR antagonists. The results suggest that FMRP plays a key role in the control of signaling at the recurrent glutamatergic synapses in the hippocampus. The absence of this control causes the synaptically activated group I mGluRs to elicit translation-dependent epileptogenic activities.

SYNCHRONIZATION OF INHIBITORY NEURONES IN THE GUINEA-PIG HIPPOCAMPUS IN VITRO

1. Intracellular recordings were obtained from pyramidal, granule and hilar cells in transverse slices of guinea‐pig hippocampus to examine synaptic interactions between GABAergic neurones. 2. In the presence of the convulsant compound 4‐aminopyridine (4‐AP), after fast excitatory amino acid (EAA) neurotransmission was blocked pharmacologically, large amplitude inhibitory postsynaptic potentials (IPSPs) occurred rhythmically (every 4‐8 s) and synchronously in all principal cell populations (triphasic synchronized IPSPs). In the presence of the GABAA receptor blocker picrotoxin (PTX), a large amplitude IPSP continued to occur spontaneously in all principal cells simultaneously (monophasic synchronized IPSP). 3. Burst firing occurred simultaneously in a group of hilar neurones (synchronized bursting neurones) coincident with triphasic synchronized IPSPs in principal cells. After PTX was added, the bursts and the underlying depolarizing synaptic potentials were completely suppressed in some of the synchronized bursting neurones (type I hilar neurones), while others (type II hilar neurones) continued to fire in bursts coincident with monophasic synchronized IPSPs in principal cells. Intense hyperpolarization blocked burst firing and revealed underlying attenuated spikes of less than 10 mV, but did not uncover any underlying depolarizing synaptic potentials. 4. In type II hilar neurones, during sufficient hyperpolarization, spontaneous activity consisted of attenuated spikes. With depolarization, the small spikes began to trigger full size action potentials. These data suggest the presence of electrotonically remote spike initiation sites. 5. The morphology of synchronized bursting neurones was revealed by intracellular injection of the fluorescent dye Lucifer Yellow. Attempts to inject dye into one type II hilar neurone often resulted in the labelling of two to four cells (dye coupling). Dye coupling was not observed in type I hilar neurones. 6. These findings indicate that excitatory interactions synchronizing the firing of GABAergic neurones can occur in the absence of fast EAA neurotransmission. GABAergic neurones can become synchronized via their recurrent collaterals through the depolarizing action of synaptically activated GABAA receptors. In addition, a subpopulation of GABAergic neurones can become synchronized by a mechanism probably involving electrotonic coupling.

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.

Different firing patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus.

1. Intracellular recordings, taken from CA1 pyramidal cells in guinea‐pig hippocampal slices, were used to examine the origins of repetitive and burst firing in these cells. Single action potentials were elicited by depolarizing current injection at somatic recording sites. In contrast, current injection during intradendritic recordings initiated burst firing in the dendrites. Burst firing could be elicited in the soma by direct depolarization of distal apical dendrites (> 150 microns from the cell body layer) with large extracellular polarizing electrodes. 2. Intracellular recordings were taken simultaneously from the apical dendrites and pyramidal cell somata with the intention of impaling the same neurone with both electrodes. Paired dendrite‐soma recordings confirmed that rhythmic single action potentials were generated at the cell soma, whereas bursts of action potentials were initiated in the distal apical dendrites (> 150 microns from the cell body layer). Fast spikes in the dendrite often triggered fast spikes in the soma, but not all fast spikes in the dendritic burst were ‘relayed’ to the soma. 3. In paired recordings, when a dendritic action potential failed to elicit a full somatic action potential, a ‘d‐spike’ was commonly recorded in the soma. Somatic d‐spikes were uniform all‐or‐none responses that could be shown, in some cases, to trigger the full somatic action potentials. 4. Attenuated spikes could be recorded in the dendrites, triggered by action potentials initiated at the cell soma. Dendritic responses to somatic stimulation sometimes varied in amplitude, but always showed a direct correspondence with somatic action potentials. 5. Dendritic recordings taken closer to the pyramidal cell bodies (< 150 microns from the cell body layer) showed a ‘transitional’ region where single action potentials rather than burst discharges could be evoked. After‐potentials of these single spikes differed from those associated with somatic spikes in that proximal dendritic spikes had depolarizing after‐potentials. The observed shift from after‐hyperpolarization to depolarizing after‐potentials in intradendritic recordings taken progressively further from the cell body corresponds to the change from repetitive to burst firing. 6. The results indicate that activity of the CA1 pyramidal cell soma, presumably a reflection of its output, can be either burst or repetitive firing. Somatic ‘bursts,’ unlike the burst discharges seen in the apical dendrites or the burst discharges reported in CA3 cells, are not initiated locally. Rather, they appear to be simply a rapid spike‐for‐spike response by the soma to the fast spikes that form part of the apical dendritic burst.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Synaptic mechanisms underlying interictal spike initiation in a hippocampal network

The intrinsic bursting capability of hippocampal neurons is well established. Recent experimental data also imply that CA3 neurons have mutual chemical excitatory interactions. Our previous simulations have shown how these two properties of the hippocampal CA3 region suffice to account for the synchronized burst discharges that occur in the presence of penicillin. Electrotonic interactions via gap junctions have also been described in the CA3 region, but their contribution to synchronization is not clear. We now show that a network of cells connected only by electrotonic junctions does not reproduce the experimental data on synchronization. In combination with chemical synapses, electrotonic junctions can prevent synchronized discharge, increase the degree of synchronization, or prolong the latency from stimulus to discharge. The effect electrotonic junctions have on synchronization of cellular bursting depends intimately on the density and strength of the chemical synapses.

Extracellular signal-regulated kinase 1/2 is required for the induction of group I metabotropic glutamate receptor-mediated epileptiform discharges.

Transient stimulation of group I metabotropic glutamate receptors (mGluRs) induces persistent prolonged epileptiform discharges in hippocampal slices via a protein synthesis-dependent process. At present, the signaling process underlying the induction of these epileptiform discharges remains unknown. We examined the possible role of extracellular signal-regulated kinases (ERK1 and ERK2) because these kinases can be activated by group I mGluRs, and their activation may regulate gene expression and alter protein synthesis. The group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 50 μm) induced activation of ERK1/2 in hippocampal slices. 2-(2-Diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) (50 μm) a specific inhibitor of mitogen-activated protein kinase kinase (MEK), suppressed ERK1/2 activation by DHPG. PD98059 or another MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (10 μm), also prevented the induction of the prolonged epileptiform discharges by DHPG. In the presence of ionotropic glutamate receptor inhibitors and tetrodotoxin (blockers), DHPG-induced epileptiform discharges were suppressed, whereas ERK1/2 activation persisted. Protein kinase C inhibitors (2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide, 1 μm; or chelerythrine, 10 μm) did not prevent the generation of DHPG-induced epileptiform discharges, nor did they suppress the activation of ERK1/2 by DHPG in slices pretreated with the blockers. Genistein (30 μm), a broad-spectrum tyrosine kinase inhibitor, suppressed the DHPG-induced epileptiform discharges and the ERK1/2 activation in the presence of blockers. Induction of DHPG-mediated epileptiform discharges was also suppressed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 μm), an Src-family tyrosine kinase inhibitor. The study shows that group I mGluRs activate ERK1/2 through a tyrosine kinase-dependent process and that this activation of ERK1/2 is necessary for the induction of prolonged epileptiform discharges in the hippocampus.

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. ImGluR(V), a voltage-gated cationic current activated by group I mGluR agonists in CA3 pyramidal cells in the hippocampus, is a possible candidate. ImGluR(V) activation is associated with group I mGluR agonist-elicited epileptiform discharges. For ImGluR(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 ImGluR(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 ImGluR(V) in CA3 pyramidal cells. Similar to group I mGluR-mediated prolonged epileptiform discharges, persistent ImGluR(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 ImGluR(V) is an intrinsic plasticity mechanism associated with group I mGluR-mediated epileptogenesis.

Excitatory synaptic potentials dependent on metabotropic glutamate receptor activation in guinea‐pig hippocampal pyramidal cells.

1. Intracellular and extracellular recordings of CA1 and CA3 neurones were performed in guinea‐pig hippocampal slices to examine synaptic activities dependent on metabotropic glutamate receptors (mGluRs). 2. Long burst activities were elicited by 4‐aminopyridine in the presence of ionotropic glutamate receptor and GABAA receptor blockers (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and 3‐(RS‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid, and picrotoxin). Long bursts were also elicited by alpha‐dendrotoxin. 3. Long bursts consisted of a 5‐25 s depolarization with overriding action potentials and occurred rhythmically at intervals ranging from 1 to 20 min. Long bursts were generated in a population of CA3 neurones and the synchronized output elicited long bursts in CA1 cells. Depolarizing potentials underlying long bursts in CA1 cells had a reversal potential of ‐14.8 +/‐ 5.1 mV. 4. Long burst‐associated depolarizations in CA1 neurones were suppressed by local application of L‐(+)‐2‐amino‐3‐phosphonopropionic acid (L‐AP3) and of the phenylglycine derivatives (+)‐alpha‐methyl‐4‐carboxyphenylglycine ((+)‐MCPG), S‐4‐carboxyphenylglycine (S‐4CPG) and S‐4‐carboxy‐3‐hydroxyphenylglycine (S‐4C3HPG). (‐)‐MCPG or atropine application did not affect the long burst‐associated depolarization. 5. Bath perfusion of (+)‐MCPG (0.5 mM), S‐4CPG (0.5 mM), S‐4C3HPG (0.5 mM) or L‐AP3 (1 mM) blocked the occurrence of long bursts. 6. The results suggest that the long burst‐associated depolarizations are synaptic potentials dependent on mGluR activation. Activation of mGluRs may also be involved in the generation of synchronized long bursts in the CA3 region.