A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations—a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1–induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits

Dopamine D4 Receptor Activation Increases Hippocampal Gamma Oscillations by Enhancing Synchronization of Fast-Spiking Interneurons

<div><h3>Background</h3><p>Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R) in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well.</p> <h3>Methodology/Principal Findings</h3><p>To address this hypothesis we use rat hippocampal slices and kainate-induced gamma oscillations. Local field potential recordings as well as intracellular recordings of pyramidal cells, fast-spiking and non-fast-spiking interneurons were carried out. We show that D4R activation with the selective ligand PD168077 increases gamma oscillation power, which can be blocked by the D4R-specific antagonist L745,870 as well as by the antipsychotic drug Clozapine. Pyramidal cells did not exhibit changes in excitatory or inhibitory synaptic current amplitudes, but inhibitory currents became more coherent with the oscillations after application of PD168077. Fast-spiking, but not non-fast spiking, interneurons, increase their action potential phase-coupling and coherence with regard to ongoing gamma oscillations in response to D4R activation. Among several possible mechanisms we found that the NMDA receptor antagonist AP5 also blocks the D4R mediated increase in gamma oscillation power.</p> <h3>Conclusions/Significance</h3><p>We conclude that D4R activation affects fast-spiking interneuron synchronization and thereby increases gamma power by an NMDA receptor-dependent mechanism. This suggests that converging deficits on fast-spiking interneurons may lead to decreased network function and thus aberrant gamma oscillations and cognitive decline in schizophrenia.</p> </div

D4 receptor activation causes reduction in high-voltage outward current in fast-spiking interneurons.

<p>Voltage steps recorded in fast-spiking interneurons (with 1 µM TTX present, 10 mV steps from −90 mV to 0 mV). <b>A.</b> Traces of current responses in control conditions (no kainate). <b>B.</b> Traces of current responses in the presence of PD168077. <b>C</b>. Traces of differences (digital subtraction of trace in B-A). <b>D</b>. Summary current vs. voltage plot across experiments. Black represents control, grey PD168077. *** P<0.001 (two-way ANOVA).</p

D4 receptor activation increases gamma oscillation power.

<p>LFP recordings of kainate-induced gamma oscillation and dopamine D4R agonist (PD168077, 100 nM) without and with prior application of D4R antagonist (L745,870, 500 nM or Clozapine (2 µM). <b>A.</b> Example traces of LFP recordings, showing an increase with D4R activation <b>B.</b> Power spectra of traces shown in A (kainate recording in black, subsequent recording with PD168077 in grey). <b>C.</b> Example traces of LFP recordings, showing the D4 effect is blocked with application of clozapine. <b>D.</b> Power spectra of traces shown in C. (kainate recording in black, clozapine as dotted line, and PD168077 in grey). <b>E.</b> Summary bar diagram of power (in % relative to initial kainate power, mean ± SEM). D4R activation significantly increases LFP gamma power, which is prevented by prior application of D4R antagonist or Clozapine. * P<0.05, ** P<0.01, *** P<0.001 (in unpaired t-tests).</p

D4 receptor modulation of gamma oscillations is NMDAR-dependent.

<p>EPSC recordings recorded from fast-spiking interneurons and the effect of NMDA receptor antagonists on LFP gamma power modulation by PD168077 <b>A</b>. Example traces of EPSCs (clamped to −70 mV, 50 µM picrotoxin, 50 µM AP5). <b>B</b>. Aggregated amplitudes across experiments plotted in an empirical cumulative distribution function (kainate recording in black, subsequent recording with PD168077 in grey). Note that the 2 graphs overlap to a large extent. <b>C.</b> Example traces of LFP recordings showing oscillations from (top to bottom) KA, the addition of AP5, the addition of PD 168077. <b>D.</b> Summary bar-diagram of LFP gamma power (in % relative to initial kainate power, means ± SEM). Addition of NMDA receptor antagonist AP5 does not have an effect on the gamma power but completely blocks the increase produced by D4R activation.</p

Histamine H3 receptor activation decreases kainate-induced hippocampal gamma oscillations in vitro by action potential desynchronization in pyramidal neurons

The study of rhythmic electrical activity in slice preparations has generated important insights into neural network function. While the synaptic mechanisms involved in the generation of in vitro network oscillations have been studied widely, little is known about the modulatory influence exerted on rhythmic activity in neuronal networks by neuropeptides and biogenic amines. Gamma oscillations play an important role in cognitive processes and are altered or disrupted in disorders such as Alzheimer's disease (AD) and schizophrenia. Given the importance of gamma oscillations for learning, memory and cognition processes as well as the recent interest in histamine H3 receptors in the development of pro-cognitive drugs to treat disorders such as AD and schizophrenia, it is relevant to study the impact of histaminergic mechanisms on network gamma oscillations. Here we show for the first time a modulation of gamma oscillation by histaminergic mechanisms. Selective activation of the H3 receptor by R-α-methylhistamine significantly reduces the power of kainate-induced gamma oscillations, but not carbachol-induced gamma oscillations, in the rat hippocampal slice preparation without affecting oscillation frequency. This effect is neither caused by a decrease in excitatory or inhibitory postsynaptic currents, nor a decrease in cellular excitability. Instead, we find that the decrease in oscillation power following H3 receptor activation results from a desynchronization of pyramidal neuron action potential firing with regard to the local field potential oscillation cycle. Our data provide a possible mechanism of action for histamine in regulating gamma oscillations in the hippocampal network

Spike-phase coupling in non-fast spiking interneurons is unaffected by D4 receptor activation.

<p><b>A</b>. Example traces of concomitant recordings of non-fast spiking interneurons and LFP oscillations before and after the addition of PD168077. <b>B</b>. Circular histograms based on recordings similar to those in A, indicating the number of action potentials discharged in each phase from −π to π. The radial axis indicates the number of action potentials. The higher and lower histograms represent recordings before and after PD168077 application, respectively. <b>C</b>. Summary bar-diagrams representing mean peak coherence and resultant vector length, respectively (mean ± SEM; kainate recording in black, subsequent recording with PD168077 in grey).</p

D4 receptor activation increases coherence of pyramidal cell IPSCs.

<p>Intracellular recordings of IPSCs in pyramidal cells (clamped to 0 mV) with ongoing gamma oscillations. <b>A.</b> Example traces of IPSC recordings. <b>B.</b> Summary bar-diagram showing the amplitude of IPSCs (mean ± SEM). <b>C.</b> Power spectrum of IPSCs constructed from the traces shown in A. (kainate recording in black, subsequent recording with PD168077 in grey). <b>D.</b> Summary bar-diagram of LFP gamma power normalized to kainate. <b>E.</b> Coherence spectrum of IPSCs versus LFP recordings from recordings shown in A (kainate recording in black, subsequent recording with PD168077 in grey). <b>F.</b> Summary bar-diagram of peak coherence values across experiments (mean ± SEM).</p