Patric K. Stanton

Resistance of the immature hippocampus to seizure-induced synaptic reorganization

Temporal lobe epilepsy is a common form of epilepsy in human adults and is associated with a unique pattern of damage in the hippocampus. The damage includes cell loss of the CA3 and CA4 areas and synaptic growth (sprouting) of mossy fibers in the supragranular layer of the dentate gyrus. Experimental evidence indicates that in adult rats the excitatory amino acid, kainic acid, induces a similar pattern of changes in hippocampal circuitry associated with alterations in perforant path excitation and inhibition. It has been suggested that, in humans, this type of damage may be a result of seizures early in life. In this study we examined the effects of kainic acid-induced status epilepticus on synaptic reorganization and paired-pulse electrophysiology in developing rats and adults. Kainic acid induced more severe seizures in 15-day-old rat pups than in adults. In contrast to adult rats, these seizures did not produce CA3/CA4 neuronal loss, mossy fiber sprouting or changes in paired-pulse excitation or inhibition in the hippocampus of rat pups tested 2-4 weeks after status epilepticus. Our results provide evidence that the immature hippocampus may be more resistant to seizure-induced changes than the mature hippocampus.

LTD, LTP, and the sliding threshold for long-term synaptic plasticity

LTD of synaptic transmission is a form of long-term synaptic plasticity with the potential to be as significant as LTP to both the activity-dependent development of neural circuitry and adult memory storage. In addition, interactions between LTP and LTD and the dynamic regulation of the gain of synaptic plasticity mechanisms are also very important. In particular, the computational ability of LTD to properly counterbalance LTP may be essential to maintaining synaptic strengths in the linear range, and to maximally sharpen the ability of synapses to compute and store frequency-based information about the phase relation between synapses. Experimental data confirm the presence of an activity-dependent sliding threshold with the expected properties. That is, when levels of neuronal activity are high, indicating circumstances increasing the likelihood of inducing LTP, compensatory changes cause the suppression of LTP and an enhanced likelihood of LTD. Conversely, we would predict that low levels of synaptic activity would shift the threshold in favor of greater LTP and less LTD, a hypothesis which has yet to be tested. The sliding threshold for LTP and LTD also has implications for underlying cellular mechanisms of both forms of long-term synaptic plasticity. If the thresholds for LTP and LTD are tightly and reciprocally co-regulated, that could imply that at least one component of LTD is a true depotentiation caused by reversal of a change mediating LTP. If so, the intuitively simplest hypothesis is that phosphorylation of AMPA glutamate receptors causes LTP of synaptic e.p.s.p.s, while dephosphorylation of the same site or sites causes depotentiation LTD. Of course, this hypothesis would refer only to a postsynaptic component of both LTP and LTD. There has been a recent report that, in neonatal rat hippocampus, a form of LTD that is expressed developmentally earlier than LTP appears to have a postsynaptic induction site, but is expressed as decreased presynaptic transmitter release (Bolshakov and Siegelbaum, 1994). Whether these properties will be retained as LTD matures is unknown, as is the likelihood that, if a component of LTP is expressed presynaptically, depotentiation of that presynaptic component can also occur. Equally unclear is the persistence of LTD relative to LTP. The few rigorous long-term anatomical studies available suggest that the latest phases of LTP may be expressed as changes in dendritic spine shapes and/or synaptic morphology. While heterosynaptic LTD has been reported to have a duration of weeks in vivo (Abraham et al., 1994), we do not know whether LTP-induced morphological changes that take many days to appear can be reversed in an activity-dependent manner. An important feature of the consolidation of memories may turn out to be the slow development of LTP that is resistant to reversal by LTD. While we still at an earlier stage in our understanding of the mechanisms underlying LTD compared to LTP, some things are becoming clear. LTD is induced by afferent neuronal activity that is relatively ineffective in exciting the postsynaptic cell--an anti-hebbian condition. This property, coupled with the hebbian properties of LTP and the dynamic nature of membrane conductances, necessarily confers upon synapses the ability to compute and store the results of a covariance function. However, the role of such a computation in processing and/or memory is unclear. In addition, LTD appears to require the activation of NMDA and metabotropic subtypes of glutamate receptors, release of Ca2+ from intracellular stores, and an increase in intracellular [Ca2+] that is lower than that necessary to induce LTP. The early evidence is consistent with some overlap of targets for modification by LTP and LTD, with some forms of LTD likely to be a reversal, or depotentiation, of previous LTP, perhaps through dephosphorylation of AMPA receptors.

Low-frequency stimulation of the kindling focus delays basolateral amygdala kindling in immature rats

Stimulation of deep brain sites is a new approach for treatment of intractable seizures. In adult rats, low-frequency stimulation (LFS; 1-3 Hz) of the kindling site interferes with the course of kindling epileptogenesis. In this study we determined whether the LFS will be effective against the fast kindling in the basolateral amygdala in immature, 15 day old rats. LFS (15 min of 1 Hz stimulation) was applied after each of the 1 s, 60 Hz kindling stimulus. LFS suppressed afterdischarge duration and seizure stage throughout the course of kindling, which indicates a strong antiepileptogenic potential. As the kindling and LFS stimulation patterns are similar to those used for induction of long-term potentiation and long-term depression (LTD), respectively, LTD or depotentiation may play a role in the mechanism of action.

Commissural synapses, but not mossy fiber synapses, in hippocampal field CA3 exhibit associative long-term potentiation and depression.

When CA3 commissural afferents received low-frequency (weak) stimuli synchronized with a train of mossy fiber bursts (strong), associative long-term potentiation (LTP) was induced at mixed commissural and associational synapses on hippocampal CA3 pyramidal cells in vitro. In contrast, a weak mossy fiber input did not potentiate when given in phase with commissural/associational bursts. Furthermore, commissural/associational synapses receiving low-frequency stimuli out-of-phase with strong rhythmic mossy fiber input showed associative long-term depression (LTD), whereas mossy fiber synaptic strengths were not depressed when they received weak inputs out-of-phase with a strong commissural/associational input. Thus, both associative LTP and associative LTD can be induced at commissural/associational synapses, but not at mossy fiber synapses.

2-Amino-3-phosphonopropionic acid, an inhibitor of glutamate-stimulated phosphoinositide turnover, blocks induction of homosynaptic long-term depression, but not potentiation, in rat hippocampus

Recently, we have reported an associative long-term depression (LTD) of synaptic strength in hippocampal field CA1 that is produced when a low-frequency test input is negatively correlated in time with a high-frequency conditioning input. We have also found that pairing of synaptic activity with postsynaptic hyperpolarization is sufficient to induce LTD. We report here that 2-amino-3-phosphonopropionate (AP3), a selective inhibitor of phosphoinositide (PI) turnover mediated by the metabotropic glutamate receptor, blocks the induction of associative LTD in hippocampal field CA1, but does not impair the induction of LTP. Our data suggest that metabotropic glutamate receptor activation is involved in the induction of LTD.

A role for N-methyl-D-aspartate receptors in norepinephrine-induced long-lasting potentiation in the dentate gyrus.

SummaryMechanisms of action of norepinephrine (NE) on dentate gyrus granule cells were studied in rat hippocampal slices using extra- and intracellular recordings and measurements of stimulus and amino acid-induced changes in extracellular Ca2+ and K+ concentration. Bath application of NE (10–50 μM) induced long-lasting potentiation of perforant path evoked potentials, and markedly enhanced high-frequency stimulus-induced Ca2+ influx and K+ efflux, actions blocked by β-receptor antagonists and mimicked by β agonists. Enhanced Ca2+ influx was primarily postsynaptic, since presynaptic Δ [Ca2+]0 in the stratum moleculare synaptic field was not altered by NE. Interestingly, the potentiation of both ionic fluxes and evoked population potentials were antagonized by the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV). Furthermore, NE selectively enhanced the Δ[Ca2+]0, Δ[K+]0 and extracellular slow negative field potentials elicited by iontophoretically applied NMDA, but not those induced by the excitatory amino acid quisqualate. These results suggest that granule cell influx of Ca2+ through NMDA ionophores is enhanced by NE via β-receptor activation. In intracellular recordings, NE depolarized granule cells (4.8±1.1 mV), and increased input resistance (RN) by 34±6.5%. These actions were also blocked by either the β-antagonist propranolol or specific β1-blocker metoprolol. Moreover, the depolarization and RN increase persisted for long periods (93±12 min) after NE washout. In contrast, while NE, in the presence of APV, still depolarized granule cells and increased RN, APV made these actions quickly reversible upon NE washout (16±9 min). This suggested that NE induction of long-term, but not short-term, plasticity in the dentate gyrus requires NMDA receptor activation. NE may be enhancing granule cell firing by some combination of blockade on the late Ca2+-activated K+ conductance and depolarization of granule cells, both actions that can bring granule cells into a voltage range where NMDA receptors are more easily activated. Furthermore, NE also elicited activity-independent long-lasting depolarization and RN increases, which required functional NMDA receptors to persist.

Long-term depression of presynaptic release from the readily releasable vesicle pool induced by NMDA receptor-dependent retrograde nitric oxide

Postsynaptic alterations are currently believed to be able to fully account for NMDA-receptor-dependent long-term depression (LTD) and long-term potentiation of synaptic strength, although there is also evidence supporting changes in presynaptic release. Using dualphoton laser scan microscopy of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43) to directly visualize presynaptic vesicular release at Schaffer collateral–CA1 excitatory synapses in hippocampal slices, we demonstrate reduced vesicular release associated with LTD. Selective loading, by hypertonic shock, of the readily releasable vesicle pool (RRP) showed that LTD of release is a selective modification of release from the RRP. Presynaptic LTD of RRP release required activation of NMDA receptors, production and extracellular diffusion of the intercellular messenger NO, and activation of cGMP-dependent protein kinase.

Lowering of extracellular pH suppresses low-Mg2+-induces seizures in combined entorhinal cortex-hippocampal slices

Lowering [Mg2+]o induces epileptiform bursting in hippocampus and entorhinal cortex (EC), presumably by activation of N-methyl-d-aspartate (NMDA) receptors. Since increasing [H+]o has been shown to reduce NMDA receptor activation, we hypothesized that this could contribute to anticonvulsant actions of acidic pH. To test this, we studied the effects of raising extracellular PCO2 (20.6%, pH = 6.7) or lowering extracellular pH (6.7 or 6.2) on low-Mg2+-induced epileptiform discharges. Lowering the pH to 6.7 by either means increased the interval between seizure-like events (SLEs), decreased the maximal amplitude of SLEs, and, if the site of seizure generation was at a distance from the recording site, acidification slowed the rate of seizure propagation. In contrast, the duration of SLEs was unaffected by acidic pH or high PCO2. Raising PCO2 or lowering pH to 6.7 also blocked early (8–10 min) but not late (> 20 min) phases of status-like discharges. All effects of the extracellular pH changes were fully reversible. Further lowering of extracellular pH to 6.2 completely and reversibly blocked both SLEs and status-like discharges. Our data show that the effects of high PCO2 and low pH on seizures in the EC in vitro may be dose-dependent and consistent with induction by proton blockade of NMDA receptors. Thus, blockade of NMDA currents by protons may be an important component of the anticonvulsant action of extracellular acidosis. The results also suggest that acidosis may be a desirable property for new antiepileptic treatments.

Sex-specific KCC2 expression and GABAA receptor function in rat substantia nigra

GABA(A) receptor activation by muscimol has sex and age specific effects on substantia nigra reticulata (SNR)-mediated control of generalized seizures. GABA(A) receptor agonists depolarize or hyperpolarize neurons depending upon the level of expression of the neuronal specific potassium chloride contransporter KCC2. We studied KCC2 mRNA expression in the SNR as a function of sex and age and correlated KCC2 expression with the in vivo and in vitro effects of muscimol. Methods included in situ hybridization, gramicidin-perforated patch clamp and fura-2 AM imaging of acute SNR slices. KCC2 mRNA expression increased between postnatal days (PN) 15 and 30 in both sexes, and reached adult levels in males by PN30. Female PN15 and PN30 SNR neurons contained more KCC2 mRNA compared with age-matched males. In male PN14-17 rats, bath application of the GABA(A) receptor agonist muscimol in acute SNR slices depolarized neurons and increased intracellular calcium concentration ([Ca(2+)](i)). Furthermore, acute in vivo administration of muscimol upregulated, whereas blockade of L-type voltage sensitive calcium channels with nifedipine downregulated KCC2 mRNA. In contrast, in female PN14-17 rats, bath application of muscimol hyperpolarized SNR neurons and did not alter [Ca(2+)](i). In vivo muscimol administration acutely downregulated KCC2 mRNA expression whereas nifedipine had no effect. The lower expression of KCC2 mRNA in infantile male SNR neurons may explain why muscimol-induced depolarization and [Ca(2+)](i) increases occur only in males. Consequently, GABA(A) receptor activation selectively upregulates the expression of calcium-regulated genes, such as KCC2, in male SNR, promoting the sexual differentiation of the SNR.

Nitric‐oxide‐guanylyl‐cyclase‐dependent and ‐independent components of multiple forms of long‐term synaptic depression

Long‐term depression (LTD) of synaptic strength is induced by glutamate‐triggered increases in postsynaptic [Ca2+], through either influx or release from intracellular stores. Induction of LTD has also been reported to require release of Ca2+ from presynaptic stores and activation of presynaptic Ca2+/calmodulin‐dependent protein kinase II. This finding leads to the hypothesis that the intercellular messenger nitric oxide (NO) may be a means by which postsynaptic Ca2+ triggers changes expressing LTD in presynaptic terminals. We report that bath application of the oxadiazoloquinoxalone derivative ODQ (5 μM), a selective inhibitor of NO‐sensitive guanylyl cyclase (NOGC), markedly attenuated (90%) the magnitude of LTD induced by low‐frequency stimulation (LFS; 1 Hz/15 min) of Schaffer collateral‐CA1 synapses in hippocampal slices in vitro. Both the NO donor S‐nitroso‐N‐acetylpenicillamine (100 μM) and the membrane‐permeant cyclic guanine 3′,5′‐monophosphate (cGMP) analogue 8‐(4‐chlorophenylthio) guanosine (8‐pCPT)‐cGMP (50 μM) enhanced the magnitude of LTD, which is consistent with the hypothesis that activation of NOGC plays a role in the induction of LTD. Nicotinamide (20 mM), an inhibitor of NO‐activated ADP ribosyltransferase, did not impair the induction of LTD. In contrast to de novo LTD, the reversal of long‐term potentiation by LFS (depotentiation) was only partially blocked (55%) by ODQ, and heterosynaptic LTD was not impaired at all, suggesting that there are both NOGC‐dependent and ‐independent forms of LTD. Because postsynaptic intracellular infusion of ODQ (500 μM) failed to block the induction of LTD, we conclude that activation of presynaptic NOGC is a necessary step in the induction of an NOGC‐dependent component of LTD. Hippocampus 7:286–295, 1997.u2003© 1997 Wiley‐Liss, Inc.