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Neuroscience

Categories
+ Basic technology
+ Behavioral neuroscience
+ Cellular mechanisms
- Development
Electroporation
Explant culture
Histological staining
Immunofluorescence
Morphogenesis
Neuron
Retinal culture
Spatial navigation
+ Nervous system disorders
+ Neuroanatomy and circuitry
+ Peripheral nervous system
+ Sensory and motor systems
Protocols in Past Issues

Electroporation of Whole-Mount Postnatal Rodent Retinas for Advanced Functional Assays
CH Chien-Ting Huang
TC Tzu-Jen Chen
YS Yu-Lin Su
CT Cai-Chieh Tseng
PC Pin-Chun Chen
CW Chih-Tien Wang
Q&A 0 Q&A
257 Views
Jan 20, 2026

To study gene function in regulating rodent retinal waves during development, an efficient method for gene delivery into whole-mount retinas is required while preserving circuit functionality for physiological studies. We present an optimized electroporation protocol for developing rodent retinal explants. The procedure includes the fabrication of horizontally aligned platinum electrodes and the placement of retinal explants between them to generate a uniform electric field for high transfection efficiency. The entire process—dissection and electroporation—can be completed within 1–2 h. Successful transfection is verified by fluorescence microscopy, and physiological assays such as patch-clamp recordings and live imaging can be performed within 1–4 days following electroporation. This rapid and reliable protocol enables functional analysis for a specific gene in regulating retinal waves and can be adapted to other organotypic slice cultures.

MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos
MU Madoka K. Utsumi
TA Taichi Akahoshi
KO Kotaro Oka
KH Kohji Hotta
Q&A 0 Q&A
1820 Views
Oct 20, 2022

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light–oxygen–voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A (Ciona robusta), a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera.


Graphical abstract:




Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation
Mariko Kinoshita-Kawada Mariko Kinoshita-Kawada
HH Hiroshi Hasegawa
TH Tsunaki Hongu
SY Shigeru Yanagi
YK Yasunori Kanaho
IM Ichiro Masai  [...]
JW Jane Y. Wu
+ 5 Authors
Q&A 0 Q&A
7050 Views
Sep 20, 2019
Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an “open-book” spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and “closed-book” spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

In utero Electroporation of Mouse Cerebellar Purkinje Cells
Yukari H. Takeo Yukari H. Takeo
Q&A 0 Q&A
16370 Views
Jun 5, 2016
In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.