Studying the Integration of Adult-born Neurons

1. Virus Injection

High-titer engineered retrovirus (1×10⁹ unit/ml) is produced by co-transfection of retroviral vectors and VSVG into HEK293T cells, followed by ultracentrifugation of viral supernatant. For sources and production methods, see the excellent JoVE demonstration (3).

Note: Young adult (4-6 weeks old) female C57Bl/6 mice (Charles River) are housed under standard conditions. All procedures follow the National Research Council’s Guide for the care and use of laboratory animals under a protocol approved by the Stony Brook University IACUC.

1. Thaw 2ul of frozen retrovirus per animal on ice.
2. Anaesthetize (100 μg ketamine + 10 μg xylazine per gram body weight) and mount the animal on a stereotaxic frame (Steolting). Remove the hair on the head and wipe the skin with 70% ethanol.
3. Expose the skull and drill four shallow holes using a dental drill (0.6mm drill bit) at the following coordinates:
   • anterioposterior = -2 mm from bregma; lateral = ±1.6 mm; ventral = 2.5 mm; anterioposterior = -3 mm from bregma; lateral = ±2.6 mm; ventral = 3.2 mm.
   • anterioposterior = -2 mm from bregma; lateral = ±1.6 mm; ventral = 2.5 mm; anterioposterior = -3 mm from bregma; lateral = ±2.6 mm; ventral = 3.2 mm.
4. Mount a 1μl Hamilton, flat-tip syringe and inject 0.5 μl retrovirus per site at a rate of 0.25 μl/min into the dentate gyrus. (See the above coordinates for ventral depth.) Pause 2 minutes after each injection before slowly withdrawing the syringe to prevent fluid backflow. Between injections, briefly rinse the external surface of the syringe tip with sterile PBS and an applicator to remove traces of blood.
5. Close the wound with sterile surgical suture material (Type P-1; Size 6-0) and return the animal to standard housing conditions until the desired time stages after injection.

2. Slice Preparation

To obtain high-quality acute slices from adult mice, we use a modified cutting solution for slice preparation (see below).

1. Pre-chill cutting solution to 0-4 °C on ice and bubble with 95% O₂-5% CO₂ for at least 30 minutes.
2. Anaesthetize and transcardially perfuse an animal with ice-cold oxygen-saturated cutting solution.
3. Quickly remove the brain into a petri-dish with filter-paper pre-wetted with cold, oxygenated cutting solution. Cut off the cerebellum and slice a mounting surface at least 1mm anterior to the (visible) injection coordinates. Glue the brain -onto a vibrotome stage and mount it into the cutting chamber filled with cold and oxygenated cutting solution.
4. Prepare coronal or horizontal slices (300-350μm) and transfer them with a large-bore pipette to an incubating chamber containing room-temperature ACSF bubbled with 95% O₂/5% CO₂.
5. Incubate the slices, bubbling continuously, at 32°C for 30-60 minutes. Return the chamber to room temperature for the duration of the recording.

3. Electrophysiological Experiments

1. Slices are transferred into the recording chamber which is continuously perfused with oxygen-saturated ACSF at 32°C – 34°C.
2. A bipolar tungsten stimulation electrode is placed in the molecular layer of the dentate gyrus using a low magnification microscope objective (10X).
3. Newborn DGCs in the sub-granular zone/ granular cell layer are identified by the expression of GFP or dTomato. Whole-cell patchclamp recording is performed on newborn neurons under high magnification (40X).
4. Firing properties of the recorded cells are obtained by injection of a series of currents under current-clamp mode.
5. Electric stimuli are delivered by the stimulation electrode via a stimulation isolator controlled by the recording software, and evoked postsynaptic currents in the newborn DGCs are recorded.

4. Data Analysis

Amplitudes of evoked postsynaptic responses are analyzed at different developmental stages of adult-born neurons.

5. Representative Results

Using the above protocol, GFP expression in newborn dentate gyrus neurons similar to Figure 1 is common. Note that newborn neurons are easily visualized and both dendrites and axons are robustly labeled. Expression in thin DGC axons and small spines depends on the quality and titer of the virus, the promoter used and the length of in vivo expression. In our hands, newborn cells and sub-cellular organelles are usually visible within a few days following injection, and spine expression can be traced from the earliest stages of development. Whole-cell recordings from newborn neurons at different time stages allow the study of unique newborn cell properties, e.g. action potentials (Figure 2a), as well as the process of synaptic integration into existing neural circuits during maturation (Figure 2b).

Figure 1 2-photon confocal image of newborn neurons in a horizontal dentate gyrus section of an adult mouse. Green cells are 21dpi (days post injection) GFP-expressing newborn DGCs labeled with pUX-GFP retrovirus.

Figure 2 a) Action potential firing properties of a newborn DGC at 21 dpi. b) representative evoked excitatory postsynaptic currents (EPSCs) recorded on a newborn DGC at 21 dpi.