Intracortical mechanisms of single pulse electrical stimulation (SPES) evoked excitations and inhibitions in humans.

Cortico-cortical evoked potentials (CCEPs) elicited by single-pulse electric stimulation (SPES) are widely used to assess effective connectivity between cortical areas and are also implemented in the presurgical evaluation of epileptic patients. Nevertheless, the cortical generators underlying the various components of CCEPs in humans have not yet been elucidated. Our aim was to describe the laminar pattern arising under SPES evoked CCEP components (P1, N1, P2, N2, P3) and to evaluate the similarities between N2 and the downstate of sleep slow waves. We used intra-cortical laminar microelectrodes (LMEs) to record CCEPs evoked by 10 mA bipolar 0.5 Hz electric pulses in seven patients with medically intractable epilepsy implanted with subdural grids. Based on the laminar profile of CCEPs, the latency of components is not layer-dependent, however their rate of appearance varies across cortical depth and stimulation distance, while the seizure onset zone does not seem to affect the emergence of components. Early neural excitation primarily engages middle and deep layers, propagating to the superficial layers, followed by mainly superficial inhibition, concluding in a sleep slow wave-like inhibition and excitation sequence.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Synaptic alterations and neuronal firing in human epileptic neocortical excitatory networks.

Epilepsy is a prevalent neurological condition, with underlying neuronal mechanisms involving hyperexcitability and hypersynchrony. Imbalance between excitatory and inhibitory circuits, as well as histological reorganization are relatively well-documented in animal models or even in the human hippocampus, but less is known about human neocortical epileptic activity. Our knowledge about changes in the excitatory signaling is especially scarce, compared to that about the inhibitory cell population. This study investigated the firing properties of single neurons in the human neocortex in vitro, during pharmacological blockade of glutamate receptors, and additionally evaluated anatomical changes in the excitatory circuit in tissue samples from epileptic and non-epileptic patients. Both epileptic and non-epileptic tissues exhibited spontaneous population activity (SPA), NMDA receptor antagonization reduced SPA recurrence only in epileptic tissue, whereas further blockade of AMPA/kainate receptors reversibly abolished SPA emergence regardless of epilepsy. Firing rates did not significantly change in excitatory principal cells and inhibitory interneurons during pharmacological experiments. Granular layer (L4) neurons showed an increased firing rate in epileptic compared to non-epileptic tissue. The burstiness of neurons remained unchanged, except for that of inhibitory cells in epileptic recordings, which decreased during blockade of glutamate receptors. Crosscorrelograms computed from single neuron discharge revealed both mono- and polysynaptic connections, particularly involving intrinsically bursting principal cells. Histological investigations found similar densities of SMI-32-immunopositive long-range projecting pyramidal cells in both groups, and shorter excitatory synaptic active zones with a higher proportion of perforated synapses in the epileptic group. These findings provide insights into epileptic modifications from the perspective of the excitatory system and highlight discrete alterations in firing patterns and synaptic structure. Our data suggest that NMDA-dependent glutamatergic signaling, as well as the excitatory synaptic machinery are perturbed in epilepsy, which might contribute to epileptic activity in the human neocortex.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Seeing beyond the spikes: reconstructing the complete spatiotemporal membrane potential distribution from paired intra- and extracellular recordings.

Although electrophysiologists have been recording intracellular neural activity routinely ever since the ground-breaking work of Hodgkin and Huxley, and extracellular multichannel electrodes have also been used frequently and extensively, a practical experimental method to track changes in membrane potential along a complete single neuron is still lacking. Instead of obtaining multiple intracellular measurements on the same neuron, we propose an alternative method by combining single-channel somatic patch-clamp and multichannel extracellular potential recordings. In this work, we show that it is possible to reconstruct the complete spatiotemporal distribution of the membrane potential of a single neuron with the spatial resolution of an extracellular probe during action potential generation. Moreover, the reconstruction of the membrane potential allows us to distinguish between the two major but previously hidden components of the current source density (CSD) distribution: the resistive and the capacitive currents. This distinction provides a clue to the clear interpretation of the CSD analysis, because the resistive component corresponds to transmembrane ionic currents (all the synaptic, voltage-sensitive and passive currents), whereas capacitive currents are considered to be the main contributors of counter-currents. We validate our model-based reconstruction approach on simulations and demonstrate its application to experimental data obtained in vitro via paired extracellular and intracellular recordings from a single pyramidal cell of the rat hippocampus. In perspective, the estimation of the spatial distribution of resistive membrane currents makes it possible to distiguish between active and passive sinks and sources of the CSD map and the localization of the synaptic input currents, which make the neuron fire. KEY POINTS: A new computational method is introduced to calculate the unbiased current source density distribution on a single neuron with known morphology. The relationship between extracellular and intracellular electric potential is determined via mathematical formalism, and a new reconstruction method is applied to reveal the full spatiotemporal distribution of the membrane potential and the resistive and capacitive current components. The new reconstruction method was validated on simulations. Simultaneous and colocalized whole-cell patch-clamp and multichannel silicon probe recordings were performed from the same pyramidal neuron in the rat hippocampal CA1 region, in vitro. The method was applied in experimental measurements and returned precise and distinctive characteristics of various intracellular phenomena, such as action potential generation, signal back-propagation and the initial dendritic depolarization preceding the somatic action potential.

Bursting of excitatory cells is linked to interictal epileptic discharge generation in humans.

Knowledge about the activity of single neurons is essential in understanding the mechanisms of synchrony generation, and particularly interesting if related to pathological conditions. The generation of interictal spikes-the hypersynchronous events between seizures-is linked to hyperexcitability and to bursting behaviour of neurons in animal models. To explore its cellular mechanisms in humans we investigated the activity of clustered single neurons in a human in vitro model generating both physiological and epileptiform synchronous events. We show that non-epileptic synchronous events resulted from the finely balanced firing of excitatory and inhibitory cells, which was shifted towards an enhanced excitability in epileptic tissue. In contrast, interictal-like spikes were characterised by an asymmetric overall neuronal discharge initiated by excitatory neurons with the presumptive leading role of bursting pyramidal cells, and possibly terminated by inhibitory interneurons. We found that the overall burstiness of human neocortical neurons is not necessarily related to epilepsy, but the bursting behaviour of excitatory cells comprising both intrinsic and synaptically driven bursting is clearly linked to the generation of epileptiform synchrony.

A covalent strategy to target intrinsically disordered proteins: Discovery of novel tau aggregation inhibitors.

Intrinsically disordered proteins (IDPs) play important roles in disease pathologies; however, their lack of defined stable 3D structures make traditional drug design strategies typically less effective against these targets. Based on promising results of targeted covalent inhibitors (TCIs) on challenging targets, we have developed a covalent design strategy targeting IDPs. As a model system we chose tau, an endogenous IDP of the central nervous system that is associated with severe neurodegenerative diseases via its aggregation. First, we mapped the tractability of available cysteines in tau and prioritized suitable warheads. Next, we introduced the selected vinylsulfone warhead to the non-covalent scaffolds of potential tau aggregation inhibitors. The designed covalent tau binders were synthesized and tested in aggregation models, and inhibited tau aggregation effectively. Our results revealed the usefulness of the covalent design strategy against therapeutically relevant IDP targets and provided promising candidates for the treatment of tauopathies.

Synthesis and characterization of new fluorescent boro-ß-carboline dyes.

The first representatives of the new fluorescent boro-ß-carboline family were synthesized by the insertion of the difluoroboranyl group into the oxaza or diaza core. The resulting compounds showed good photophysical properties with fine Stokes-shifts in the range of 38-85 nm with blue and green emission. The energetics of the excitation states and molecular orbitals of two members were investigated by quantum chemical computations suggesting effects for the improved properties of diazaborinino-carbolines over oxazaborolo-carbolines. These properties nominated this chemotype as a new fluorophore for the development of fluorescent probes. As an example, diazaborinino-carbolines were used for the specific labeling of anti-Her2 antibody trastuzumab. The fluorescent conjugate showed a high fluorophore-antibody ratio and was confirmed as a useful tool for labeling and confocal microscopy imaging of tumour cells in vitro together with the ex vivo two-photon microscopy imaging of tumour slices.

Perisomatic Inhibition and Its Relation to Epilepsy and to Synchrony Generation in the Human Neocortex.

Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated-in conjunction with the electron microscopy-that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events.