Agustin Liotta

Partial Disinhibition Is Required for Transition of Stimulus-Induced Sharp Wave–Ripple Complexes Into Recurrent Epileptiform Discharges in Rat Hippocampal Slices

Sharp wave-ripple complexes (SPW-Rs) in the intact rodent hippocampus are characterized by slow field potential transients superimposed by close to 200-Hz ripple oscillations. Similar events have been recorded in hippocampal slices where SPW-Rs occur spontaneously or can be induced by repeated application of high-frequency stimulation, a standard protocol for induction of long-lasting long-term potentiation. Such stimulation is reminiscent of protocols used to induce kindling epilepsy and ripple oscillations may be predictive of the epileptogenic zone in temporal lobe epilepsy. In the present study, we investigated the relation between recurrent epileptiform discharges (REDs) and SPW-Rs by studying effects of partial removal of inhibition. In particular, we compared the effects of nicotine, low-dose bicuculline methiodide (BMI), and elevated extracellular potassium concentration ([K(+)](o)) on induced SPW-Rs. We show that nicotine dose-dependently transformed SPW-Rs into REDs. This transition was associated with reduced inhibitory conductance in CA3 pyramidal cells. Similar results were obtained from slices where the GABAergic conductance was reduced by application of low concentrations of BMI (1-2 μM). In contrast, sharp waves were diminished by phenobarbital. Elevating [K(+)](o) from 3 to 8.5 mM did not transform SPW-Rs into REDs but significantly increased their incidence and amplitude. Under these conditions, the equilibrium potential for inhibition was shifted in depolarizing direction, whereas inhibitory conductance was significantly increased. Interestingly, the propensity of elevated [K(+)](o) to induce seizure-like events was reduced in slices where SPW-Rs had been induced. In conclusion, recruitment of inhibitory cells during SPW-Rs may serve as a mechanism by which hyperexcitation and eventually seizure generation might be prevented.

Adrenergic modulation of sharp wave‐ripple activity in rat hippocampal slices

Norepinephrine (NE) has been shown to facilitate learning and memory by modulating synaptic plasticity in the hippocampus in vivo. During memory consolidation, transiently stored information is transferred from the hippocampus into the cortical mantle. This process is believed to depend on the generation of sharp wave‐ripple complexes (SPW‐Rs), during which previously stored information might be replayed. Here, we used rat hippocampal slices to investigate neuromodulatory effects of NE on SPW‐Rs, induced by a standard long‐term potentiation (LTP) protocol, in the CA3 and CA1. NE (10–50 μM) dose‐dependently and reversibly suppressed the generation of SPW‐Rs via activation of α1 adrenoreceptors, as indicated by the similar effects of phenylephrine (100 μM). In contrast, the unspecific β adrenoreceptor agonist isoproterenol (2 μM) significantly increased the incidence of SPW‐Rs. Furthermore, β adrenoreceptor activation significantly facilitated induction of both LTP and SPW‐Rs within the CA3 network. Suppression of SPW‐Rs by NE was associated with a moderate hyperpolarization in the majority of CA3 pyramidal cells and with a reduction of presynaptic Ca2+ uptake in the stratum radiatum. This was indicated by activity‐dependent changes in [Ca2+]o and Ca2+fluorescence signals, by changes in the paired pulse ratio of evoked EPSPs and by analysis of the coefficient of variance. In the presence of NE, repeated high frequency stimulation (high‐frequency stimulation (HFS)) failed to induce SPW‐Rs, although SPW‐Rs appeared following washout of NE. Together, our data indicate that the NE‐mediated suppression of hippocampal SPW‐Rs depends on α1 adrenoreceptor activation, while their expression and activity‐dependent induction is facilitated via β1‐adrenoreceptors.

Energy Demand of Synaptic Transmission at the Hippocampal Schaffer-Collateral Synapse

Neuroenergetic models of synaptic transmission predicted that energy demand is highest for action potentials (APs) and postsynaptic ion fluxes, whereas the presynaptic contribution is rather small. Here, we addressed the question of energy consumption at Schaffer-collateral synapses. We monitored stimulus-induced changes in extracellular potassium, sodium, and calcium concentration while recording partial oxygen pressure (pO2) and NAD(P)H fluorescence. Blockade of postsynaptic receptors reduced ion fluxes as well as pO2 and NAD(P)H transients by ~50%. Additional blockade of transmitter release further reduced Na+, K+, and pO2 transients by ~30% without altering presynaptic APs, indicating considerable contribution of Ca2+-removal, transmitter and vesicle turnover to energy consumption.

In vitro seizure like events and changes in ionic concentration

BACKGROUND In vivo, seizure like events are associated with increases in extracellular K(+) concentration, decreases in extracellular Ca(2+) concentration, diphasic changes in extracellular sodium, chloride, and proton concentration, as well as changes of extracellular space size. These changes point to mechanisms underlying the induction, spread and termination of seizure like events. METHODS We investigated the potential role of alterations of the ionic environment on the induction of seizure like events-considering a review of the literature and own experimental work in animal and human slices. RESULTS Increasing extracellular K(+) concentration, lowering extracellular Mg(2+) concentration, or lowering extracellular Ca(2+) concentration can induce seizure like events. In human tissue from epileptic patients, elevation of K(+) concentration induces seizure like events in the dentate gyrus and subiculum. A combination of elevated K(+) concentration and 4-AP or bicuculline can induce seizure like events in neocortical tissue. CONCLUSIONS These protocols provide insight into the mechanisms involved in seizure initiation, spread and termination. Moreover, pharmacological studies as well as studies on mechanisms underlying pharmacoresistance are feasible.

Nonspecific effects of the gap junction blocker mefloquine on fast hippocampal network oscillations in the adult rat in vitro

It has been suggested that gap junctions are involved in the synchronization during high frequency oscillations as observed during sharp wave-ripple complexes (SPW-Rs) and during recurrent epileptiform discharges (REDs). Ripple oscillations during SPW-Rs, possibly involved in memory replay and memory consolidation, reach frequencies of up to 200 Hz while ripple oscillations during REDs display frequencies up to 500 Hz. These fast oscillations may be synchronized by intercellular interactions through gap junctions. In area CA3, connexin 36 (Cx36) proteins are present and potentially sensitive to mefloquine. Here, we used hippocampal slices of adult rats to investigate the effects of mefloquine, which blocks Cx36, Cx43 and Cx50 gap junctions on both SPW-Rs and REDs. SPW-Rs were induced by high frequency stimulation in the CA3 region while REDs were recorded in the presence of the GABA(A) receptor blocker bicuculline (5 μM). Both, SPW-Rs and REDs were blocked by the gap junction blocker carbenoxolone. Mefloquine (50 μM), which did not affect stimulus-induced responses in area CA3, neither changed SPW-Rs nor superimposed ripple oscillations. During REDs, 25 and 50 μM mefloquine exerted only minor effects on the expression of REDs but significantly reduced the amplitude of superimposed ripples by ∼17 and ∼54%, respectively. Intracellular recordings of CA3 pyramidal cells revealed that mefloquine did not change their resting membrane potential and input resistance but significantly increased the afterhyperpolarization following evoked action potentials (APs) resulting in reduced probability of AP firing during depolarizing current injection. Similarly, mefloquine caused a reduction in AP generation during REDs. Together, our data suggest that mefloquine depressed RED-related ripple oscillations by reducing high frequency discharges and not necessarily by blocking electrical coupling.

Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy

It was previously shown that the ictogenic potential of 4-aminopyridine (4-AP) was reduced in the parahippocampal region of kainate treated chronic epileptic rats. In the actual study we investigated the potential of 4-aminopyridine (50 and 100μM) to induce seizure like events (SLEs) in combined entorhinal cortex hippocampal slices from Wistar rats following pilocarpine induced status epilepticus. The potential of 4-AP to induce SLEs in the entorhinal cortex was reduced in the latent period and in slices of chronic epileptic animals with a high seizure incidence in vivo (>2seizures/24h). 4-AP induced SLEs in slices from animals with a low incidence of seizures in vivo (<2seizures/24h) in a similar manner as compared to controls. The hippocampal formation displayed no SLEs, instead short recurrent epileptiform discharges (REDs) were evoked by application of 4-AP in areas CA3 and CA1. The incidence of REDs was largest in slices from control animals. This study shows that the reduced ictogenic potential of 4-AP is not restricted to kainate treated chronic epileptic animals as it can be found in the pilocarpine model as well. The underlying mechanisms may relate to altered expression and editing of voltage gated potassium channels.

Enhanced dopamine-dependent hippocampal plasticity after single MK-801 application.

Dopaminergic hyperfunction and N-methyl-D-aspartate receptor (NMDAR) hypofunction have both been implicated in psychosis. Dopamine-releasing drugs and NMDAR antagonists replicate symptoms associated with psychosis in healthy humans and exacerbate symptoms in patients with schizophrenia. Though hippocampal dysfunction contributes to psychosis, the impact of NMDAR hypofunction on hippocampal plasticity remains poorly understood. Here, we used an NMDAR antagonist rodent model of psychosis to investigate hippocampal long-term potentiation (LTP). We found that single systemic NMDAR antagonism results in a region-specific, presynaptic LTP at hippocampal CA1-subiculum synapses that is induced by activation of D1/D5 dopamine receptors and modulated by L-type voltage-gated Ca2+ channels. Thereby, our findings may provide a cellular mechanism how NMDAR antagonism can lead to an enhanced hippocampal output causing activation of the hippocampus-ventral tegmental area-loop and overdrive of the dopamine system.

A neuronal lactate uptake inhibitor slows recovery of extracellular ion concentration changes in the hippocampal CA3 region by affecting energy metabolism.

Astrocyte-derived lactate supports pathologically enhanced neuronal metabolism, but its role under physiological conditions is still a matter of debate. Here, we determined the contribution of astrocytic neuronal lactate shuttle for maintenance of ion homeostasis and energy metabolism. We tested for the effects of α-cyano-4-hydroxycinnamic acid (4-CIN), which could interfere with energy metabolism by blocking monocarboxylate-transporter 2 (MCT2)-mediated neuronal lactate uptake, on evoked potentials, stimulus-induced changes in K+, Na+, Ca2+, and oxygen concentrations as well as on changes in flavin adenine dinucleotide (FAD) autofluorescence in the hippocampal area CA3. MCT2 blockade by 4-CIN reduced synaptically evoked but not antidromic population spikes. This effect was dependent on the activation of KATP channels indicating reduced neuronal ATP synthesis. By contrast, lactate receptor activation by 3,5-dihydroxybenzoic acid (3,5-DHBA) resulted in increased antidromic and orthodromic population spikes suggesting that 4-CIN effects are not mediated by lactate accumulation and subsequent activation of lactate receptors. Recovery kinetics of all ion transients were prolonged and baseline K+ concentration became elevated by blockade of lactate uptake. Lactate contributed to oxidative metabolism as both baseline respiration and stimulus-induced changes in Po2 were decreased, while FAD fluorescence increased likely due to a reduced conversion of FAD into FADH2 These data suggest that lactate shuttle contributes to regulation of ion homeostatsis and synaptic signaling even in the presence of ample glucose.

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism‐dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO2, field potentials and changes in extracellular potassium concentration ([K+]o).

A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence.