Christoph J. Behrens

Induction of sharp wave-ripple complexes in vitro and reorganization of hippocampal networks

Hippocampal sharp wave–ripple complexes (SPW-Rs) occur during slow-wave sleep and behavioral immobility and are thought to represent stored information that is transferred to the neocortex during memory consolidation. Here we show that stimuli that induce long-term potentiation (LTP), a neurophysiological correlate of learning and memory, can lead to the generation of SPW-Rs in rat hippocampal slices. The induced SPW-Rs have properties that are identical to spontaneously generated SPW-Rs: they originate in CA3, propagate to CA1 and subiculum and require AMPA/kainate receptors. Their induction is dependent on NMDA receptors and involves changes in interactions between clusters of neurons in the CA3 network. Their expression is blocked by low-frequency stimulation but not by NMDA receptor antagonists. These data indicate that induction of LTP in the recurrent CA3 network may facilitate the generation of SPW-Rs.

Cholinergic dysfunction in temporal lobe epilepsy

Summary:  The entorhinal cortex–hippocampus complex is believed to be the site of origin of seizure activity in the majority of patients with temporal lobe epilepsy (TLE). Both these regions are enriched with cholinergic innervation, which plays a key role in the normal control of neuronal excitability and in higher cognitive processes. In TLE, anatomical and functional changes occur in all cellular components of the local neural circuit. Thus, while it is not surprising that cholinergic functions are altered in the epileptic temporal lobe, the exact nature and role of these changes in the pathogenesis of the disease are not known. In this report, we summarize the scientific background and experimental data supporting a “cholinergic hypothesis of TLE.” We conclude that while the exact role of cholinergic dysfunction in TLE is not known, there is a firm basis for suggesting that changes in the expression of key cholinergic proteins—and the associated cholinergic dysfunction—are key factors in the basic mechanisms underlying TLE.

Effects of the GABAA receptor antagonists bicuculline and gabazine on stimulus-induced sharp wave-ripple complexes in adult rat hippocampus in vitro

Hippocampal sharp wave‐ripple‐complexes (SPW‐Rs) are characterized by slow field potential transients superimposed by ripples with a frequency of ∼200 Hz. In epileptic humans and rats frequencies of such transient network oscillations can reach up to 500 Hz potentially due to loss of functional inhibition. Therefore, we investigated whether GABAA receptor antagonists increased ripple frequency during SPW‐Rs. Within area CA3, SPW‐Rs were induced by repeated stimulation of stratum radiatum in area CA1 of adult Wistar rat hippocampal slices. Intracellular recordings showed that in approximately 50% of recorded CA3 pyramidal cells SPW‐Rs were accompanied by compound excitory postsynaptic potentials (EPSPs) of ∼10 mV superimposed by up to four action potentials (APs). The remaining cells responded with a compound inhibitory postsynaptic potential (IPSP) during SPW‐Rs. The GABAA receptor antagonists bicuculline (BMI) or gabazine (SR‐95531) led to a transition of SPW‐Rs into prolonged bursts with a significant increase in amplitude and duration reminiscent of recurrent epileptiform discharges (REDs). Ripple frequencies increased from ∼190 Hz to ∼300 Hz. In naïve slices SR‐95531 and BMI also evoked REDs with similar incidence and high frequency ripple frequencies of ∼240 Hz. Elevations in extracellular potassium concentration during REDs were approximately 20‐fold higher than those observed during SPW‐Rs. Intracellular recordings revealed bursts that were characterized by a large (> 25 mV) prolonged depolarization superimposed by up to 40 APs in close synchrony with extracellularly recorded ripples. Our results suggest that the generation of high frequency ripples, which are also observed in epileptic humans and rats, could indicate a loss of functional inhibition.

Acetylcholine-induced seizure-like activity and modified cholinergic gene expression in chronically epileptic rats

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)‐hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre‐mRNA transcripts, accompanied by a shift from membrane‐bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10–100 µm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds‐long seizure‐like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh‐induced seizure‐like events could be completely blocked by the non‐specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic‐1 receptor antagonist, pirenzepine; but were not affected by the non‐specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

Monoamines block kainate‐ and carbachol‐induced γ‐oscillations but augment stimulus‐induced γ‐oscillations in rat hippocampus in vitro

Monoamines are implicated in a cognitive processes in a variety of brain regions, including the hippocampal formation, where storage and retrieval of information are facilitated by synchronous network activities. We have investigated the effects of norepinephrine, serotonin, and dopamine on carbachol‐, kainate‐, and stimulus‐induced hippocampal γ‐oscillations employing combined extra‐ and intracellular recordings. Monoamines dose‐dependently and reversibly suppressed kainate‐ and carbachol‐induced γ‐oscillations while increasing the frequency. The effect of serotonin was mimicked by fenfluramine, which releases serotonin from presynaptic terminals. Forskolin also suppressed kainate‐ and carbachol‐induced γ‐oscillations. This effect was mimicked by 8‐Br‐cAMP and isoproterenol, an agonist of noradrenergic β‐receptor suggesting that the monoamines‐mediated suppression of these oscillations could involve intracellular cyclic adenosine 3′,5′‐cyclic monophosphate (AMP). By contrast, stimulus‐induced γ‐oscillations were dose‐dependently augmented in power and duration after monoamines application. Intracellular recordings from pyramidal cells revealed that monoamines prolonged the stimulus‐induced depolarization and membrane potential oscillations. Stimulus‐induced γ‐oscillations were also suppressed by isoproterenol, the D1 agonist SKF‐38393 forskolin, and 8‐Br‐cAMP. This suggests that the augmentation of stimulus‐induced γ‐oscillations by monoamines involves—at least in part—different classes of cells than in case of carbachol‐ and kainate‐induced γ‐oscillations.

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.

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.

Hypoxia suppresses kainate-induced gamma-oscillations in rat hippocampal slices.

Hypoxia or global ischemia causes rapid loss of consciousness and a sudden increase in spontaneous transmitter release suggesting that coordinated synaptic activity is impaired. Gamma oscillations (30–100 Hz) are thought to provide for binding of parallel processed information in the brain, contributing to cognition and formation of short-term memory. We hypothesized that &ggr;-oscillations are rapidly blocked by hypoxia and that prolonged hypoxia reduces the capability to generate such activity. In ventral hippocampal slices, kainate-induced &ggr;-oscillations reversibly declined 40 s after onset of 3 min hypoxia. Repetition of such hypoxic periods led to accumulative impairment of &ggr;-activities. By contrast, 6 min of hypoxia led to a transient anoxic depolarization after which &ggr;-oscillations remained almost completely blocked.

C-type natriuretic peptide decreases hippocampal network oscillations in adult rats in vitro

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the human brain and the cerebrospinal fluid. CNP is involved in anxiogenesis and exerts its effects through the natriuretic peptide receptor B (NPR-B), which is expressed in the hippocampus. Hippocampal network oscillations of distinct frequency bands like gamma (gamma)-oscillations and sharp wave-ripple complexes (SPW-Rs) are likely involved in various cognitive functions such as the storage of information and memory consolidation in vivo. Here, we tested the effects of CNP on distinct network oscillations in horizontal slices of rat hippocampus. We found that CNP decreased the power of stimulus- and ACh/physostigmine-induced gamma-oscillations. In contrast to stimulus-induced gamma-oscillations, CNP increased the frequency of ACh-induced, persistent network oscillations. Moreover, the peptide hormone reduced the incidence of LTP-associated SPW-Rs in area CA3 and CA1. Immunohistochemistry indicates that the peptide binds to receptors expressed on a subset of GAD 65-67-immunopositive cells in addition to binding to principal and other presumably non-neuronal cells. CNP caused a hyperpolarization of CA3 neurons increased their input resistance and decreased inhibitory conductance. Together, our data suggest that the effects of CNP on synchronized hippocampal network oscillations might involve effects on hippocampal interneurons.