Different functions of hyperpolarization-activated cation channels for hippocampal sharp waves and ripples in vitro.

Hyperpolarization-activated currents (I(h)) affect multiple neuronal functions including membrane potential, intrinsic firing properties, synaptic integration and frequency-dependent resonance behavior. Consistently, I(h) plays a key role for oscillations at the cellular and network level, including theta and gamma oscillations in rodent hippocampal circuits. Little is known, however, about the contribution of I(h) to a prominent memory-related pattern of network activity called sharp-wave-ripple complexes (SPW-R). Here we report that pharmacological suppression of I(h) induces specific changes in SPW-R in mouse hippocampal slices depending on the specific drug used and the region analyzed. Spontaneous generation of the events was reduced by blocking I(h) whereas the amplitude was unaffected or increased. Interestingly, the superimposed ripple oscillations at ∼200 Hz persisted with unchanged frequency, indicating that I(h) is not critical for generating this rhythmic pattern. Likewise, coupling between field oscillations and units was unchanged, showing unaltered recruitment of neurons into oscillating assemblies. Control experiments exclude a contribution of T-type calcium channels to the observed effects. Together, we report a specific contribution of hyperpolarization-activated cation currents to the generation of sharp waves in the hippocampus.

Deletion of connexin45 in mouse neurons disrupts one-trial object recognition and alters kainate-induced gamma-oscillations in the hippocampus.

Neuronal gap junctions, allowing fast intercellular electrotonic signal transfer, have been implicated in mechanisms governing learning and memory processes. We have examined conditional neuron-directed (Cx45fl/fl:Nestin-Cre) connexin45 deficient mice in terms of behavioral and electrophysiological correlates of learning and memory. Behavioral habituation to a novel environment and motor learning were not changed in these mice. Novel object recognition after delays of up to 60min was impaired in neuronal Cx45 deficient mice. However, object-place recognition was not significantly different from controls. Analysis of enhanced green fluorescent reporter protein expression controlled by the endogenous mouse Cx45 promoter in the brain of neuronal Cx45 deficient mice suggested that Cx45 is expressed in the perirhinal cortex and the CA3 subregion of the hippocampus. The neuronal Cx45 deficient mice were also examined for aberrations in the generation and synchronization of network oscillations in the hippocampus. General excitability, synaptic short time plasticity, and spontaneous high-frequency oscillations (sharp-wave ripples) in the hippocampus were not different from controls. However, bath stimulation of hippocampal slices with kainate induced significantly lower gamma-oscillation amplitudes in the CA3, but not in the CA1 subfield of the neuronal Cx45 deficient mice. Additionally, they exhibited a significantly larger full width half maximum of the frequency distribution in the CA1 subfield as compared to the controls. In conclusion, the neuron-directed deletion of Cx45 impaired one-trial novel object recognition and altered kainate-induced gamma-oscillations possibly via the disruption of inter-neuronal gap junctional communication in the hippocampus or perirhinal cortex.

No simple brake--the complex functions of inhibitory synapses.

Synaptic inhibition can be viewed as a counterbalance of synaptic excitation. However, multiple recent studies at the cellular and network level show that inhibition serves a variety of additional, highly specific functions in the mammalian nervous system. At the molecular and cellular level, inhibitory synapses express diverse postsynaptic reversal potentials, kinetics, plasticity, and pharmacological modulation. This heterogeneity corresponds to the complexity of inhibition at the network level, where interneurons are now perceived as diverse and highly specific organizers of coherent activity patterns. We review some important new developments in the molecular, cellular and network physiology of inhibition. It turns out that understanding inhibition is a key to understanding neuronal network behaviour and, ultimately, may provide important clues for the development of novel therapeutic strategies in neuro-psychiatric diseases.

Region- and pattern-specific effects of glutamate uptake blockers on epileptiform activity in rat brain slices.

Many epileptic syndromes develop into pharmaco-resistant forms, calling for the development of new anticonvulsant strategies. The transmitter glutamate serves a double role as excitatory transmitter and as precursor for GABA, thus interfering with glutamate uptake may therefore exert complex effects on excitation-inhibition-balance in epileptic networks. In the present study we tested the effect of two different glutamate uptake blockers on acutely induced epileptiform activity in hippocampal-entorhinal cortex slices from adult rats: dihydrokainate (DHK) which blocks predominantly glial glutamate uptake, and threo-beta-benzyloxyaspartic acid (TBOA) which blocks both glial and neuronal glutamate uptake. Three different models were used to induce epileptiform discharges: (i) increasing NMDA receptor-mediated excitation by omitting Mg(2+)-ions; (ii) blocking potassium channels by 4-aminopyridine; (iii) reducing GABA(A) receptor-mediated inhibition by penicillin. Application of DHK or TBOA markedly reduced the frequency of epileptiform discharges in CA1 in the low magnesium and the 4-AP model while pathological activity was increased in the penicillin-model. In contrast, frequency of epileptiform discharges in EC was consistently increased by DHK and TBOA. Effects of DHK were more easily reversible than those of TBOA. Thus glutamate uptake blockers exert variable effects on epileptiform activity, depending on brain region and on the mechanism of ictogenesis.

Synaptic transmission is impaired prior to plaque formation in amyloid precursor protein-overexpressing mice without altering behaviorally-correlated sharp wave-ripple complexes.

One of the hallmarks of Alzheimer's disease is the accumulation of amyloid plaques in brains of affected patients. Several recent studies provided evidence that soluble oligomer forms of amyloid-beta (Abeta) rather than plaques determine cognitive decline. In vitro studies using artificial Abeta oligomer preparations suggest that such pathophysiology is caused by a specific impairment of synaptic function. We examined whether synaptic deficits occur before deposition of insoluble fibrillar Abeta by analyzing brain slices taken from young Tg2576 mice overexpressing mutant amyloid precursor protein. Excitatory synaptic transmission in the hippocampal CA1 region was strongly impaired before plaque development, suggesting a dissociation of an early synaptic impairment, probably caused by soluble oligomeric amyloid-beta, from subsequent plaque formation. At higher age neurotransmission was also decreased in wild type mice, paralleling a cognitive decline of normal aged animals. Memory formation in rats is accompanied by distinct hippocampal network oscillations. It has recently been shown that hippocampal gamma oscillations, a network correlate of exploratory behavior, are impaired in amyloid precursor protein (APP)-overexpressing mice. We determined whether sharp wave-ripple complexes, which contribute to memory consolidation during slow wave-sleep, are modified in Tg2576 mice. Interestingly, neither sharp waves nor superimposed ripples were changed at pre-plaque or plaque stages. During aging, however, there was a strong reduction of sharp wave frequency and ripple energy in wild type and APP-overexpressing animals. This indicates that the reported changes in network oscillations following APP-overexpression are specific for gamma oscillations, whereas aging has a more general effect on network properties. Taken together our data suggest that non-fibrillar forms of Abeta--possibly Abeta oligomers--specifically interfere with synaptic function in Tg2576, but do not globally alter memory-related network properties. We propose that mechanisms leading to Abeta-related cognitive decline are different from those related to aging.

Fast effects of glucocorticoids on memory-related network oscillations in the mouse hippocampus.

Transient or lasting increases in glucocorticoids accompany deficits in hippocampus-dependent memory formation. Recent data indicate that the formation and consolidation of declarative and spatial memory are mechanistically related to different patterns of hippocampal network oscillations. These include gamma oscillations during memory acquisition and the faster ripple oscillations (approximately 200 Hz) during subsequent memory consolidation. We therefore analysed the effects of acutely applied glucocorticoids on network activity in mouse hippocampal slices. Evoked field population spikes and paired-pulse responses were largely unaltered by corticosterone or cortisol, respectively, despite a slight increase in maximal population spike amplitude by 10 microm corticosterone. Several characteristics of sharp waves and superimposed ripple oscillations were affected by glucocorticoids, most prominently the frequency of spontaneously occurring sharp waves. At 0.1 microm, corticosterone increased this frequency, whereas maximal (10 microm) concentrations led to a reduction. In addition, gamma oscillations became slightly faster and less regular in the presence of high doses of corticosteroids. The present study describes acute effects of glucocorticoids on sharp wave-ripple complexes and gamma oscillations in mouse hippocampal slices, revealing a potential background for memory deficits in the presence of elevated levels of these hormones.

Enhanced synaptic excitation-inhibition ratio in hippocampal interneurons of rats with temporal lobe epilepsy.

A common feature of all epileptic syndromes is the repetitive occurrence of pathological patterns of synchronous neuronal activity, usually combined with increased neuronal discharge rates. Inhibitory interneurons of the hippocampal formation control both neuronal synchronization as well as the global level of activity and are therefore of crucial importance for epilepsy. Recent evidence suggests that changes in synaptic inhibition during temporal lobe epilepsy are rather specific, resulting from selective death or alteration of interneurons in specific hippocampal layers. Hence, epilepsy-induced changes have to be analysed separately for different types of interneurons. Here, we focused on GABAergic neurons located at the border between stratum radiatum and stratum lacunosum-moleculare of hippocampal area CA1 (SRL interneurons), which are included in feedforward inhibitory circuits. In chronically epileptic rats at 6-8 months after pilocarpine-induced status epilepticus, frequencies of spontaneous and miniature inhibitory postsynaptic currents were reduced, yielding an almost three-fold increase in excitation-inhibition ratio. Consistently, action potential frequency of SRL interneurons was about two-fold enhanced. Morphological alterations of the interneurons indicate that these functional changes were accompanied by remodelling of the local network, probably resulting in a loss of functional inhibitory synapses without conceivable cell death. Our data indicate a strong increase in activity of interneurons in dendritic layers of the chronically epileptic CA1 region. This alteration may enhance feedforward inhibition and rhythmogenesis and--together with specific changes in other interneurons--contribute to seizure susceptibility and pathological synchronization.

Layer-specific potentiation of evoked IPSCs in rat hippocampal CA1 pyramidal cells by lanthanum.

Lanthanum (La3+) potentiates or depresses GABAA receptors (GABAAR) in a subunit-dependent manner. Such differential modulators may help to discriminate between-layer-specific inhibitory synaptic inputs to individual neurons, which use different molecular GABAAR isoforms. Here, we show that inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells are potentiated by La3+ (100 microM) when they are evoked by stimulation in stratum oriens. In contrast, stimulation in stratum radiatum yields IPSCs which are not sensitive towards La3+. These data point towards an input-specific molecular and functional diversity of inhibitory synapses at CA1 pyramidal cells.

High frequency oscillations in the dentate gyrus of rat hippocampal slices induced by tetanic stimulation.

Tetanic stimulation induces high-frequency network oscillations in area CA1 and in the subiculum of rat hippocampal slices. Here, we describe the effects of similar tetanic stimulation in the molecular layer of the dentate gyrus. We found field potential oscillations in the dentate granule cell layer which shared several properties with tetanically induced oscillations in CA1, including delayed onset, duration, progressive slowing of frequency within the oscillations and sensitivity to blockers of GABA(A) receptors, NMDA receptors and metabotropic glutamate receptors. However, the mean frequency of the oscillations in the dentate is approximately 100 Hz, much higher than tetanic oscillations in CA1 and, in contrast to CA1, dentate high-frequency oscillations are sensitive to antagonists of AMPA-receptors. Oscillation frequency was decreased by metabotropic glutamate receptor antagonists and increased by antagonists of AMPA-receptors as well as the gap junction blocker carbenoxolone. The oscillations can be observed in the whole dentate gyrus-CA3-network and are tightly correlated between the dentate gyrus and area CA3. Thus, tetanic stimulation in the dentate elicits a new pattern of network oscillations with coherence in the dentate-CA3-network which may affect the processing of afferent information in the hippocampus.

Tetanus toxin induces long-term changes in excitation and inhibition in the rat hippocampal CA1 area.

Intrahippocampal tetanus toxin induces a period of chronic recurrent limbic seizures in adult rats, associated with a failure of inhibition in the hippocampus. The rats normally gain remission from their seizures after 6-8 weeks, but show persistent cognitive impairment. In this study we assessed which changes in cellular and network properties could account for the enduring changes in this model, using intracellular and extracellular field recordings in hippocampal slices from rats injected with tetanus toxin or vehicle, 5 months previously. In CA1 pyramidal neurones from toxin-injected rats, the slope of the action potential upstroke was reduced by 32%, the fast afterhyperpolarisation by 32% and the slow afterhyperpolarisation by 54%, suggesting changes in voltage-dependent conductances. The excitatory postsynaptic potential slope was reduced by 60% and the population synaptic potential slope was reduced at all stimulus intensities, suggesting a reduced afferent input in CA1. Paired-pulse stimulation showed an increase of the excitability ratio and an increase of cellular excitability only for the second pulse, suggesting a reduced inhibition. The polysynaptic inhibitory postsynaptic potential was reduced by 34%, whereas neither the inhibitory postsynaptic potential at subthreshold stimulus intensities,nor the pharmacologically isolated monosynaptic inhibitory postsynaptic potential were different in toxin-injected rats, suggesting a reduced synaptic excitation of interneurones. Stratum radiatum stimuli in toxin-injected rats, and not in controls, evoked antidromic activation of CA1 neurones, demonstrating axonal sprouting into areas normally devoid of CA1 pyramidal cell axons.We conclude that this combination of enduring changes in cellular and network properties, both pro-epileptic (increased recurrent excitatory connectivity, reduced recurrent inhibition and reduced afterhyperpolarisations) and anti-epileptic (impaired firing and reduced excitation), reaches a balance that allows remission of seizures, perhaps at the price of persistent cognitive impairment.

GAD and GABA transporter (GAT-1) mRNA expression in the developing rat hippocampus.

Synaptic inhibition in the mammalian central nervous system is mostly mediated by GABA (gamma-aminobutyric acid). Inhibitory interneurons can be identified by staining for glutamate decarboxylase (GAD), the key enzyme which produces the transmitter. After release, GABA is removed from the extracellular space by specific transporters which are localized at the presynaptic endings of interneurons, in adjacent glial processes and, possibly, also in the postsynaptic target cell membranes. The GABAergic system undergoes profound functional and structural changes during the first 2 weeks of postnatal development, including migration of interneurons and changes in the level of expression and subcellular distribution of GABA transporters. We therefore analyzed the distribution of mRNA coding for GAD and GAT-1 (the main neuronal GABA transporter) in the developing rat hippocampus. Our data show that both transcripts are present in putative interneurons from the first postnatal day and exhibit a largely similar distribution throughout postnatal ontogenesis, with some specific differences in certain hippocampal subfields. Quantification of stained somata confirmed the postnatal redistribution of putative interneurons in the area dentata from dendritic layers towards the hilus. We also found a general staining of principal cell layers for both probes, which differs with postnatal age and between GAD and GAT-1 mRNA. Together, our data reveal a profound reorganization of the GABAergic system in the rat hippocampus during the first weeks of postnatal development.

Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain.

The vesicular protein synaptobrevin contributes to two mutually exclusive complexes in mature synapses. Synaptobrevin tightly interacts with the plasma membrane proteins syntaxin and SNAP 25 forming the SNARE complex as a prerequisite for exocytotic membrane fusion. Alternatively, synaptobrevin binds to the vesicular protein synaptophysin. It is unclear whether SNARE complex formation is diminished or facilitated when synaptobrevin is bound to synaptophysin. Here we show that the synaptophysin-synaptobrevin complex is increased in adult rat brain after repeated synaptic hyperactivity in the kindling model of epilepsy. Two days after the last kindling-induced stage V seizure the relative amount of synaptophysin-synaptobrevin complex obtained by co-immunoprecipitation from cortical and hippocampal membranes was increased twofold compared to controls. By contrast the relative amounts of various synaptic proteins as well as that of the SNARE complex did not change in membrane preparations from kindled rats compared to controls. The increased amount of synaptophysin-synaptobrevin complex in kindled rats supports the idea that this complex represents a reserve pool for synaptobrevin enabling synaptic vesicles to adjust to an increased demand for synaptic efficiency. We conclude that the synaptophysin-synaptobrevin interaction is involved in activity-dependent plastic changes in adult rat brain.

Plasticity of rat central inhibitory synapses through GABA metabolism.

1. The production of the central inhibitory transmitter GABA (gamma-aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity. 2. We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA-transaminase (GABA-T) was blocked by gamma-vinyl-GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3-mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole-cell patch clamp technique. 3. Elevated intra-synaptic GABA levels after block of GABA-T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations. 4. The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs. 5. We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up-regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity-dependent regulation of GABA synthesis in mammals.

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Efficacy of background GABA uptake in rat hippocampal slices.

GABA uptake is crucial for the termination of inhibitory synaptic events. In addition, GABA transporters may also control the level of diffusely distributed GABA in the extracellular space. We analysed this function by superfusing rat hippocampal slices with different concentrations of GABA. Whole-cell patch clamp recordings of CA1 pyramidal cells revealed small increases in chloride conductance at 5-10 microM GABA which increased dramatically upon addition of the GABA uptake blocker tiagabine. Tiagabine alone induced a significant chloride conductance indicating that spontaneous release of GABA in hippocampal slices is neutralized by GAT-1, the main hippocampal GABA transporter. Thus, GAT-1 clears the extracellular space in the hippocampus from diffusely distributed GABA with high efficacy.

Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus.

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

Ripple (approximately 200-Hz) oscillations in temporal structures.

Spontaneous network oscillations near 200 Hz have been described in the hippocampus and parahippocampal regions of rodents and humans. During the last decade the characteristics and the mechanisms behind these field "ripples" have been studied extensively, mainly in rodents. They occur during rest or slow-wave sleep and provide a very fast, short-lasting (approximately 50 msec) rhythmic and synchronous activation of specific projection cells and interneurons. Ripples are frequently triggered by a massive synaptic activation from the hippocampal CA3 subfield, which is called a sharp wave. Recent evidence suggests that ripples have a specific task in memory processing-namely, that they convey information stored in the hippocampus to the cortex where it can be preserved permanently. Network mechanisms involved in ripple oscillations may be relevant for understanding pathologic synchronization processes in temporal lobe epilepsy.

Presence of gamma-aminobutyric acid transporter mRNA in interneurons and principal cells of rat hippocampus.

After release, neurotransmitters are removed from the extracellular space by high-affinity uptake. Specific sodium-dependent transporters serve this function for the inhibitory transmitter gamma-aminobutyric acid (GABA). However, it is largely unknown to which proportion GABA is taken up by GABAergic interneurons, glia cells or principal neurons. We analyzed the distribution of mRNA for the main GABA-transporter subtype in the hippocampus, GAT-1, in adult rats. Most interneurons were strongly stained for GAT-1 mRNA, indicating re-uptake by the GABA-releasing cells. Surprisingly, prominent signals for GAT-1 were also found throughout the principal cell layers (granule and pyramidal cells). These data indicate that GABA transporters may be present in non-GABAergic projection cells of the rat hippocampus which contribute to the clearance of GABA from the extracellular space.

Acute effects of gamma-vinyl-GABA on low-magnesium evoked epileptiform activity in vitro.

Vigabatrin (gamma-vinyl-GABA, VGB) is a gamma-aminobutyric acid (GABA) derivative designed to boost synaptic inhibition by inhibiting the degradation of GABA in brain tissue. Indeed, VGB shows potent anti-convulsant activity in animal models of epilepsy and in humans with complex partial seizures. However, details of the mechanism of action of VGB are not well understood and the systemic effects include possible pro-convulsant actions. We therefore analysed the effects of VGB in rat brain slices in the low-Mg(2+) model in vitro. VGB at 100 microM-5 mM showed a concentration- and time-dependent reduction of interictal-like events in the hippocampal CA1 region. Likewise, VGB suppressed epileptiform discharges in the medial entorhinal cortex (mEC), which are known to resist conventional anti-convulsants. In contrast, evoked population spikes in CA1 (which became repetitive after washout Mg(2+)) were not altered by VGB. Our data show that VGB is efficient against epileptiform discharges in temporal structures including pharmacoresistant patterns of activity. The waveform of evoked population spikes in this in vitro model is no indicator for the anti-convulsant properties of drugs.

Expression of Kv1 potassium channels in mouse hippocampal primary cultures: development and activity-dependent regulation.

Excitability and discharge behavior of neurons depends on the highly variable expression pattern of voltage-dependent potassium (Kv) channels throughout the nervous system. To learn more about distribution, development, and activity-dependent regulation of Kv channel subunit expression in the rodent hippocampus, we studied the protein expression of members of the Kv1 subfamily in mouse hippocampus in situ and in primary cultures. In adult hippocampus, Kv1 (1-6) channel alpha-subunits were present, whereas at postnatal day 2, none of these proteins could be detected in CA1-CA3 and dentate gyrus. Kv1.1 was the first channel to be observed at postnatal day 6. The delayed postnatal expression and most of the subcellular distribution observed in hippocampal sections were mimicked by cultured hippocampal neurons in which Kv channels appeared only after 10 days in vitro. This developmental upregulation was paralleled by a dramatic increase in total K(+) current, as well as an elevated GABA release in the presence of 4-aminopyridine. Thus, the developmental profile, subcellular localization, and functionality of Kv1 channels in primary culture of hippocampus closely resembles the in situ situation. Impairing secretion by clostridial neurotoxins or blocking activity by tetrodotoxin inhibited the expression of Kv1.1, Kv1.2, and Kv1.4, whereas the other Kv1 channels still appeared. This activity-dependent depression was only observed before the initial appearance of the respective channels and lost after they had been expressed. Our data show that hippocampal neurons in culture are a convenient model to study the developmental expression and regulation of Kv1 channels. The ontogenetic regulation and the activity-dependent expression of Kv1.1, Kv1.2, and Kv1.4 indicate that neuronal activity plays a crucial role for the development of the mature Kv channel pattern in hippocampal neurons.