This protocol describes a high-efficiency Ca2+-phosphate transfection method with low cell toxicity. The Ca2+-phosphate transfection method is widely used in transfecting neurons because of its low cell toxicity and simplicity in use, but the efficiency is typically low (approximately 1-5%). To solve this problem we have developed a new Ca2+-phosphate transfection protocol that increases the efficiency by 10-fold (< or = 60%), while maintaining low cell toxicity. First, it is critical to have gentle mixing of the DNA-Ca2+ solution with phosphate buffer to form a homogeneous snowlike precipitate (particle size 1-3 microm). Second, the precipitate should be dissolved using a slightly acidic culture medium to reduce cell toxicity. The high efficiency of this new protocol makes it possible to transfect single autaptic neurons as well as mature neurons (15-82 days in vitro) for gene functional analysis. The total time required for the protocol is 2-4 h (including 45 min-3 h incubation time).

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

The restoration of light response with complex spatiotemporal features in retinal degenerative diseases towards retinal prosthesis has proven to be a considerable challenge over the past decades. Herein, inspired by the structure and function of photoreceptors in retinas, we develop artificial photoreceptors based on gold nanoparticle-decorated titania nanowire arrays, for restoration of visual responses in the blind mice with degenerated photoreceptors. Green, blue and near UV light responses in the retinal ganglion cells (RGCs) are restored with a spatial resolution better than 100 µm. ON responses in RGCs are blocked by glutamatergic antagonists, suggesting functional preservation of the remaining retinal circuits. Moreover, neurons in the primary visual cortex respond to light after subretinal implant of nanowire arrays. Improvement in pupillary light reflex suggests the behavioral recovery of light sensitivity. Our study will shed light on the development of a new generation of optoelectronic toolkits for subretinal prosthetic devices.

Exploring efficient and earth-abundant electrocatalysts for water splitting is crucial for various renewable energy technologies. In this work, iron (Fe)-doped nickel phosphide (NiP) nanosheet arrays supported on nickel foam (NiFeP NSAs/NF) are fabricated through a facile hydrothermal method, followed by phosphorization. The electrochemical analysis demonstrates that the NiFeP NSAs/NF electrode possesses high electrocatalytic activity for water splitting. In 1.0 M KOH, the NiFeP NSAs/NF electrode only needs overpotentials of 106 mV at 10 mA cm and 270 mV at 20 mA cm to drive the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), respectively. Furthermore, the assembled two-electrode (NiFeP NSAs/NF∥NiFeP NSAs/NF) alkaline water electrolyzer can produce a current density of 10 mA cm at 1.61 V. Remarkably, it can maintain stable electrolysis over 20 h. Thus, this work undoubtedly offers a promising electrocatalyst for water splitting.

Background: Cofilin rods are associated with Alzheimer disease, but their pathological significance is unclear. Results: Time-lapse imaging revealed that cofilin rods inhibit the movement of mitochondria and early endosomes. Cofilin rods reduce dendritic spines and impair synaptic transmission. Cofilin rods are discovered in senile rat brains. Conclusion: Cofilin rods block intracellular transport and induce synaptic loss. Significance: Our work identifies a signaling pathway underlying neurodegeneration and brain aging.

Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABA A receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 μM, 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 μmol in 5 μl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs. Cyclothiazide (CTZ) was originally known as a diuretic drug with antihypertensive effects (Julius et al. 1962;Schvartz et al. 1962). It was later found that CTZ is a potent blocker of AMPA receptor desensitization Trussell et al. 1993;Yamada & Tang, 1993;Zorumski et al. 1993;Barnes-Davies & Forsythe, 1995;Mennerick & Zorumski, 1995). CTZ also increases presynaptic glutamate release (Diamond & Jahr, 1995;Bellingham & Walmsley, 1999;Ishikawa & Takahashi, 2001). Our recent work has further demonstrated that CTZ can directly inhibit GABA A receptors as shown by both whole-cell and single-channel experiments (Deng & Chen, 2003). Thus, CTZ has a unique characteristic in acting simultaneously on two prominent synaptic transmission systems: it significantly enhances excitatory glutamatergic neurotransmission while suppressing inhibitory GABAergic neurotransmission. The net effect of CTZ on a neural network will be a significant tilt of the excitationinhibition balance toward hyperexcitation. We therefore hypothesize that CTZ may work as an epileptogenic agent to induce epileptiform activity in central neurons.To test our hypothesis, we treated hippocampal CA1-CA3 cultures with CTZ either in short-term duration but high concentration (1-2 h, 20-50 μm), or chronically with low concentration (2-10 days, 5 μm). In both conditions, CTZ consistently induced ro...

Multibranched pi-conjugated materials and hyperbranched polymers have received considerable interest in both academic research and industrial applications because of their unusual molecular structures and properties. In this communication, we provide an efficient approach to synthesize two novel pi-conjugated multibranch compounds, which have good solubility and are nanosize (about 5 nm). The double bonds of the multibranch compounds are synthesized using solvent-free Wittig-Horner-Emmons reactions. The reaction conditions are simple and mild. Their optical properties are discussed too.

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