transcription - dependent neuronal plasticity is evolutionarily conserved from invertebrates to mammals ( frank and greenberg , 1994 ) . accordingly , the neuronal activity - dependent gene expression program , which underlies adaptation - related brain functions , is likely generic , meaning that many of the activity - responsive genes are shared between species and neuron types ( discussed by bading , 2013 ) . these genes are poorly characterized in humans , especially in comparison to mice whose activity - regulated transcriptome , implicating 1,000 genes , is known ( kim et  al . , 2010 , , 2011 , spiegel et  al . , 2014 , zhang et  al . , 2007 , zhang et  al . , 2009 ) . it is important as well as exciting to fill this gap in knowledge , since , as for phenotypic differences between species in general ( necsulea and kaessmann , 2014 , wilson and odom , 2009 ) , evolutionary gene regulation and expression changes may , to a large extent , determine the dissimilarities in brain development and function of humans compared to other species ( bae et  al . , 2015 , cognitive abilities , which in humans have evolved to a perplexing species - specific phenotype distinction , are in part reliant on synaptic activity - dependent gene regulation ( bading , 2013 , west and greenberg , 2011 ) . in humans , the orthologs of rodent activity - regulated genes are expressed with a species - specific temporal profile during brain development ( liu et  al . , 2012 ) . whether the human synaptic activity - regulated gene program itself has distinctive features is unknown , however , mostly because obtaining human neurons for experimental use is ethically problematic . here we determine the gene expression response to synaptic activity in human neurons derived from induced pluripotent stem cells ( ipscs ) to find out if the human genetic background expands the repertoire of known activity - regulated genes . studies of the human neuronal adaptive gene program require an experimental system where human neurons are synaptically connected . we differentiated ipscs ( see the experimental procedures ) to human ipsc - derived ( hipscd ) neurons , and we confirmed the generation of postmitotic neuronal cells by expression analysis of multiple markers , rna sequencing ( rna - seq ) , and comparison of the hipscd neuron transcriptomes to previously published hipscd neuron and human fetal brain gene expression profiles ( figure  s1 ; table s1 ) . to directly assess cellular phenotype on a functional level , we performed whole - cell patch - clamp recordings from hipscd neurons differentiated for 7 or 10  weeks from hipscd neuronal precursor cells ( npcs ) ( hereafter 7- or 10-week hipscd neurons ) . at earlier time points , , we recorded from 7-week hipscd neurons cultured together with mouse ( post - natal day [ p]0 ) primary hippocampal neurons for the last 10  days ( hereafter hipscd neuron / mouse primary neuron co - culture or mixed - species culture ) . the hipscd neurons in the co - cultures were distinguished by their expression of egfp under the control of the human synapsin i ( syn1 ) promoter ( psyn1 ) delivered by lentiviral infection of npcs ( see also figures s1c , s1d , and 3 g ) . passive properties ( membrane capacitance [ 3438 pf ] , resistance [ 9001,350 m ] , and resting membrane potential [ vrest , 43 to 48  mv ] ) of hipscd neurons were not affected by co - culture with mouse neurons ( in figures denoted by hs  + mm , hipscd neuron - only culture is denoted by hs ) , and they were not significantly different between cells in the 7- and 10-week hipscd neuron - only cultures ( figures 1a1c ) . action potential ( ap ) amplitudes and numbers were improved when evoked from 70  mv instead of vrest ( figures 1d1f ) , which reflects their relatively depolarized state . almost all hipscd neurons in all cultures analyzed showed spontaneous postsynaptic currents that could be divided into two groups , with rapid or slow decay time constants ( figures 1g1i ) . they were blocked in egfp - expressing ( egfp ) cells by either nbqx ( 5  m ) , a blocker of ampa / kainate - type glutamate receptors ( 7-week hipscd neuron - only cultures or mixed - species cultures , n  = 4/4 for each ) , or by the gaba type a ( gabaa ) receptor antagonists bicuculline ( bic , 50  m ) or gabazine ( 5  m ) ( 7-week hipscd neuron - only cultures , n  = 7/7 ; mixed - species cultures , n  = 5/5 ) , respectively . this confirms the presence of functional synapses containing postsynaptic ampa / kainate or gabaa receptors in our hipscd neurons . to verify the existence of functional nmda , ampa , and gabaa receptors as well as voltage - gated ca channels ( vgccs ) in hipscd neurons , we used ca imaging . in almost all egfp and egfp - non - expressing ( egfp ) cells in the 7-week hipscd neuron - only cultures , large ca responses were evoked by nmda , applied with its co - agonist glycine , or by ampa , applied with an inhibitor of ampa receptor desensitization , cyclothiazide ( figure  1j ) . gaba also increased ca levels ( figure  1k ) , indicating an excitatory , depolarizing effect . ca influx in response to ampa , nmda , and gaba presumably involves vgccs , and their presence in hipscd neurons was confirmed by robust increases in ca in response to depolarization with a high - concentration k solution ( figure  1k ) . thus , 7  weeks of differentiation of human ipsc - derived npcs is sufficient to obtain hipscd neurons that exhibit functional ampa , nmda , and gabaa receptors as well as vgccs . having established culture systems generating synaptically connected hipscd neurons , we sought a method to experimentally induce robust excitatory synaptic activity in these cultures to  study activity - induced transcription in hipscd neurons . in 10-day cultures of mouse primary hippocampal neurons , synaptic activity - dependent gene expression can be induced by disinhibition of the neuronal network with bic , which triggers synchronous bursts of ap firing along with ampa and nmda receptor- and vgcc - mediated ca signals , whose frequency is increased by coapplication of bic with the k channel blocker 4-aminopyridine ( 4ap ) ( arnold et  al . , 2005 , hardingham et  al . , 2001 ) . bic ( 50  m ) applied together with 4ap ( 250  m ) ( bic/4ap ) activated large recurring bursts of synaptic activity in both egfp and egfp cells in our 7-week mixed - species cultures ( figures 2a and 2b ) and , to a lesser extent , in 7-week hipscd neuron - only cultures ( figure  2c ) . egfp cells in 7-week hipscd neuron - only cultures and mixed - species cultures showed , respectively , a 4.4-fold and a 10.8-fold increase in synaptic charge influx after bic/4ap treatment ( figure  2e ) . in the 10-week hipscd neuron cultures , bic/4ap induced larger synaptic bursts than in the 7-week hipscd neuron - only cultures ( figures 2c and 2d ) , and it caused recurring membrane potential oscillations ( 18.9  1.9  mv ; 1.73  0.44/min ; n  = 8) , which were abolished by nbqx ( n  = 3 ; data not shown ) . synaptic activation caused by bic/4ap was accompanied by recurrent depolarizations and/or aps in all cultures assessed . analyses of ca transients activated downstream of bic/4ap - induced synaptic activity revealed large , recurrent increases in ca levels with a sustained increase in baseline ca levels in both egfp and egfp cells in the 7-week co - cultures ( figure  2f ) , similar to responses seen in mouse primary hippocampal neurons ( hardingham et  al . , 2001 ) . recurrent ca responses to bic/4ap also were apparent in 7- and 10-week hipscd neuron - only cultures but without a sustained increase in baseline ca levels and with a smaller amplitude in the 7-week than in the 10-week cultures ( figures 2g2i ) . the depolarizing effect of gaba ( see above ) and the lack of response to bic ( figure  s2a ) in 7-week hipscd neuron - only cultures may explain their weaker responses to bic/4ap compared to 10-week hipscd neurons , where gabaa receptors likely have undergone the developmental switch from excitatory to inhibitory ( ben - ari et  al . , 2007 ) . these results demonstrate that bic/4ap induces robust recurrent increases in ampa / kainate receptor - mediated synaptic and ap activity along with ca influx into hipscd neurons in the mixed - species or hipscd neuron - only cultures . the addition of mouse primary neurons to the 7-week hipscd neuron cultures or the extra 3  weeks of differentiation of hipscd neuron - only cultures greatly increase hipscd neuron responses to bic/4ap . we used species - specific rt - qpcr on human orthologs of three prototypical activity - responsive mouse immediate - early ( ie ) genes , npas4 , fos , and arc , to assess if enhancement of synaptic input to hipscd neurons induces human ie genes . increases in human npas4 , fos , and arc mrna levels in hipscd neurons after a 1-hr bic/4ap treatment were easily detectable in both hipscd neurons cultured with or without mouse primary neurons ( figures 3a and 3b ) . all the inductions peaked 1  hr after bic/4ap addition and dropped almost to the basal levels by 4  hr ( figures 3c and 3d ) . in the 7-week co - cultures , the increases in the levels of all of the three assessed human ie gene mrnas in response to bic/4ap were abolished in the presence of tetrodotoxin ( ttx , 1  m ) , an na channel blocker that prevents bic/4ap - induced ca signals by inhibiting ap firing ( hardingham et  al . , 2002 ) , proving that expression of npas4 , fos , and arc is activity - regulated in hipscd neurons ( figure  3e ) . the bic/4ap - induced increase in npas4 mrna levels was reduced by the inhibition of l - type vgccs with nifedipine ( nif , 10  m ) or by two nmda receptor antagonists , 2-amino-5-phosphonovalerate ( apv , 50  m ) and mk801 ( mk , 10  m ) ( apv / mk ) . induction of npas4 and fos was more strongly inhibited by the combination of nif , apv , and  mk ( nif / apv / mk ) , revealing a potential synergistic role of vgccs and nmda receptors ( figure  3e ) . in the 10-week hipscd neuron - only cultures , the induction of npas4 and fos was severely reduced in the presence of ttx , nif , apv / mk , or nif / apv / mk , although the effect of nif on fos was comparatively weaker ( figure  3f ) . arc induction was reduced by blocking vgccs and/or nmda receptors in both cultures ( figures 3e and 3f ) . finally , co - culture experiments with hipscd neurons produced from a distinct ipsc line ( hips 3 , see the experimental procedures ) produced nearly identical results ( figures s2b and  s2c ) . to determine the proportion of hipscd neurons responding to bic/4ap treatment with ie gene induction and to confirm the response at the protein level , we used immunostaining for yet another classical ie gene product , junb . the co - culture showed junb protein expression after 2  hr of enhanced excitatory synaptic input ( figure  3 g ) . these results demonstrate that strong induction of excitatory synaptic activity - dependent ie gene expression in human cells can be evoked by a simple bic/4ap treatment of hipscd cultures . also , they show that nmda receptors and vgccs are involved in triggering synaptic activity - induced increases in ie gene mrna levels in hipscd neurons . we performed rna - seq to examine the synaptic activity - regulated gene program in hipscd neurons . rna levels of 50 or 42 human genes were significantly changed ( benjamini - hochberg adjusted p [ padj ]  < 0.1 ) in hipscd neurons 1  hr after bic/4ap addition to the 7-week mixed - species cultures ( figure  4a ; table s2 ) or to the 10-week hipscd neuron cultures ( table s2 ) , respectively . rna levels of 37 human genes were significantly changed in both culture systems ( figure  4b ) . the expression fold change values of all of the obtained 55 genes between the two cultures were nearly equivalent ( pearson s r [ r ]  = 0.96 ; figure  4c ) , supporting the validity of the mixed - species rna - seq analysis ( see the supplemental experimental procedures ) . we assigned a human gene to be a putative ie gene ( hereafter ie  gene ) if the significant expression level change ( padj  < 0.1 ) in one dataset was supported by a change with an unadjusted p  less than 0.01 in the other or if the change was more than 2-fold with the padj less than 0.05 in at least one of the datasets . altogether we identified 47 human synaptic activity - regulated ie  genes , all with upregulated rna levels ( table s3 ) . rna - seq results with the mixed - species samples were validated for hipscd neurons derived from two different ipsc lines with the ncounter method using species - specific probes ( figures s2d and s3s5 ) . we categorized the 47 human ie genes according to the data available for their rodent orthologs ( figure  4d ) . the rodent orthologs of 41 of the human ie genes ( 87.2% ) were significantly upregulated after bic/4ap treatment in mouse primary neurons according to our rna - seq and ncounter analysis , and they have been reported to be neuronal activity regulated in other studies using different experimental systems and time points ( kim et  al . , 1997 , saha et  al . , 2011 , spiegel et  al . , 2014 , zhang et  al . , 2007 , zhang et  al . , 2009 ) . the orthologs of three human ie genes ( c11orf96 , mir22hg , and socs3 ) have not been previously identified as activity - regulated genes in rodents , although socs3 was only regulated in one of the two ipsc lines used here . three of the human ie genes , bre - as1 , linc00473 , and znf331 , are exceptional because they do not have rodent orthologs . bre - as1 and linc00473 rna levels were detected to be strongly ( 15-fold ) and znf331 rna levels weakly ( 1.5-fold ) upregulated in hipscd neurons with rna - seq after 1  hr of bic/4ap treatment , and all three genes were confirmed to be activity induced with ncounter analysis in hipscd neurons derived from both of the ipsc lines used here ( figures s3 and s5 ) . also , their synaptic activity - induced expression was blocked or severely reduced in the presence of ttx or nif / apv / mk ( figure  s2d ) . bre - as1 is a noncoding rna gene antisense to the bre ( brain and reproductive organ - expressed ) gene . linc00473 is a primate - specific long noncoding rna gene that has been documented to be duplicated along with only three other genes in a patient with delayed speech and language development ( decipher 277616 ; firth et  al . , 2009 ) . the most significantly over - represented gene ontology ( go ) terms among the human synaptic activity - regulated ie genes were all associated with transcription factor activity ( figure  4d ; table s4 ; 24 genes , 51.1% , encode transcription factors ) . also , camp response elements ( cres ) or cre half site ( crehs)-containing sequences and serum response elements ( sres ) were significantly enriched in the proximal promoter regions of human ie genes ( table s4 ) , suggesting that many of them are targets of the well - established neuronal activity - dependent transcriptional regulators , cre - binding protein ( creb ) and sre - interacting proteins ( bading et  al . , 1993 ) . next , we compared the synaptic activity - regulated human ie  genes to their mouse orthologs using the data from the co - cultures  where rna of the two species is collected from cells stimulated in identical culture conditions . rapid human rna level changes after enhancement of glutamatergic synaptic activity were paralleled by similar changes of the respective mouse orthologs ( figure  4e ; expression change correlation , r  = 0.73 ) . based on induction strength , half of the ie gene ortholog pairs grouped together ( figures 4f and 4 g ) . for example , egr4/egr4 , fos / fos , fosb / fosb , junb / junb , npas4/npas4 , and nr4a1/nr4a1 were strongly induced in human cells as well as in mouse cells . to classify genes in terms of temporal expression profile regardless of induction strength , we performed hierarchical clustering of human ie genes together with their mouse orthologs , if present , based on rna levels scaled between 0 and 1 from untreated and 1- or 4-hr bic/4ap - treated mixed - species cultures ( figure  4h ) . relative to their upregulated expression levels at the 1-hr time point , ie gene rna levels at the 4-hr time point were either decreased ( termed here transient ) , unchanged ( termed sustained ) , or further increased ( termed amplified ) . this analysis revealed that half of the examined mouse genes , but only 17% of the human ie genes , belonged to the amplified cluster . instead , 55.3% of the human ie genes grouped into the transient set of genes and 27.7% had the sustained rna expression profile . examples of transient human ie genes with amplified or sustained mouse orthologs include gadd45b / gadd45b and nr4a2/nr4a2 or fosb / fosb and npas4/npas4 , respectively . human hic1 , encoding hypermethylated in cancer 1 , grouped to the sustained cluster and mouse hic1 grouped to the transient cluster . hic1/hic1 is the only relatively strongly induced ie gene in both cell types ( human , 5.9-fold ; mouse , 4.5-fold ) that had a more sustained increase in expression levels in hipscd neurons compared to mouse neurons ( figure  4i ) . these results establish that overall the hipscd neuron synaptic activity - responsive ie  gene program is generic . however , it involves genes that do not have orthologs in the mouse , and many of the human ie  genes are in hipscd neurons upregulated more transiently than their mouse orthologs in mouse primary hippocampal neurons . we next asked if the three synaptic activity - regulated human ie  genes that do not have mouse orthologs ( see figure  5a for ncounter confirmation of rna - seq results ) represent loci regulated by neuronal activity via conserved mechanisms or are induced by cell - type or species - specific mechanisms . to answer this , we conducted luciferase reporter assays for the regulation of bre - as1 , linc00473 , and znf331 promoters in mouse primary hippocampal and cortical neurons . the promoters of these genes contain cre and crehs cis elements ( figure  5b ) that may direct the induction of promoter activity upon excitatory synaptic signaling , and , moreover , the linc00473 promoter ( plinc00473 ) contains a bhlh - pas transcription factor response element ( pasre ; figure  5b ) that could permit the enhancement of transcriptional activity after the immediate response . the assays showed increases in normalized luciferase levels 6  hr after bic/4ap addition with the bre - as1 promoter ( pbre - as1 ) and plinc00473 and two of the cloned  znf331 promoter - containing regions ( pznf331 ii and iii , covering three and two 5 exons , respectively ; figures 5b and s6a ) . these results reveal that bre - as1 , linc00473 , and znf331 are under the control of promoters that are responsive to synaptic activity in mouse neurons . this demonstrates that they are targets of evolutionarily conserved synapse - to - nucleus signaling mechanisms , most probably involving nuclear ca / calmodulin kinases ( bading , 2013 ) . to investigate if synaptic activity - dependent regulation of pbre - as1 , plinc00473 , and pznf331 iii is sensitive to nuclear ca / calmodulin inhibition and interference with cre - dependent or pasre - mediated transcription , we used co - transfection of constructs encoding for one of the following proteins : ( 1 )  cambp4 , a nuclear - localized inhibitor of ca / calmodulin signaling ( wang et  al . ( 2 ) icer1 ( icer ) , an inhibitor of creb family proteins ( de cesare and sassone - corsi , 2000 ) ; or ( 3 ) npas4 without the transcription activation domain ( deltad - npas4 ) , a dominant - negative regulator of bhlh - pas transcription factors ( pruunsild et  al . , 2011 ) . we found that luciferase expression under the control of pbre - as1 , plinc00473 , and pznf331 iii after 6  hr of bic/4ap treatment was significantly reduced by cambp4 ( figure  5c ) . overexpression of icer led to diminished bic/4ap - induced luciferase levels with pbre - as1 and plinc00473 , but not with pznf331 iii . the dominant - negative bhlh - pas factor deltad - npas4 considerably lowered bic/4ap - induced transcription only from plinc00473 ( figure  5c ) . additionally , pznf331 iii activity was significantly reduced by cambp4 in unstimulated conditions , indicating that basal nuclear ca / calmodulin activity regulates transcription from this promoter . collectively , these results prove  that the promoters of human bre - as1 , linc00473 , and the most 3 promoter - containing region of znf331 are regulated  by the conserved ca - signaling pathway that activates nuclear  ca / calmodulin kinases in neurons . pbre - as1 and plinc00473 are probably induced by creb , and the transcriptional activity from plinc00473 is likely to be increased by recruitment of the bhlh - pas transcription factor npas4 and arnt2 heterodimer to the pasre . rna levels of 428 or 215 human genes were detected to be significantly changed ( padj  < 0.1 ) with rna - seq 4  hr after bic/4ap addition to the 7-week hipscd neuron / mouse primary hippocampal neuron co - culture ( figure  6a ; table s5 ) or to the 10-week hipscd neuron - only culture ( table s5 ) , respectively . although the overlap of significantly regulated genes was smaller between the two culture conditions than detected for the 1-hr time point ( figures 6b and 4b ) , the mean fold change values of the total of these 524 genes correlated strongly between the datasets ( figure  6c , r  = 0.88 ) . using the same two criteria as for defining the human ie genes , we designated 218 human genes to belong to the later phase of the synaptic activity - responsive genomic program in hipscd neurons . from these we excluded 20 genes that we had designated to be ie genes , and we acquired a set of 198 human synaptic activity - regulated late - response ( lr ) genes ( table s6 ; figure  6d ) . the majority of the human lr genes ( 167 genes , 84.3% ) have mouse orthologs that were correspondingly regulated in mouse primary neurons in this study and/or have been shown previously to be activity regulated ( balik et  al . , 2013 , kim et  al . , 2010 , maze et  al . , 2015 , mo et  al . , 2015 , saha et  al . , 2011 , spiegel et  al . , 2014 , , 2006 , zhang et  al . , 2007 , zhang et  al . , 2009 ) . the mouse orthologs of 22 or nine human lr genes that were up- or downregulated , respectively , in hipscd neurons have not been described to be  neuronal activity regulated or have been found to be regulated  in the opposite direction in mice by others and/or by us ( table  s7 ) . the only significantly enriched go categories among the 150 upregulated human lr genes were synapse ( go:0045202 , false  discovery rate [ fdr]b&y  = 2.56e03 ) and synapse part ( go:0044456 , fdrb&y  = 2.74e03 , figure  6d ) . this suggests that a key consequence of synaptic activity - dependent gene expression in hipscd neurons could be the adjustment of the composition and function of synapses . rna level changes of the human lr genes and their mouse orthologs in the 7-week mixed - species cultures were positively correlated ( r  = 0.46 ; figure  6e ) . however , the correlation between human and mouse gene rna level changes 4  hr after the bic/4ap addition was significantly weaker than that at the 1-hr time point ( r  = 0.46 versus r  = 0.73 , z  = 2.5 , p  = 0.012 , fisher z - transformation ) . furthermore , the levels of a number of human lr genes were changed hipscd neuron specifically , suggesting that the late phase of the transcriptional response to synaptic activity depends more on the cell type and/or the species than the early phase . we also noted that the human lr genes include the orthologs for 26 mouse excitatory neuron - specifically induced genes , like erf , homer1 , and scg2 , and ten mouse inhibitory neuron - specifically induced genes , like nab1 and hivep2 ( spiegel et  al . , 2014 ) , indicating that the activity - regulated gene program described here for hipscd neurons originates from both excitatory and inhibitory cells . it seems likely that the neuron type , including possible effects determined by the species , defines the complete set of lr genes that are regulated by synaptic activity . it is uncertain whether synaptic activity - induced transcription in hipscd neurons reflects that of the human brain in  vivo . ( 2010 ) to find out if the promoters of human genes regulated by synaptic activity in hipscd neurons have a neuron - enriched open chromatin - associated transcription initiation mark h3k4me3 in the human prefrontal cortex in  vivo during development and in the adult . this is indicative of the genes accessibility for activity - dependent regulation in the human brain in  vivo . the neuron - enriched h3k4me3 promoter mark was present on significantly more genes than expected by chance in all hipscd neuron synaptic activity - regulated gene groups , except the hipscd neuron - specifically downregulated genes ( figure  7a ) . this reveals that the genes detected here to be synaptic activity regulated in hipscd neurons are enriched for genes that are more apt to transcription in the human brain than in other tissues . we next explored whether the synaptic activity - controlled gene program described here includes human genes associated with regulatory changes compared to other mammals , by examining if hipscd neuron activity - regulated genes have human - specific promoter or enhancer h3k27ac and/or h3k4me2 gains during human brain cortex development in  vivo , according to the data from reilly et  al . significantly more genes than expected by chance were positive for a human - specific epigenetic regulatory gain only among the genes that were hipscd neuron - specifically upregulated by 4  hr of bic/4ap - induced activity ( figure  7b ) . this infers that expression of a relatively large proportion of the genes shown here to be responsive to synaptic activity only in hipscd neurons is probably regulated in a human - specific manner also in the brain in  vivo . finally , we focused on hic1/hic1 to study if regulatory dna sequence dissimilarities of species rather than the cell type could explain temporal differences in gene expression in response to synaptic activity between hipscd neurons and mouse primary neurons . we chose hic1/hic1 because it was the only relatively strongly induced ie gene that was upregulated more transiently in mouse neurons compared to hipscd neurons , a result that we confirmed with species - specific rt - qpcr ( figures 7c and s6b ) . the promoter of hic1/hic1 is not well conserved among vertebrates in general , but it contains several almost identical regions potentially encompassing important regulatory sequences ( figure  7e ) . we searched these conserved sequences for cis elements that are bound by transcription factors in human cells using the encyclopedia of dna elements ( encode ) chromatin immunoprecipitation sequencing ( chip - seq ) data , and we found that the only orthologous binding motif relevant to activity - controlled gene regulation that is not present in the rodent hic1 promoter is the ie protein egr1-binding sequence located 1,000  bp upstream of the human hic1 refseq transcript 5 end ( figures 7d and 7e ) . this suggests that , upon synaptic activity , mrna levels of mouse hic1 may not be sustained because egr1 can not bind to the mouse hic1 promoter . to test this , we  generated a luciferase reporter construct with the mouse hic1 promoter , and , for comparison , we inserted the egr1 site in it by changing two nucleotides according to the human hic1  promoter . consistent with our hypothesis , luciferase assays in mouse primary neurons revealed that the mouse hic1 promoter humanized with the egr1 motif produced a moderate rise in luciferase levels after 6  hr of bic/4ap treatment , whereas the wild - type promoter did not ( figure  7f ) . this result supports the idea that a simple dna element alteration in the promoter could lead to a difference in the temporal profile of  hic1 expression in response to synaptic activity in mice compared to its ortholog s expression profile in humans . this study reveals three ways in which the human genetic background influences neuronal adaptogenomics compared to that of mice : ( 1 ) it expands the repertoire of human synaptic activity - regulated genes with lineage - specific genes , ( 2 ) it permits synaptic activity - dependent regulation of some human genes whose orthologs are not activity - regulated in mice , and ( 3 ) it can gene - selectively change the temporal profile of synaptic activity - responsive transcriptional activity . we identify three human genes , bre - as1 , linc00473 , and znf331 , that lack mouse orthologs but are regulated by synaptic  activity via conserved signaling mechanisms to be included in the human activity - responsive adaptive gene program . linc00473 , which belongs to the core set of hipscd neuron synaptic activity - regulated genes ( figure  s7 ) , has been shown to be a primate - specific camp pathway - responsive noncoding rna  that negatively regulates ie genes ( reitmair et  al . , 2012 ) . znf331 and also hic1/hic1 , the only relatively strongly induced ie gene that had a more sustained induction in humans compared to mice , encode transcriptional repressors . interestingly , a number of synaptic activity - controlled ie genes were upregulated more transiently in hipscd neurons than their mouse counterparts in primary neurons ( e.g. , egr4/egr4 , fosb / fosb , and npas4/npas4 ) , possibly reflecting lineage - specific ie gene regulation by linc00473 , znf331 , and hic1 . more transient upregulation of some of the ie genes in hipscd neurons can not be explained by a lack of sustained bic/4ap - induced activity and mechanisms amplifying transcription , since the distinct temporal regulation of synaptic activity - controlled genes between hipscd neurons and mouse primary neurons was gene selective . specifically , along with the distinct temporal profiles , the upregulation of several orthologous ie genes in both human and mouse cells  was commonly transient ( e.g. , arc / arc , btg2/btg2 , and nr4a1/nr4a1 ) or commonly amplified after the initial response ( e.g. , csrnp1/csrnp1 , fosl2/fosl2 , and rgs2/rgs2 ) . we therefore propose that genetic differences gene - selectively cause some of the orthologous human and mouse ie genes to have different expression kinetics after synaptic activity . the mechanisms probably involve both regulation in trans , including regulation by lineage - specific activity - responsive genes as discussed above , and regulation in cis , as indicated by our result with the hic1 promoter that suggests a role for a species - specific cis - regulatory element . such an interpretation also is supported by a recent study that used a high concentration of extracellular kcl to stimulate neurons ( qiu et  al . , 2016 ) . in rodents a large fraction of neuronal activity - induced ie genes are transcription factors ( lanahan and worley , 1998 ) . our data confirm that genes encoding orthologs to these also are enriched among the synaptic activity - regulated ie genes in humans . the induced transcription factors are in turn expected to regulate genes that produce the adaptive response . in mouse excitatory and inhibitory neurons the adaptive transcriptional responses are different ( spiegel et  al . , 2014 ) , indicating that neuron type - specific activity - dependent gene regulation occurs . we found that the mouse orthologs of 15.7% of the hipscd neuron late - response genes have no evidence for neuronal activity - dependent regulation . it could be that , because the hipscd neurons are relatively immature , this cell type - specific part of the response is developmental stage specific rather than species specific . however , the genes upregulated only in hipscd neurons were over - represented by genes that have a neuron - enriched h3k4me3 promoter mark in the human brain throughout development as well as in the adult . second , this suggests that a considerable fraction of hipscd neuron - specifically synaptic activity - regulated genes are accessible for regulation during the course of the entire lifespan and are unlikely to be exclusively responsive to activity during neuronal maturation . besides , the genes that were upregulated only in hipscd neurons displayed enrichment for genes that are accompanied by human - specific epigenetic gains in the human brain ( reilly et  al . , 2015 ) , suggesting that they might include genes whose regulation , possibly including activity - dependent regulation , is species specific . our data suggest that acquisition of lineage - specific synapse - to - nucleus signaling target genes and differences in synaptic activity - dependent regulation of common targets contribute to the phenotypic differences of humans compared to mice . first , one of the genes whose upregulation by synaptic activity was specific to hipscd neurons was adra1b ( table s7 ; figure  s7 ) , which is associated with attention deficit / hyperactivity disorder ( mick et  al . , 2010 ) . if adra1b is activity regulated in  vivo , it could be involved in the regulation of mood , cognition , and behavior as an activity - induced component of the monoaminergic system in humans that is not synaptic activity dependent in mice ( but see kobori et  al . , second , we detected camta1 , which is associated with human episodic memory performance ( huentelman et  al . , 2007 ) and intellectual disability ( thevenon et  al . , 2012 ) , to be upregulated by activity in hipscd neurons but not in mouse neurons ( table s7 ) . this is  exciting because camta1 is linked to a conserved noncoding sequence with accelerated evolution in humans ( prabhakar et  al . , 2006 ) , has several putative enhancer regions with human - specific epigenetic gains ( reilly et  al . , 2015 ) , and knockdown of camta1 in the mouse hippocampus specifically alters long - term memory ( bas - orth et  al . , 2016 ) , making camta1 a good candidate for a factor that adjusts episodic memory in a human - specific , synaptic activity - dependent manner . third , hic1 , the gene with a prolonged increase in mrna levels after synaptic activity in hipscd neurons compared to mouse neurons ( figure  s7 ) , negatively regulates expression of reelin receptor genes ( dubuissez et  al . , 2013 ) . since reelin is essential for the lamination of the cortex and has roles in neuronal maturation and synaptic functions ( frster et  al . , 2010 ) , hic1 may be involved in brain cortex development . moreover , miller - dieker syndrome ( mds ) with lissencephaly as part of the phenotype is caused by haploinsufficiency of the genomic locus encompassing hic1 ( yingling et  al . although heterozygous loss of the pafah1b1 ( alias lis1 ) and ywhae ( alias 14 - 3 - 3 ) genes is believed to cause lissencaphaly in mds , there is evidence that it also can result from a deletion not involving these but the hic1 gene ( decipher 250068 ; firth et  al . , collectively , these examples accentuate that some of the potentially human lineage - specifically regulated genes uncovered in this study take part in brain functions or developmental processes that could account for the superior cognitive abilities of humans compared to other mammals . thus , the evolution of synaptic activity - regulated adaptogenomics may have driven the evolution of cognition . in conclusion , this study demonstrates that the generic transcriptional response to evolutionarily conserved synapse - to - nucleus signaling has lineage - specific features in human neurons . this confirms that inherent genetic differences have an impact on synaptic activity - dependent transcription , providing a mechanism of how they may tune adaptive , plasticity - related brain functions . hips d1 was generated from healthy human fibroblasts with an inducible polycistronic lentiviral reprogramming vector encoding for klf4 , myc , pou5f1 , and sox2 ( horschitz et  al . , 2015 ) . hips 3 was generated from healthy human melanocytes with inducible lentiviral reprogramming vectors encoding for klf4 , myc , and pou5f1 ( utikal et  al . , 2009 ) . we differentiated ipscs to npcs using a slightly modified spin embryoid body protocol ( kim et  al . , 2011 ) with dual smad inhibition ( chambers et  al . , 2009 ) , as specified in the supplemental experimental procedures . differentiation of npcs was initiated by plating 50,000 cells / cm on poly - ornithine- ( 0.1  mg / ml ) and laminin- ( 10  g / ml ) ( both sigma - aldrich ) coated ( po / lam ) dishes in npc medium ( see the supplemental experimental procedures ) without egf and fgf2 . after 1  week with medium changes every other day , cells were replated 75,000 cells / cm on po / lam dishes or coverslips in  neuronal differentiation ( nd ) medium ( neurobasal , glutamax [ 1  mm ] , b27-vitamin a [ 2% ] , pen / strep [ 1:200 ; all thermo fisher scientific ] , ascorbic acid [ 200  m , sigma - aldrich ] , and bdnf [ 10  ng / ml , peprotech ] ) . differentiation was allowed to proceed for up to 9 more weeks with medium changes three times per week . p0 mouse primary hippocampal cells , 75,000 cells / cm , were added to hipscd neurons in nd medium for the last 10  days of differentiation ; 10  days in  vitro is sufficient for inducing ap bursting in mouse primary neuron cultures with bic/4ap ( arnold et  al . , rna - seq was performed on four biological replicates of untreated and 1-hr bic/4ap - treated mixed - species or hipscd neuron - only culture samples and three biological replicates of 4-hr bic/4ap - treated mixed - species or hipscd neuron - only culture samples ( geo : gse88773 ) . tophat2 ( version 2.0.9 ) ( kim  et  al . , 2013 ) , htseq - count ( htseq version 0.6.0 or 0.6.1 ) ( anders et  al . , 2015 ) , and deseq2 ( version 1.8.1 ) ( love et  al . , 2014 ) were used for  rna - seq read mapping , counting , and differential gene expression analysis , respectively . the co - culture sample reads were filtered for either human or mouse reads as specified in the supplemental experimental procedures . whole - cell patch - clamp recordings were made at 32c from cells plated on  glass coverslips and secured with a platinum ring in a recording chamber ( open access chamber-1 , science products ) with heated in - line perfusion ( 32c , tc324b , warner instruments ) running constantly at 2  ml / min . two samples were compared with two - tailed student s or welch s t test . if indicated , holm - sidak p adjustment was applied . multiple samples were compared with one - way anova and dunnett s or tukey s test , two - way anova and tukey s test , or two - way anova on ranks and holm - sidak test . in rna - seq data analysis ,
summarylong - term adaptive responses in the brain , such as learning and memory , require synaptic activity - regulated gene expression , which has been thoroughly investigated in rodents . 
 using human ipsc - derived neuronal networks , we show that the human and the mouse synaptic activity - induced transcriptional programs share many genes and both require ca2 + -regulated synapse - to - nucleus signaling . 
 species - specific differences include the noncoding rna genes bre - as1 and linc00473 and the protein - coding gene znf331 , which are absent in the mouse genome , as well as several human genes whose orthologs are  either not induced by activity or are induced with different kinetics in mice . 
 these results indicate that lineage - specific gain of genes and dna regulatory elements affects the synaptic activity - regulated gene program , providing a mechanism driving the evolution of human cognitive abilities .