wash the coverslips ( 12 mm or 22 mm round , german glass - carolina assistant brand ) overnight in concentrated nitric acid ( hno3 ) in a dedicated glass jar or beaker . remove the coverslips from nitric acid and wash extensively if plating neurons on 12 mm coverslips directly place coverslips in a sterile 35 mm dish and proceed to section 2 . imaging chambers are constructed by drilling a 15 mm hole in the bottom of 35 mm petri dishes ( remove all burrs ) and attaching the cleaned coverslips with a 3:1 mixture of paraffin and petroleum jelly . melt the paraffin / petroleum jelly mixture in a conical tube inside a boiling water bath . use a small paint brush and coat the underside of the dish around the 15 mm hole . make sure to keep stirring the paraffin / petroleum jelly mixture as it will separate . this usually results in chambers glued together with a higher concentration of petroleum jelly , which will become viscous when dishes are placed in the incubator , resulting in coverslip detachment . place the dish upside down on a flat tray and place the coverslip over the hole . turn the dishes over and sterilize both the insides of the covers and the bottoms of chambers with uv light . coat coverslips or glass regions of chambers with 1.0mg / ml poly - d - lysine ( 30kda ) in borate buffer ( 0.1 m sodium borate , ph 8.5 ) for one hour . we usually use cleaned coverslips within one month of preparation . prepare the dissection medium ( dm ) by adding appropriate amounts of 10x hbss and 100x hepes to tissue culture grade water . the day before the dissection prepare the plating medium ( pm ) and serum - free medium ( sfm ) . pm consists of neurobasal medium , b27 supplement , 2 mm glutamine , 0.3% glucose , 37.5 mm nacl and 5% fetal bovine serum ( fbs ) . sfm consists of neurobasal medium , b27 supplement , 2 mm glutamine , 0.3% glucose and 37.5 mm nacl . make only enough for the dissection and store in a tissue culture incubator overnight with the cap slightly ajar so that the temperature and co2 content of the medium equilibrates . we find the cultures do better at a more physiological osmolality ( neurobasal osmolality is typically 205 - 245mosm ) . if long term cultures are to be prepared , perform this part of the protocol two to three weeks before continuing with the cortical or hippocampal dissections . prepare glial medium ( gm ) with mem , 0.3% glucose , penicillin / streptomycin and 10% horse serum . mince the cortices with a clean , sterile razor blade as fine as possible and remove chopped tissue with a plastic pipette to a 50 ml conical tube containing 12 ml of cold dm . add trypsin and dnase to final concentrations of 0.25% ( 1.5 ml ) and 0.1% ( 1.5 ml ) , respectively . remove the tube containing the cortical tissue and clean thoroughly with 70% ethanol before bringing into the tissue culture hood . pipet cortical tissue up and down with a 10 ml pipette approximately 10 - 15 times , or until most chunks disappear . return the tube to 37c in a water bath for another 10 minutes with intermittent swirling . thoroughly clean the tube with 70% ethanol and bring it back to the tissue culture hood . pipet the cortical tissue up and down with a 5 ml pipette approximately 10 - 15 times , or until the chunks disappear . add 15 ml of warm gm and centrifuge at 200xg ( 1000rpm ) for 10 minutes . discard supernatant , resuspend the pelleted cells in 20 ml of fresh gm and count with a hemocytometer . after one day and every 2 - 3 subsequent days in culture , dislodge loose cells by knocking the flask against your hand . remove the medium along with any dislodged cells and replace with 15 ml of fresh gm . glia can be harvested after 1 - 2 weeks of growth in the flasks , when they are about 70 - 100% confluent . to prepare individual coverslips coated with glia , place 6 nitric acid cleaned and sterilized 25 mm round coverslips in a 10 cm dish , and place 3 dots of the 3:1 mixture of paraffin / petroleum jelly onto each coverslip in a triangular pattern with a small paint brush . coat the coverslips with 0.1 mg/ ml poly - d - lysine ( 30kda ) in borate buffer for one hour , then wash extensively ( 3 - 5x ) with sterile tissue culture grade deionized water and let dry . remove the glial - containing flask from incubator , discard the medium and rinse with 5 ml of pre - warmed trypsin / edta solution . remove the trypsin / edta solution from the flask and pipette 3 ml of fresh pre - warmed trypsin / edta into the flask . incubate the flask for 1 minute at 37c before adding 5 ml of gm to stop the trypsinization . remove the glia from flask by repeated pipetting 10 - 15 times , and then transfer the media to a 15 ml conical tube . remove the supernatant and add 10 ml of gm , count cells , and plate 5x10 cells in 12.5 ml of gm per 10 cm dish containing the coverslips . the day before the neuron dissection , remove the gm and replace with sfm ( section 2.2 ) . use this glial - conditioned sfm in step 4.12 when flooding cortical or hippocampal cultures . remove appropriate amounts of nucleofection solutions ( lonza ) , combine , and warm to room temperature before starting the dissection . since nucleofection solution has a limited lifetime when combined , we only combine the needed amount for each preparation ( 100 l per transfection ) .   euthanize a pregnant mouse at e15.5 with co2 ( day of plug is e0.5 ) and remove the uterus to a 10 cm petri dish . remove fetuses and decapitate into cold dm ( section 2.1 ) . remove the entire brain into a separate dish of cold dm and with a bent tungsten needle , remove both neocortices . remove the meninges with microforceps and place cortices into new dish of cold dm . with a small iris or wecker scissors , remove the cortex or hippocampus and place in a 1.5 ml eppendorf tube filled with 1.0 ml of cold dm . keep this tube on ice . after dissecting all cortices or hippocampi , add 110 l of 2.5% trypsin to the eppendorf tube containing the tissue and place tube in a 37c incubator for 20 minutes . remove the supernatant and wash cortices or hippocampi with 1.0 ml pm ( section 2.2 ) by gently inverting the eppendorf tube . triturate the chunks 15 times with a p1000 pipette , and remove supernatant / cells to a new 15 ml conical tube containing 4 ml of pm , leaving any chunks that remain in eppendorf tube . spin the 15 ml tube at 20xg ( 350rpm ) for 7 minutes with the brake off . discard the supernatant and add 100 l of premixed , room temperature nucleofection solution ( lonza ) for each transfection . remove 100 l of the nucleofection solution / cell mixture to each new eppendorf tube and add the appropriate amount of dna . for long term cultures we generally use 1 - 2g of dna per transfection . however , this only labels a small < 10% proportion of neurons in the culture . we generally use 5 - 10g of dna per transfection if want higher transfection efficiency for short term culture . we have used up to a total of 40g of dna when transfecting with two different plasmids . add cell suspension / dna to the cuvette ( lonza ) and electroporate the cells in the nucleofector ( lonza ) , using program o-005 ( mouse cns neurons ) . working quickly , add 500 l of pre - warmed and equilibrated pm to the cuvette and remove solution / cells to a new 1.5 ml eppendorf tube . count the cells with a hemocytometer and plate at 3 - 5x10 cells / cm for young cultures , or 5 - 10x10 cells / cm for long - term cultures . for short - term cultures , flood the 35 mm culture dishes with 2.0 ml of warmed , co2-equilibrated sfm after one hour of plating . if using coverslips , we remove one - half of the pm and replace it with sfm , then repeat two more times . either flooding the imaging chambers or washing the coverslips results in a very low serum content ( < 0.5% ) . short - term cultures do not need to be cultured with a glial feeder layer and do not need to be re - fed .   for long - term cultures , we remove a glia covered coverslip containing three dots of paraffin / petroleum jelly and invert it over the 15 mm hole in the 35 mm dish , one hour after initial plating . two milliliters of the conditioned sfm from the glial dish is then added to the imaging chamber . to feed the long - term cultures , we remove one - third of the sfm every 2 - 3 days and replace it with fresh , pre - warmed and co2-equilibrated sfm . figure 1 . living hippocampal neurons in successive stages of development . paired images of representative living hippocampal neurons each of these cells has been transfected with egfp - tubulin and dsred2 in pcax vectors . the neurons were imaged at the following days in vitro ( div ) : stage 1(1div ) , stage 2 ( 1div ) , stage 3 ( 2div ) , stage 4 ( 11div ) and stage 5 ( 32div ) . wash the coverslips ( 12 mm or 22 mm round , german glass - carolina assistant brand ) overnight in concentrated nitric acid ( hno3 ) in a dedicated glass jar or beaker . remove the coverslips from nitric acid and wash extensively if plating neurons on 12 mm coverslips directly place coverslips in a sterile 35 mm dish and proceed to section 2 . imaging chambers are constructed by drilling a 15 mm hole in the bottom of 35 mm petri dishes ( remove all burrs ) and attaching the cleaned coverslips with a 3:1 mixture of paraffin and petroleum jelly . melt the paraffin / petroleum jelly mixture in a conical tube inside a boiling water bath . use a small paint brush and coat the underside of the dish around the 15 mm hole . make sure to keep stirring the paraffin / petroleum jelly mixture as it will separate . this usually results in chambers glued together with a higher concentration of petroleum jelly , which will become viscous when dishes are placed in the incubator , resulting in coverslip detachment . place the dish upside down on a flat tray and place the coverslip over the hole . turn the dishes over and sterilize both the insides of the covers and the bottoms of chambers with uv light . coat coverslips or glass regions of chambers with 1.0mg / ml poly - d - lysine ( 30kda ) in borate buffer ( 0.1 m sodium borate , ph 8.5 ) for one hour . prepare the dissection medium ( dm ) by adding appropriate amounts of 10x hbss and 100x hepes to tissue culture grade water . the day before the dissection prepare the plating medium ( pm ) and serum - free medium ( sfm ) . pm consists of neurobasal medium , b27 supplement , 2 mm glutamine , 0.3% glucose , 37.5 mm nacl and 5% fetal bovine serum ( fbs ) . sfm consists of neurobasal medium , b27 supplement , 2 mm glutamine , 0.3% glucose and 37.5 mm nacl . make only enough for the dissection and store in a tissue culture incubator overnight with the cap slightly ajar so that the temperature and co2 content of the medium equilibrates . we find the cultures do better at a more physiological osmolality ( neurobasal osmolality is typically 205 - 245mosm ) . if long term cultures are to be prepared , perform this part of the protocol two to three weeks before continuing with the cortical or hippocampal dissections . prepare glial medium ( gm ) with mem , 0.3% glucose , penicillin / streptomycin and 10% horse serum . mince the cortices with a clean , sterile razor blade as fine as possible and remove chopped tissue with a plastic pipette to a 50 ml conical tube containing 12 ml of cold dm . add trypsin and dnase to final concentrations of 0.25% ( 1.5 ml ) and 0.1% ( 1.5 ml ) , respectively . remove the tube containing the cortical tissue and clean thoroughly with 70% ethanol before bringing into the tissue culture hood . pipet cortical tissue up and down with a 10 ml pipette approximately 10 - 15 times , or until most chunks disappear . return the tube to 37c in a water bath for another 10 minutes with intermittent swirling . thoroughly clean the tube with 70% ethanol and bring it back to the tissue culture hood . pipet the cortical tissue up and down with a 5 ml pipette approximately 10 - 15 times , or until the chunks disappear . add 15 ml of warm gm and centrifuge at 200xg ( 1000rpm ) for 10 minutes . discard supernatant , resuspend the pelleted cells in 20 ml of fresh gm and count with a hemocytometer . after one day and every 2 - 3 subsequent days in culture , dislodge loose cells by knocking the flask against your hand . remove the medium along with any dislodged cells and replace with 15 ml of fresh gm . glia can be harvested after 1 - 2 weeks of growth in the flasks , when they are about 70 - 100% confluent . to prepare individual coverslips coated with glia , place 6 nitric acid cleaned and sterilized 25 mm round coverslips in a 10 cm dish , and place 3 dots of the 3:1 mixture of paraffin / petroleum jelly onto each coverslip in a triangular pattern with a small paint brush . coat the coverslips with 0.1 mg/ ml poly - d - lysine ( 30kda ) in borate buffer for one hour , then wash extensively ( 3 - 5x ) with sterile tissue culture grade deionized water and let dry . remove the glial - containing flask from incubator , discard the medium and rinse with 5 ml of pre - warmed trypsin / edta solution . remove the trypsin / edta solution from the flask and pipette 3 ml of fresh pre - warmed trypsin / edta into the flask . incubate the flask for 1 minute at 37c before adding 5 ml of gm to stop the trypsinization . remove the glia from flask by repeated pipetting 10 - 15 times , and then transfer the media to a 15 ml conical tube . remove the supernatant and add 10 ml of gm , count cells , and plate 5x10 cells in 12.5 ml of gm per 10 cm dish containing the coverslips . exchange the medium with fresh pre - warmed gm every 2 - 3 days . the day before the neuron dissection , remove the gm and replace with sfm ( section 2.2 ) . use this glial - conditioned sfm in step 4.12 when flooding cortical or hippocampal cultures . remove appropriate amounts of nucleofection solutions ( lonza ) , combine , and warm to room temperature before starting the dissection . since nucleofection solution has a limited lifetime when combined , we only combine the needed amount for each preparation ( 100 l per transfection ) .   euthanize a pregnant mouse at e15.5 with co2 ( day of plug is e0.5 ) and remove the uterus to a 10 cm petri dish . remove fetuses and decapitate into cold dm ( section 2.1 ) . remove the entire brain into a separate dish of cold dm and with a bent tungsten needle , remove both neocortices . remove the meninges with microforceps and place cortices into new dish of cold dm . with a small iris or wecker scissors , remove the cortex or hippocampus and place in a 1.5 ml eppendorf tube filled with 1.0 ml of cold dm . keep this tube on ice . after dissecting all cortices or hippocampi , add 110 l of 2.5% trypsin to the eppendorf tube containing the tissue and place tube in a 37c incubator for 20 minutes . remove the supernatant and wash cortices or hippocampi with 1.0 ml pm ( section 2.2 ) by gently inverting the eppendorf tube . repeat the wash twice , leaving 1 ml of pm in the tube . triturate the chunks 15 times with a p1000 pipette , and remove supernatant / cells to a new 15 ml conical tube containing 4 ml of pm , leaving any chunks that remain in eppendorf tube . spin the 15 ml tube at 20xg ( 350rpm ) for 7 minutes with the brake off . discard the supernatant and add 100 l of premixed , room temperature nucleofection solution ( lonza ) for each transfection . remove 100 l of the nucleofection solution / cell mixture to each new eppendorf tube and add the appropriate amount of dna . for long term cultures we generally use 1 - 2g of dna per transfection . however , this only labels a small < 10% proportion of neurons in the culture . we generally use 5 - 10g of dna per transfection if want higher transfection efficiency for short term culture . we have used up to a total of 40g of dna when transfecting with two different plasmids . add cell suspension / dna to the cuvette ( lonza ) and electroporate the cells in the nucleofector ( lonza ) , using program o-005 ( mouse cns neurons ) . working quickly , add 500 l of pre - warmed and equilibrated pm to the cuvette and remove solution / cells to a new 1.5 ml eppendorf tube . count the cells with a hemocytometer and plate at 3 - 5x10 cells / cm for young cultures , or 5 - 10x10 cells / cm for long - term cultures . for short - term cultures , flood the 35 mm culture dishes with 2.0 ml of warmed , co2-equilibrated sfm after one hour of plating . if using coverslips , we remove one - half of the pm and replace it with sfm , then repeat two more times . either flooding the imaging chambers or washing the coverslips results in a very low serum content ( < 0.5% ) . short - term cultures do not need to be cultured with a glial feeder layer and do not need to be re - fed .   for long - term cultures , we remove a glia covered coverslip containing three dots of paraffin / petroleum jelly and invert it over the 15 mm hole in the 35 mm dish , one hour after initial plating . two milliliters of the conditioned sfm from the glial dish is then added to the imaging chamber . to feed the long - term cultures , we remove one - third of the sfm every 2 - 3 days and replace it with fresh , pre - warmed and co2-equilibrated sfm . figure 1 . living hippocampal neurons in successive stages of development . paired images of representative living hippocampal neurons are shown as both a differential interference contrast image and a corresponding fluorescent micrograph . each of these cells has been transfected with egfp - tubulin and dsred2 in pcax vectors . the neurons were imaged at the following days in vitro ( div ) : stage 1(1div ) , stage 2 ( 1div ) , stage 3 ( 2div ) , stage 4 ( 11div ) and stage 5 ( 32div ) . this protocol for culturing embryonic hippocampal and cortical mouse neurons was developed as a modification of the banker protocol , which uses rat neurons . this protocol works equally well for both hippocampal and neocortical neurons and is similar to a protocol published by meberg and miller . generally , we use hippocampal neurons for long - term culture because they are well characterized and a more established model system . furthermore , they are likely to contain a more homogeneous population of neurons than neocortex . however , neocortical neurons cultured using this protocol also survive and differentiate similarly ( unpublished data ) . dissection of neocortex also results in substantially more neurons ( 1.5x10 neurons per pair of cortices ) than hippocampal dissection ( 2.5x10 neurons per pair of hippocampi ) , which makes it a better choice of material for western blotting , for example . as with any primary culture , it is essential to minimize the time that it takes from the death of the animal to the plating of the cells . it will generally take 10 - 20 dissections to become consistently fast at dissection and plating . also , when working with the lonza nucleofector , it is critical to work quickly during the electroporation procedure , as the viability of the neurons decreases rapidly if they are left in the nucleofection buffer . much of our imaging is conducted with total internal reflectance fluorescence microscopy ( tirfm ) . this type of microscopy is only capable of imaging several hundred nanometers beyond the coverslip . therefore , the areas of the neurons that we frequently image , the axonal growth cone and dendritic spines , need to be adhered directly to the coverslip . thus , we use low density cultures that require glial feeding for long - term culture . we have used higher density cultures ( > 2x10 cells / cm ) , without glial feeder layers , for long - term cultures and found that they survive very well with little feeding . however , the dendritic spines of these neurons are oftentimes too far away from the substrate to image in tirfm , although they can be readily detected with wide - field microscopy or confocal microscopy . in most of our studies we transfect neurons before plating , and have imaged fluorescently - labeled proteins for up to three months in culture . this long - term expression of fluorescently - labeled proteins gives us confidence that by using low concentrations of dna ( 1 - 2g ) we are not producing overexpression artifacts in the neurons . however , this procedure can also be used to study overexpression of proteins if high amounts of dna are used ( 10 - 20g ) . the plasmids that we use to transfect neurons usually contain egfp or mcherry fusion proteins , although we also label the neuronal cytoplasm with dsred2 or egfp alone . we prefer plasmids that contain a -actin promoter with a cmv enhancer and -globin poly - a tail ( pcaggs or pcax plasmids ) , due to the relatively high levels of expression , and the fact that they are well tolerated by the neurons in both short- and long - term culture . generally , proteins begin to express within about 4 hours of plating and reach levels sufficient for imaging within 10 - 24hrs . we have successfully used cmv - promoter - driven plasmids in short term cultures , but have found that they can cause high levels of overexpression that kill neurons in long - term culture . nevertheless , we have found that the glial conditioning of low density cultures helps with the survival of neurons transfected with cmv - promoter driven plasmids , compared to higher density ( non - glial fed ) cultures .
hippocampal and cortical neurons have been used extensively to study central nervous system ( cns ) neuronal polarization , axon / dendrite outgrowth , and synapse formation and function . 
 an advantage of culturing these neurons is that they readily polarize , forming distinctive axons and dendrites , on a two dimensional substrate at very low densities . 
 this property has made them extremely useful for determining many aspects of neuronal development . 
 furthermore , by providing glial conditioning for these neurons they will continue to develop , forming functional synaptic connections and surviving for several months in culture . in this protocol 
 we outline a technique to dissect , culture and transfect embryonic mouse hippocampal and cortical neurons . 
 transfection is accomplished by electroporating dna into the neurons before plating via nucleofection . 
 this protocol has the advantage of expressing fluorescently - tagged fusion proteins early in development ( ~4 - 8hrs after plating ) to study the dynamics and function of proteins during polarization , axon outgrowth and branching . 
 we have also discovered that this single transfection before plating maintains fluorescently - tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron ( > 2 months in culture ) . 
 thus , this methodology is useful for studying protein localization and function throughout cns development with little or no disruption of neuronal function .