The dysfunction of the small-conductance calcium-activated K⁺ channel SK3 has been described as one of the factors responsible for the progress of psychoneurological diseases, but the molecular basis of this is largely unknown. This report reveals through use of immunohistochemistry and computational tomography that long-term increased expression of the SK3 small-conductance calcium-activated potassium channel (SK3-T/T) in mice induces a notable bilateral reduction of the hippocampal area (more than 50 %). Histological analysis showed that SK3-T/T mice have cellular disarrangements and neuron discontinuities in the hippocampal formation CA1 and CA3 neuronal layer. SK3 overexpression resulted in cognitive loss as determined by the object recognition test. Electrophysiological examination of hippocampal slices revealed that SK3 channel overexpression induced deficiency of long-term potentiation in hippocampal microcircuits. In association with these results, there were changes at the mRNA levels of some genes involved in Alzheimer's disease and/or linked to schizophrenia, epilepsy, and autism. Taken together, these features suggest that augmenting the function of SK3 ion channel in mice may present a unique opportunity to investigate the neural basis of central nervous system dysfunctions associated with schizophrenia, Alzheimer's disease, or other neuropsychiatric/neurodegenerative disorders in this model system. As a more detailed understanding of the role of the SK3 channel in brain disorders is limited by the lack of specific SK3 antagonists and agonists, the results observed in this study are of significant interest; they suggest a new approach for the development of neuroprotective strategies in neuropsychiatric/neurodegenerative diseases with SK3 representing a potential drug target.

Background

Multi-electrode arrays (MEAs) allow non-invasive multi-unit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEA's surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging.

New Method

In this paper, we present a novel approach utilizing micro-contact printing (μCP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-μCP (accurate positioning micro-contact printing).

Comparison with existing Methods

Other approaches presented in the literature using μCP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning.

Conclusion

Here we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.

Spontaneous bursting activity in cultured neuronal networks is initiated by leader neurons, which constitute a small subset of first-to-fire neurons forming a sub-network that recruits follower neurons into the burst. While the existence and stability of leader neurons is well established, the influence of stimulation on the leader-follower dynamics is not sufficiently understood. By combining multi-electrode array recordings with whole field optical stimulation of cultured Channelrhodopsin-2 transduced hippocampal neurons, we show that fade-in photo-stimulation induces a significant shortening of intra-burst firing rate peak delay of fol-lower electrodes after offset of the stimulation compared to unperturbed spontaneous activity. Our study shows that optogenetic stimulation can be used to change the dynamical fine structure of self-organized network bursts.

Many diverse studies have shown that a mechanical displacement of the axonal membrane accompanies the electrical pulse defining the action potential (AP). We present a model for these mechanical displacements as arising from the driving of surface wave modes in which potential energy is stored in elastic properties of the neuronal membrane and cytoskeleton while kinetic energy is carried by the axoplasmic fluid. In our model, these surface waves are driven by the travelling wave of electrical depolarization characterizing the AP, altering compressive electrostatic forces across the membrane. This driving leads to co-propagating mechanical displacements, which we term Action Waves (AWs). Our model allows us to estimate the shape of the AW that accompanies any travelling wave of voltage, making predictions that are in agreement with results from several experimental systems. Our model can serve as a framework for understanding the physical origins and possible functional roles of these AWs.

In the axons of cultured hippocampal neurons, actin forms various structures, including bundles, patches (involved in the preservation of neuronal polarity), and a recently reported periodic ring-like structure. Nevertheless, the overlaying organization of actin in neurons and in the axon initial segment (AIS) is still unclear, due mainly to a lack of adequate imaging methods. By harnessing live-cell stimulated emission depletion (STED) nanoscopy and the fluorescent probe SiR-Actin, we show that the periodic subcortical actin structure is in fact present in both axons and dendrites. The periodic cytoskeleton organization is also found in the peripheral nervous system, specifically at the nodes of Ranvier. The actin patches in the AIS co-localize with pre-synaptic markers. Cytosolic actin organization strongly depends on the developmental stage and subcellular localization. Altogether, the results of this study reveal unique neuronal cytoskeletal features.

We present a novel experimental paradigm "In vitro closed loop optical network electrophysiology (ivCLONE)". This seminar note gives an overview of the basics of optical neurostimulation, network electrophysiology and closed loop electrophysiology. Moreover, the notes discuss how combination of aforementioned techniques would help us to address network-level phenomenon and how single neuron properties are related to collective network dynamics.

Synchronized bursting is found in many brain areas and has also been implicated in the pathophysiology of neuropsychiatric disorders such as epilepsy, Parkinson’s disease, and schizophrenia. Despite extensive studies of network burst synchronization, it is insufficiently understood how this type of network wide synchronization can be strengthened, reduced, or even abolished. We combined electrical recording using multi-electrode array with optical stimulation of cultured channelrhodopsin-2 transducted hippocampal neurons to study and manipulate network burst synchronization. We found low frequency photo-stimulation protocols that are sufficient to induce potentiation of network bursting, modifying bursting dynamics, and increasing interneuronal synchronization. Surprisingly, slowly fading-in light stimulation, which substantially delayed and reduced light-driven spiking, was at least as effective in reorganizing network dynamics as much stronger pulsed light stimulation. Our study shows that mild stimulation protocols that do not enforce particular activity patterns onto the network can be highly effective inducers of network-level plasticity.

Central neurons operate in a regime of constantly fluctuating conductances, induced by thousands of presynaptic cells. Channelrhodopsins have been almost exclusively used to imprint a fixed spike pattern by sequences of brief depolarizations. Here we introduce continuous dynamic photostimulation (CoDyPs), a novel approach to mimic in-vivo like input fluctuations noninvasively in cells transfected with the weakly inactivating channelrhodopsin variant ChIEF. Even during long-term experiments, cultured neurons subjected to CoDyPs generate seemingly random, but reproducible spike patterns. In voltage clamped cells CoDyPs induced highly reproducible current waveforms that could be precisely predicted from the light-conductance transfer function of ChIEF. CoDyPs can replace the conventional, flash-evoked imprinting of spike patterns in in-vivo and in-vitro studies, preserving natural activity. When combined with non-invasive spike-detection, CoDyPs allows the acquisition of order of magnitudes larger data sets than previously possible, for studies of dynamical response properties of many individual neurons.

Dynamic oscillatory coherence is believed to play a central role in flexible communication between brain circuits. To test this communication-through-coherence hypothesis, experimental protocols that allow a reliable control of phase-relations between neuronal populations are needed. In this modeling study, we explore the potential of closed-loop optogenetic stimulation for the control of functional interactions mediated by oscillatory coherence. The theory of non-linear oscillators predicts that the efficacy of local stimulation will depend not only on the stimulation intensity but also on its timing relative to the ongoing oscillation in the target area. Induced phase-shifts are expected to be stronger when the stimulation is applied within specific narrow phase intervals. Conversely, stimulations with the same or even stronger intensity are less effective when timed randomly. Stimulation should thus be properly phased with respect to ongoing oscillations (in order to optimally perturb them) and the timing of the stimulation onset must be determined by a real-time phase analysis of simultaneously recorded local field potentials (LFPs). Here, we introduce an electrophysiologically calibrated model of Channelrhodopsin 2 (ChR2)-induced photocurrents, based on fits holding over two decades of light intensity. Through simulations of a neural population which undergoes coherent gamma oscillations—either spontaneously or as an effect of continuous optogenetic driving—we show that precisely-timed photostimulation pulses can be used to shift the phase of oscillation, even at transduction rates smaller than 25%. We consider then a canonic circuit with two inter-connected neural populations oscillating with gamma frequency in a phase-locked manner. We demonstrate that photostimulation pulses applied locally to a single population can induce, if precisely phased, a lasting reorganization of the phase-locking pattern and hence modify functional interactions between the two populations.

In the presented thesis work, an “Optical Network Electrophysiology “ system that combines optical stimulation using optogenetic tools and multisite neuronal recording using microelectrode arrays was developed and its applicability to address questions related to neuronal network dynamics was demonstrated. The system was used to modify the intrinsic collective dynamics of a cultured neuronal network potentially maximizing spike synchronization using mild whole field photostimulation. It offers an attractive alternative to stimulation paradigms that externally control neuronal networks. Another application of the system was to drive neurons in a naturalistic in-vivo like fashion where fluctuating light waveforms where used to put neurons in “fluctuation driven regime”. This regime is crucial to characterize basic computational properties of neurons such as frequency-input curves, spike triggered average and correlation gain.