This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.
In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells.
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.
In the United Kingdom, mice are housed, bred, and treated according to the guidelines approved by the Home Office under the Animal (Scientific Procedures) Act 1986.
1. Preparation: DNA Solution and Needles
2. Preparation of the Surgery
3. Surgery Before DNA Injection and Electroporation
No microscope is required for visualization.
4. Injection of DNA and Electroporation
5. Surgery Post-electroporation
6. Post-surgery
Check the behavior of the mice to assess pain, suffering or distress and weigh the animals 24 h and 48 h after the surgery. If needed, analgesics can be administered to minimize pain and discomfort.
7. Tissue Processing
Collect the electroporated embryos or pups at the embryonic or postnatal stages required for the experiment.
– For analysis at embryonic stages (for example to study cell proliferation or migration):
Euthanize mother via cervical dislocation and collect the embryos. After decapitation, select the brains that have been properly electroporated, as indicated by the amount and location of the fluorescent signal, visualized across the skull using a fluorescent binocular. Dissect the brain out of the skull and fix overnight in 4% PFA and then place in 20% sucrose / PBS overnight. Embed in OCT compound, freeze at -80 °C and section using a cryostat.
– For analysis at postnatal stages (for example to study dendrites and spines):
Anesthetize pups or adult mice with intraperitoneal injection of pentobarbitone (40-60 mg/kg) and perform transcardial perfusion with PBS, followed by 4% PFA in PBS. Dissect the brains out of the skull and post-fix in 4% PFA overnight. After washings in PBS, section the brains using a vibratome (100 μm sections for dendrite analysis). Mount the sections in Aqua Poly/mount using 0.16-0.19 mm thick coverslips to image dendrites and spines.
8. Representative results
Figure 3 shows examples of electroporated cells in the cerebral cortex (Figures 3A, B), in the CA1 (Figures 3C, D) and in the dentate gyrus of the hippocampus (Figures 3E, F). Wild-type mice were electroporated at E14.5 with a GFP construct (pCA-b-EGFPm5 silencer 3) and brains were harvested at postnatal day (P) 14. By injecting a small volume of DNA solution (0.5 μl or less of a solution at 1 μg/μl), a few cells are labeled, which allows the visualization of the dendritic arborization of isolated GFP+ cells (Figure 3) as well as their spines at higher magnification (Figure 4).
Figure 1. Schematic representation of electroporation protocols that can be used to study dendrites and spines. (A) Electroporation of a GFP construct to compare dendrites and spines in wild-type and mutant mice. (B) Electroporation of GFP-shRNA (GFP is expressed from the same construct) to compare dendrites and spines in mice electroporated with a shRNA construct specific for a gene of interest (X) or a control shRNA. (C) IUE can be used together with a Cre inducible system in order to restrict expression of the shRNA to the desirable time period. In this experiment, a vector expressing a form of the Cre recombinase which can be activated by 4-hydroxytamoxifen (CAG-ERT2CreERT2; 1 μg/μl) 10, is electroporated together with a vector expressing a specific shRNA in a Cre dependent manner (1 μg/μl, and with a recombination indicator (CALNL-GFP construct, GFP expression inducible by Cre; 1 μg/μl) 10. The efficiency of the knockdown can be improved by increasing the concentration of the shRNA as well as increasing CAG-ERT2CreERT2 concentration.
Figure 2. Spatial control of electroporation. This figure shows where to position the paddle electrodes according to the DNA injection site in order to target the cerebral cortex or the hippocampus.
Figure 3. Visualization of the dendritic arbor of in utero electroporated cells in the cerebral cortex and hippocampus. (A, B) Coronal sections showing GFP+ pyramidal cells in the cerebral cortex, (C-D) pyramidal cells in CA1 of the hippocampus and (E, F), granule cells in the dentate gyrus at P14. A GFP construct (pCA-b-EGFPm5 silencer 3) was electroporated at E14.5. Higher magnification pictures (B, D, F) show that IUE is an efficient method to visualize dendrites. Scale bars represent 50 μm (B, D and F), 150 μm (A, C, E).
Figure 4. Visualization of dendritic spines in P14 neurons that were electroporated in utero at E14.5 with a GFP expressing construct. (A, B) High magnification images of spines from basal dendrites of hippocampal pyramidal neurons. Scale bars represent 5 μm (A) and 2 μm (B).
IUE is a powerful tool to manipulate gene expression not only in space but also in time. We show here that this technique can be used to visualize and genetically manipulate dendrites and spines in the cerebral cortex and hippocampus of mice. Besides the advantages previously cited, it is worth noting that IUE, in contrast to Golgi method, can be combined with immunohistochemistry or in situ hybridization, which allows for example to phenotype the electroporated cells. It is also important to mention that this procedure does not induce evident brain malformations despite its relative invasiveness. In addition, at the cellular level, IUE does not modify the electrophysiological properties of the electroporated neurons13. While our demonstration focuses on the visualization of dendrite and spine morphologies, IUE of cortical or hippocampal neurons at E14.5 could also be used to study other developmental events such as axon formation and guidance. In addition, the same kind of protocol could be implemented at other stages of embryonic development to target different populations. For example, a developmentally very late cortical electroporation paradigm at E18.5 can be performed to drive expression in astrocytic progenitors 1. Similarly, while an electroporation of the hippocampus at E14.5 allows to target CA1-CA3 pyramidal neuron progenitors and dentate granule cell progenitor at the same time, a late hippocampal electroporation (E18.5 or early postnatal) would allow to target different dentate granule progenitors 14. In this case, the injected volume of DNA can be increased as well as the intensity of current.
Transgenes introduced by IUE appear to remain episomal and are therefore lost from cells following successive cell divisions. In postmitotic cells such as neurons, however, the episomal transgenes remain active for months after electroporation allowing long-term studies 13,15. In our study, we have observed bright GFP+ cells up to 7 weeks after birth (the latest time point we analyzed) indicating that embryonic targeting of cortical or hippocampal neuronal precursors using IUE results in persistent expression of the transgene from early developmental time points up to adulthood.
A current limitation of the technique is that it is difficult to exert a fine control over the total number of electroporated cells. However, by decreasing the injected volume of DNA solution, we have shown that it is possible to label a few cells and to visualize the dendritic arborization of isolated GFP+ cells as well as their spines. The dimension of the transfected area could also be adjusted by modifying the parameters of the electroporation such as intensity of current and number of pulses or the diameter of the electroporation paddles.
Altogether IUE is a method that is easy to implement, rapid and efficient to study dendrites and spines in vivo.
The authors have nothing to disclose.
The authors would like to thank Dr. Kathleen Mathers, Dr. Jean-Philippe Mocho and Dr. Yolanda Saavedra Torres for their help to perform in utero electroporation under aseptic procedures, and Hayley Wood for her help to prepare the drawings.
E.P. was supported by a long-term Federation of European Biochemical Societies (FEBS) fellowship and a Medical Research Council (MRC) career development fellowship, M.A.H. by a Wellcome Trust grant to Elizabeth Fisher and Victor Tybulewicz (080174/B/06/Z), H.W. by an EMBO long-term fellowship and R.A. by an MRC studentship. This work was supported by a project grant from the Wellcome Trust (086947/Z/08/Z) and by a Grant-in-Aid from the Medical Research Council (U117570528) to F.G.
Name of the reagent | Company | Catalogue number | Yorumlar | |||
Preparation of needles and DNA solution for injection | ||||||
Endofree Plasmid Maxi Kit | Qiagen | 12362 | ||||
Fast Green | Sigma | F-7258 | ||||
Borosilicate glass capillaries 1.0 mm O.D. x 0.58 mm I.D. |
Harvard Apparatus | 30-0016 | ||||
Microloader tips | Eppendorf | 5242956003 | For Eppendorf pipettes 0.5 μl-10 μl / 2-20 μl | |||
Material for surgery | ||||||
Extra thin Iris scissors | Fine Science Tools | 14088-10 | ||||
Curved Forceps | Fine Science Tools | 91197-00 | ||||
Ring forceps | Fine Science Tools | 11103-09 | ||||
Needle holder | Fine Science Tools | 12002-12 | ||||
Graefe Forceps | Fine Science Tools | 11050-10 | ||||
Vicryl absorbable suture | Ethicon Inc (Johnson & Johnson) | W9074 | ||||
Sterile drapes 30cm x 45 cm | Buster | 141765 | ||||
Sterile swabs | Shermond | HUBY-340 | ||||
Cotton buds | Clean Cross Co., Ltd | 1860 | ||||
Buprenorphine (Vetergesic) | Alstoe Animal Health | |||||
Clorhexidine | Vetasept | XHG007 | ||||
Eye gel Viscotears | Novartis | |||||
Isoflurane | Abbott Laboratories | B506 | ||||
Pentoject, Pentobarbitone Sodium 20% | Animalcare | |||||
Electroporation | ||||||
Electroporator | BTX | ECM830 | ||||
Platinum Tweezertrode 5mm | BTX, Harvard Apparatus | 45-0489 | ||||
Femtojet microinjector | Eppendorf | 5247000030 | ||||
Foot Control for Femtojet Microinjector | Eppendorf | 5247623002 | ||||
Capillary holder | Eppendorf | 5176190002 | ||||
Tissue processing | ||||||
Paraformaldehyde | Sigma | P6148 | ||||
Sucrose | VWR (Prolabo) | 27480.294 | ||||
Microscope slides | ThermoScientific (Menzel-Gläser) | J1800AMNZ | ||||
Coverslips | Menzel-Gläser | 22 x 50 mm #1,5 | ||||
Aqua Poly/mount | Polysciences, Inc | 18606 |