Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.

BACKGROUND: Exogenous gene introduction by transfection is one of the most important approaches for understanding the function of specific genes at the cellular level. Electroporation has a long-standing history as a versatile gene delivery technique in vitro and in vivo. However, it has been underutilized in vitro because of technical difficulty and insufficient transfection efficiency. NEW METHOD: We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses. Here, we describe the technique and compare it with existing methods. RESULTS: Our method achieves a high transfection efficiency (∼80 %) in both excitatory and inhibitory neurons with no detectable side effects on their function. We demonstrate this method is capable of transferring at least three different genes into a single neuron. In addition, we demonstrate the ability to transfect different genes into neighboring cells. COMPARISON WITH EXISTING METHODS: The majority of existing methods use fine-tipped glass electrodes (i.e. > 10 MΩ) and apply high voltage (10 V) pulses with high frequency (100 Hz) for 1 s. These parameters contribute to practical difficulties thus lowering the transfection efficiency. Our unique method minimizes electrode clogging and therefore procedure duration, increasing transfection efficiency and cellular viability. CONCLUSIONS: Our modifications, relative to current methods, optimize electroporation efficiency and cell survival. Our approach offers distinct research strategies not only in elucidating cell-autonomous functions of genes but also for assessing genes contributing to intercellular functions, such as trans-synaptic interactions.

Specific Neuroligin3-αNeurexin1 signaling regulates GABAergic synaptic function in mouse hippocampus.

Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.