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 .