Lucia Wittner

Perturbed Chloride Homeostasis and GABAergic Signaling in Human Temporal Lobe Epilepsy

Changes in chloride (Cl−) homeostasis may be involved in the generation of some epileptic activities. In this study, we asked whether Cl− homeostasis, and thus GABAergic signaling, is altered in tissue from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Slices prepared from this human tissue generated a spontaneous interictal-like activity that was initiated in the subiculum. Records from a minority of subicular pyramidal cells revealed depolarizing GABAA receptor-mediated postsynaptic events, indicating a perturbed Cl− homeostasis. We assessed possible contributions of changes in expression of the potassium–chloride cotransporter KCC2. Double in situ hybridization showed that mRNA for KCC2 was absent from ∼30% of CaMKIIα (calcium/calmodulin-dependent protein kinase IIα)-positive subicular pyramidal cells. Combining intracellular recordings with biocytin-filled electrodes and KCC2 immunochemistry, we observed that all cells that were hyperpolarized during interictal events were immunopositive for KCC2, whereas the majority of depolarized cells were immunonegative. Bumetanide, at doses that selectively block the chloride-importing potassium–sodium–chloride cotransporter NKCC1, produced a hyperpolarizing shift in GABAA reversal potentials and suppressed interictal activity. Changes in Cl− transporter expression thus contribute to human epileptiform activity, and molecules acting on these transporters may be useful antiepileptic drugs.

The human K-complex represents an isolated cortical down-state.

Down But Not Out The K-complex, a defining characteristic of slow wave sleep, is the largest spontaneously occurring component of the healthy human electroencephalogram (EEG) but little is known about its physiological characteristics in the human cortex. Cash et al. (p. 1084) investigated the intracortical origin of K-complexes in humans undergoing surgery for epileptic seizures. In simultaneous subdural EEG and intracortical multisite microelectrode recordings, K complexes represented cortical downstates reflecting a decrease in neural firing. These down-states are a fundamental mode of cortical operation that have been well studied in animals and may contribute to sleep preservation and memory consolidation. A characteristic electroencephalogram pattern seen during sleep is accompanied by a steep decline in neural activity. The electroencephalogram (EEG) is a mainstay of clinical neurology and is tightly correlated with brain function, but the specific currents generating human EEG elements remain poorly specified because of a lack of microphysiological recordings. The largest event in healthy human EEGs is the K-complex (KC), which occurs in slow-wave sleep. Here, we show that KCs are generated in widespread cortical areas by outward dendritic currents in the middle and upper cortical layers, accompanied by decreased broadband EEG power and decreased neuronal firing, which demonstrate a steep decline in network activity. Thus, KCs are isolated “down-states,” a fundamental cortico-thalamic processing mode already characterized in animals. This correspondence is compatible with proposed contributions of the KC to sleep preservation and memory consolidation.

Laminar analysis of slow wave activity in humans

Brain electrical activity is largely composed of oscillations at characteristic frequencies. These rhythms are hierarchically organized and are thought to perform important pathological and physiological functions. The slow wave is a fundamental cortical rhythm that emerges in deep non-rapid eye movement sleep. In animals, the slow wave modulates delta, theta, spindle, alpha, beta, gamma and ripple oscillations, thus orchestrating brain electrical rhythms in sleep. While slow wave activity can enhance epileptic manifestations, it is also thought to underlie essential restorative processes and facilitate the consolidation of declarative memories. Animal studies show that slow wave activity is composed of rhythmically recurring phases of widespread, increased cortical cellular and synaptic activity, referred to as active- or up-state, followed by cellular and synaptic inactivation, referred to as silent- or down-state. However, its neural mechanisms in humans are poorly understood, since the traditional intracellular techniques used in animals are inappropriate for investigating the cellular and synaptic/transmembrane events in humans. To elucidate the intracortical neuronal mechanisms of slow wave activity in humans, novel, laminar multichannel microelectrodes were chronically implanted into the cortex of patients with drug-resistant focal epilepsy undergoing cortical mapping for seizure focus localization. Intracortical laminar local field potential gradient, multiple-unit and single-unit activities were recorded during slow wave sleep, related to simultaneous electrocorticography, and analysed with current source density and spectral methods. We found that slow wave activity in humans reflects a rhythmic oscillation between widespread cortical activation and silence. Cortical activation was demonstrated as increased wideband (0.3-200 Hz) spectral power including virtually all bands of cortical oscillations, increased multiple- and single-unit activity and powerful inward transmembrane currents, mainly localized to the supragranular layers. Neuronal firing in the up-state was sparse and the average discharge rate of single cells was less than expected from animal studies. Action potentials at up-state onset were synchronized within +/-10 ms across all cortical layers, suggesting that any layer could initiate firing at up-state onset. These findings provide strong direct experimental evidence that slow wave activity in humans is characterized by hyperpolarizing currents associated with suppressed cell firing, alternating with high levels of oscillatory synaptic/transmembrane activity associated with increased cell firing. Our results emphasize the major involvement of supragranular layers in the genesis of slow wave activity.

Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus. We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the axon loses parvalbumin or immunoreactivity for parvalbumin. The hyperinnervation of axon initial segments might be a compensatory change in the inhibitory network, but at the same time may lead to a more effective synchronization of granule cell firing that could contribute to the generation or amplification of epileptic seizures.

Changes in the distribution and connectivity of interneurons in the epileptic human dentate gyrus

The distribution, size, dendritic morphology and synaptic connections of calbindin-, calretinin- and substance P receptor-positive interneurons and pathways have been examined in control and epileptic human dentate gyrus. In the epileptic dentate gyrus, calbindin-containing interneurons are preserved, but their dendrites become elongated and spiny, and several cell bodies appear hypertrophic. The relative laminar distribution of calretinin-containing cells did not change, but their number was considerably reduced. The calretinin-positive axonal bundle at the top of the granule cell layer originating from the supramammillary nucleus expanded, forming a dense network in the entire width of the stratum moleculare. Substance P receptor-immunopositive cells were partially lost in epileptic samples, and in addition, the laminar distribution and dendritic morphology of the surviving cells differed considerably from the controls. In the control human dentate gyrus, the majority of substance P receptor-positive cells can be seen in the hilus, while most are present in the stratum moleculare in the epileptic tissue. Their synaptic input is also changed. The extent of individual pathological abnormalities correlates with each other in most cases. Our data suggest, that although a large proportion of inhibitory interneurons are preserved in the epileptic human dentate gyrus, their distribution, morphology and synaptic connections differ from controls. These functional alterations of inhibitory circuits in the dentate gyrus are likely to be compensatory changes with a role to balance the enhanced excitatory input in the region.

Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy

The distribution, morphology, synaptic coverage and postsynaptic targets of calbindin-containing interneurons and afferent pathways have been analyzed in the control and epileptic CA1 region of the human hippocampus. Numerous calbindin-positive interneurons are preserved even in the strongly sclerotic CA1 region. The morphology of individual cells is altered: the cell body and dendrites become spiny, the radially oriented dendrites disappear, and are replaced by a large number of curved, distorted dendrites. Even in the non-sclerotic epileptic samples, where pyramidal cells are present and calbindin-immunoreactive interneurons seem to be unchanged, some modifications could be observed at the electron microscopic level: they received more inhibitory synaptic input, and the calbindin-positive excitatory afferents - presumably derived from the CA1, the CA2 and/or the dentate gyrus - are sprouted. In the strongly sclerotic tissue, with the death of pyramidal cells, calbindin-positive terminals (belonging to interneurons and the remaining excitatory afferents) change their targets. Our data suggest that an intense synaptic reorganization takes place in the epileptic CA1 region, even in the non-sclerotic tissue, before the death of considerable numbers of pyramidal cells. Calbindin-positive interneurons participate in this reorganization: they show plastic changes in response to epilepsy. The enhanced inhibition of inhibitory interneurons may result in the disinhibition of pyramidal cells or in an abnormal synchrony in the output region of the hippocampus.

The epileptic human hippocampal cornu ammonis 2 region generates spontaneous interictal-like activity in vitro

The dentate gyrus, the cornu ammonis 2 region and the subiculum of the human hippocampal formation are resistant to the cell loss associated with temporal lobe epilepsy. The subiculum, but not the dentate gyrus, generates interictal-like activity in tissue slices from epileptic patients. In this study, we asked whether a similar population activity is generated in the cornu ammonis 2 region and examined the electrophysiological and neuroanatomical characteristics of human epileptic cornu ammonis 2 neurons that may be involved. Hippocampal slices were prepared from postoperative temporal lobe tissue derived from epileptic patients. Field potentials and multi-unit activity were recorded in vitro using multiple extracellular microelectrodes. Pyramidal cells were characterized in intra-cellular records and were filled with biocytin for subsequent anatomy. Fluorescent immunostaining was made on fixed tissue against the chloride-cation cotransporters sodium-potassium-chloride cotransporter-1 and potassium-chloride cotransporter-2. Light and electron microscopy were used to examine the parvalbumin-positive perisomatic inhibitory network. In 15 of 20 slices, the hippocampal cornu ammonis 2 region generated a spontaneous interictal-like activity, independently of population events in the subiculum. Most cornu ammonis 2 pyramidal cells fired spontaneously. All cells fired single action potentials and burst firing was evoked in three cells. Spontaneous excitatory postsynaptic potentials were recorded in all cells, but hyperpolarizing inhibitory postsynaptic potentials were detected in only 27% of the cells. Two-thirds of cornu ammonis 2 neurons showed depolarizing responses during interictal-like events, while the others were inhibited, according to the current sink in the cell body layer. Two biocytin-filled cells both showed a pyramidal-like morphology with axons projecting to the cornu ammonis 2 and cornu ammonis 3 regions. Expression of sodium-potassium-chloride cotransporter-1 and potassium-chloride cotransporter-2 was reduced in some cells of the epileptic cornu ammonis 2 region, but not to an extent corresponding to the proportion of cells in which hyperpolarizing postsynaptic potentials were absent. Numbers of parvalbumin-positive inhibitory cells and axons were shown to be decreased in the epileptic tissue. Electron microscopy showed the preservation of somatic inhibitory input of cornu ammonis 2 cells, and confirmed the loss of parvalbumin from the interneurons rather than their death. An extra excitatory input (partly coming from sprouted mossy fibres) was demonstrated to innervate cornu ammonis 2 cell bodies. Our results show that the cornu ammonis 2 region of the sclerotic human hippocampus can generate an independent epileptiform activity. Inhibitory and excitatory signalling were functional but modified in epileptic cornu ammonis 2 pyramidal cells. Overexcitation and the altered functional properties of perisomatic inhibitory network, rather than a modified chloride homeostasis, may account for the perturbed gamma-aminobutyric acid-ergic signalling and the generation of interictal-like activity in the human epileptic cornu ammonis 2 region.

Simultaneous activation of gamma and theta network oscillations in rat hippocampal slice cultures

Hippocampal activity in vivo is characterized by concurrent oscillations at theta (4–15 Hz) and gamma (20–80 Hz) frequencies. Here we show that cholinergic receptor activation (methacholine 10–20 nm) in hippocampal slice cultures induces an oscillatory mode of activity, in which the intrinsic network oscillator (located in the CA3 area) expresses simultaneous theta and gamma network oscillations. Pyramidal cells display synaptic theta oscillations, characterized by cycles consisting of population EPSP‐IPSP sequences that are dominated by population IPSPs. These rhythmic IPSPs most probably result from theta‐modulated spiking activity of several interneurons. At the same time, the majority of interneurons consistently display synaptic gamma oscillations. These oscillatory cycles consist of fast depolarizing rhythmic events that are likely to reflect excitatory input from CA3 pyramidal cells. Interneurons comprising this functional group were identified morphologically. They include four known types of interneurons (basket, O‐LM, bistratified and str. lucidum‐specific cells) and one new type of CA3 interneuron (multi‐subfield cell). The oscillatory activity of these interneurons is only weakly correlated between neighbouring cells, and in about half of these (44 %) is modulated by depolarizing theta rhythmicity. The overall characteristics of acetylcholine‐induced oscillations in slice cultures closely resemble the rhythmicity observed in hippocampal field and single cell recordings in vivo. Both rhythmicities depend on intrinsic synaptic interactions, and are expressed by different cell types. The fact that these oscillations persist in a network lacking extra‐hippocampal connections emphasizes the importance of intrinsic mechanisms in determining this form of hippocampal activity.

Short and long term biocompatibility of NeuroProbes silicon probes

Brain implants provide exceptional tools to understand and restore cerebral functions. The utility of these devices depends crucially on their biocompatibility and long term viability. We addressed these points by implanting non-functional, NeuroProbes silicon probes, without or with hyaluronic acid (Hya), dextran (Dex), dexamethasone (DexM), Hya+DexM coating, into rat neocortex. Light and transmission electron microscopy were used to investigate neuronal survival and glial response. The surface of explanted probes was examined in the scanning electron microscope. We show that blood vessel disruption during implantation could induce considerable tissue damage. If, however, probes could be inserted without major bleeding, light microscopical evidence of damage to surrounding neocortical tissue was much reduced. At distances less than 100 microm from the probe track a considerable neuron loss ( approximately 40%) occurred at short survival times, while the neuronal numbers recovered close to control levels at longer survival. Slight gliosis was observed at both short and long term survivals. Electron microscopy showed neuronal cell bodies and synapses close (<10 microm) to the probe track when bleeding could be avoided. The explanted probes were usually partly covered by tissue residue containing cells with different morphology. Our data suggest that NeuroProbes silicon probes are highly biocompatible. If major blood vessel disruption can be avoided, the low neuronal cell loss and gliosis should provide good recording and stimulating results with future functional probes. We found that different bioactive molecule coatings had small differential effects on neural cell numbers and gliosis, with optimal results achieved using the DexM coated probes.

Complex Propagation Patterns Characterize Human Cortical Activity during Slow-Wave Sleep

Cortical electrical activity during nonrapid eye movement (non-REM) sleep is dominated by slow-wave activity (SWA). At larger spatial scales (∼2–30 cm), investigated by scalp EEG recordings, SWA has been shown to propagate globally over wide cortical regions as traveling waves, which has been proposed to serve as a temporal framework for neural plasticity. However, whether SWA dynamics at finer spatial scales also reflects the orderly propagation has not previously been investigated in humans. To reveal the local, finer spatial scale (∼1–6 cm) patterns of SWA propagation during non-REM sleep, electrocorticographic (ECoG) recordings were conducted from subdurally implanted electrode grids and a nonlinear correlation technique [mutual information (MI)] was implemented. MI analysis revealed spatial maps of correlations between cortical areas demonstrating SWA propagation directions, speed, and association strength. Highest correlations, indicating significant coupling, were detected during the initial positive-going deflection of slow waves. SWA propagated predominantly between adjacent cortical areas, albeit spatial noncontinuities were also frequently observed. MI analysis further uncovered significant convergence and divergence patterns. Areas receiving the most convergent activity were similar to those with high divergence rate, while reciprocal and circular propagation of SWA was also frequent. We hypothesize that SWA is characterized by distinct attributes depending on the spatial scale observed. At larger spatial scales, the orderly SWA propagation dominates; at the finer scale of the ECoG recordings, non-REM sleep is characterized by complex SWA propagation patterns.