Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis

Abstract

Background: Postnatal olfactory bulb (OB) neurogenesis involves the generation of granule and periglomerular cells by neural stem cells (NSCs) located in the walls of the lateral ventricle (LV). Recent studies show that NSCs located in different regions of the LV give rise to different types of OB neurons. However, the molecular mechanisms governing neuronal specification remain largely unknown and new methods to approach these questions are needed.

Results: In this study, we refine electroporation of the postnatal forebrain as a technique to perform precise and accurate delivery of transgenes to NSCs located in distinct walls of the LV in the mouse. Using this method, we confirm and expand previous studies showing that NSCs in distinct walls of the LV produce neurons that invade different layers of the OB. Fate mapping of the progeny of radial glial cells located in these distinct LV walls reveals their specification into defined subtypes of granule and periglomerular neurons.

Conclusions: Our results provide a baseline with which future studies aiming at investigating the role of factors in postnatal forebrain neuronal specification can be compared. Targeted electroporation of defined LV NSC populations will prove valuable to study the genetic factors involved in forebrain neuronal specification.

Figure 1

Electroporation efficiency using electrodes of different sizes. (A) Schematic representation of the position and size (10 mm and 5 mm) of the electrodes used in the first part of this study. Only the positive pole is represented. (B) Representative example of electroporated radial glial cells (RGCs) expressing high levels of GFP at 1 day post-electroporation. The right panel shows a higher magnification of the region surrounded by the dotted box. Only cells with clear RGC morphology, that is, with an end foot contacting the ventricle surface and a main apical process, were counted. DAPI (blue) was used as a nuclear counterstain. (C,D) Measurements of the electroporated area (C) and of the number of electroporated RGCs (D) when 10-mm (filled bar) or 5-mm (hatched bars) diameter electrodes were used. Please refer to Materials and methods and Additional file 1 for experimental details. Note that the use of smaller electrodes does not improve the precision of electroporation (that is, did not decrease the electroporated area size), but tend to decrease the number of electroporated cells. Error bars represent standard error of the mean. Scale bars: 50 μm and 10 μm in (B) (left and right panels, respectively). LV, lateral ventricle.

Figure 2

Electroporation efficiency using different electrode locations. (A) Schematic representation of the location of the electrodes used in the second part of this study. Only the positive pole is represented. Note the very ventral location of the tweezer-electrode cathode to target the lateral and septal wall of the lateral ventricle (LV), with no transfection of the dorsal wall. (B) Representative three-dimensional reconstruction illustrating the location and extent of the electroporated area after electroporation (EPO) with the electrode position shown in (A). Gray contours, brain; white contours, lateral ventricle; green contours, area containing electroporated cells. (C,D) Measurements of the electroporated area (C) and of the number of electroporated RGCs (D) obtained with the three electrode positions tested. Error bars represent standard error of the mean. *P < 0.05 as determined by unpaired t-test.

Figure 3

Targeted electroporation of defined lateral ventricle wallS. (A) Quantification of the percentage of GFP+ RGCs in subregions of the LV following lateral, dorsal and septal electroporation. Note the efficient targeting of distinct LV walls with different positioning of the electrodes. The most ventral regions of the lateral and septal LV walls, however, show lower numbers of electroporated cells. Error bars represent standard error of the mean. (B) Quantification of the percentage of GFP+ RGCs at defined rostro-caudal levels of the LV. (C-E) Representative overviews of the distribution of electroporated (GFP+, green) cells at two rostro-caudal levels of the LV (that is, Bregma 1.4 mm and 0.2 mm). DAPI (blue) was used as a nuclear counterstain. Scale bars: 1 mm. Ctx, cortex; EPO, electroporation; Sp, septum; Str, striatum.

Figure 4

Radial glial cells located in defined walls of the postnatal lateral ventricle produce neurons that migrate to distinct layers of the olfactory bulb. (A,B) Quantification of the percentage of GFP+ newborn neurons in defined layers of the OB at 21 dpe. The OB layers are illustrated in (B). Error bars represent standard error of the mean. (C-E) Representative overviews of the distribution of newborn neurons in the OB of laterally, dorsally, and septally electroporated animals. DAPI (blue) was used as a nuclear counterstain. Scale bars: 100 μm. GCL, granule cell layer; EPO, electroporation; GL, glomerular layer; iPL, internal plexiform layer; MCL, mitral cell layer.

Figure 5

Radial glial cells located in defined walls of the lateral ventricle specify distinct periglomerular neuron subtypes. (A-C) Representative immunostainings for the three neuronal subtype markers CR (A), TH (B), and CB (C). For all markers, a low magnification overview with DAPI as a nuclear counterstain is shown (left panel), as well as confocal pictures showing higher magnifications of GFP+ neurons positive for the three markers (right panels). (D-F) Quantification of the percentage of newborn neurons (that is, GFP+) expressing CR (D), TH (E) or CB (F), which derived from the lateral (red), dorsal (gray) or septal (blue) LV walls. Error bars represent standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001; determined by unpaired t-test. Scale bars: 50 μm, 30 μm and 10 μm in the left, middle and right panels, respectively. CB, calbindin; CR, calretinin; EPO, electroporation; TH, tyrosin hydroxylase.

Figure 6

Radial glial cells located in defined walls of the lateral ventricle produce distinct granule neuron subtypes. (A-C) Representative immunostainings for CR expression in the GCL of animals electroporated in the lateral (A), dorsal (B) or septal (C) walls of the LV. For all groups, a low magnification overview (left panel) as well as a higher magnification of the underlined region (right panel) are presented. DAPI (blue) was used as a nuclear counterstain. (D) Quantification of the percentage of newborn granule cells (GFP+) expressing CR that derived from the lateral (red), dorsal (gray) or septal (blue) LV walls. Error bars represent standard error of the mean. *P < 0.05 as determined by unpaired t-test. Scale bars: 50 μm and 10 μm in the left and right panels, respectively. CR, calretinin; EPO, electroporation.