high - titer engineered retrovirus ( 110unit / ml ) is produced by co - transfection of retroviral vectors and vsvg into hek293 t 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 .
anaesthetize ( 100 g ketamine + 10 g 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 . expose the skull and drill four shallow holes using a dental drill ( 0.6 mm 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 . 
  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 .
mount a 1l hamilton , flat - tip syringe and inject 0.5 l retrovirus per site at a rate of 0.25 l / min into the dentate gyrus .
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 .
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 . to obtain high - quality acute slices from adult mice , we use a modified cutting solution for slice preparation ( see below ) .
pre - chill cutting solution to 0 - 4 c on ice and bubble with 95% o2 - 5% co2 for at least 30 minutes .
anaesthetize and transcardially perfuse an animal with ice - cold oxygen - saturated cutting solution .
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 1 mm 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 . prepare coronal or horizontal slices ( 300 - 350m ) and transfer them with a large - bore pipette to an incubating chamber containing room - temperature acsf bubbled with 95% o2/5% co2 . incubate the slices , bubbling continuously , at 32c for 30 - 60 minutes . return the chamber to room temperature for the duration of the recording .
slices are transferred into the recording chamber which is continuously perfused with oxygen - saturated acsf at 32c - 34c .
a bipolar tungsten stimulation electrode is placed in the molecular layer of the dentate gyrus using a low magnification microscope objective ( 10x ) .
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 ) .
firing properties of the recorded cells are obtained by injection of a series of currents under current - clamp mode .
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 .
amplitudes of evoked postsynaptic responses are analyzed at different developmental stages of adult - born neurons .
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 .
high - titer engineered retrovirus ( 110unit / ml ) is produced by co - transfection of retroviral vectors and vsvg into hek293 t 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 .
anaesthetize ( 100 g ketamine + 10 g 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 . expose the skull and drill four shallow holes using a dental drill ( 0.6 mm 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 . 
  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 .
mount a 1l hamilton , flat - tip syringe and inject 0.5 l retrovirus per site at a rate of 0.25 l / min into the dentate gyrus .
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 .
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 .
to obtain high - quality acute slices from adult mice , we use a modified cutting solution for slice preparation ( see below ) . pre - chill cutting solution to 0 - 4 c on ice and bubble with 95% o2 - 5% co2 for at least 30 minutes .
anaesthetize and transcardially perfuse an animal with ice - cold oxygen - saturated cutting solution . 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 1 mm 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 .
prepare coronal or horizontal slices ( 300 - 350m ) and transfer them with a large - bore pipette to an incubating chamber containing room - temperature acsf bubbled with 95% o2/5% co2 .
slices are transferred into the recording chamber which is continuously perfused with oxygen - saturated acsf at 32c - 34c .
a bipolar tungsten stimulation electrode is placed in the molecular layer of the dentate gyrus using a low magnification microscope objective ( 10x ) .
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 ) .
firing properties of the recorded cells are obtained by injection of a series of currents under current - clamp mode .
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 .
amplitudes of evoked postsynaptic responses are analyzed at different developmental stages of adult - born neurons .
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 .
continuous neurogenesis in the hippocampus of adult mammalian brains provides a unique experimental model system to study neuronal development and integration in the mature brain .
the protocol presented here combines retroviral labeling and electrophysiological methods to study the integration of newborn dentate granule neurons in the brains of adult mice . to ensure that your experiments are successful
, we recommend the following during critical steps of the procedure : to avoid unwanted infection and inflammation , all tools should be sterilized ( by autoclaving , any type of sterilizer , or 70% ethanol ) before surgery .
use sterile pbs to wash the tip of the syringe needle before injection . for virus injection ,
animals must to be well mounted on the stereotaxic device and sources of unnecessary movement must be minimized during injection .
make sure the head is well fixed and firm , and adjust the nose position of the mouse to level bregma and lambda prior to calculating injection coordinates .
extract virus into the syringe quickly to minimize exposure to room temperatures - retroviruses are particularly temperature sensitive
. inject virus slowly at the recommended rate to minimize pressure damage . using the recommended flat - tipped syringe ( vs. common beveled tip ) 
will ensure an evenly distributed dentate gyrus infection area . before preparing slices , cutting solution and incubating acsf
must be bubbled with 95% o2 - 5% co2 for at least 30 minutes to allow oxygen to saturate the solutions .
animals should be perfused quickly and tissue maintained in cold , oxygen - saturated solution for best slice quality . for recording ,
change location of the stimulation electrode within the molecular layer of dentate gyrus as necessary . in summary
, the recommended approach offers a way to explore distinct properties and possible functional roles of adult - born neurons during early - stage integration into active , mature circuitry .
 neurogenesis occurs in adult mammalian brains in the sub - ventricular zone ( svz ) of the lateral ventricle and in the sub - granular zone ( sgz ) of the hippocampal dentate gyrus throughout life . 
 previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders , including epilepsy , schizophrenia , depression and anxiety ( 1 ) . 
 deciphering the process of normal and aberrant adult - born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.sgz adult neurogenesis mirrors embryonic and post - natal neuronal development , including stages of fate specification , migration , synaptic integration , and maturation . 
 however , full integration occurs over a prolonged , 6-week period . 
 initial synaptic input to adult - born sgz dentate granule cells ( dgcs ) is gabaergic , followed by glutamatergic input at 14 days ( 2 ) . 
 the specific factors which regulate circuit formation of adult - born neurons in the dentate gyrus are currently unknown.our laboratory uses a replication - deficient retroviral vector based on the moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells . 
 this viral technique provides high specificity and resolution for analysis of cell birth date , lineage , morphology , and synaptogenesis.a typical experiment often employs two or three viruses containing unique label , transgene , and promoter elements for single - cell analysis of a desired developmental process in vivo . 
 the following protocol describes a method for analyzing functional newborn neuron integration using a single green ( gfp ) or red ( dtomato ) fluorescent protein retrovirus and patch - clamp electrophysiology . 
Protocol
1. Virus Injection
2. Slice Preparation
3. Electrophysiological Experiments
4. Data Analysis
5. Representative Results
Discussion
Disclosures