gabaa receptors ( gabaars ) are essential mediators of inhibitory neurotransmission in the central nervous system and are critical for maintaining the correct balance of excitation and inhibition in the brain ( smith and kittler , 2010 ) .
gabaergic synapses undergo extensive synaptic plasticity that alters the strength and efficacy of synaptic inhibition ( luscher et  al . , 2011a ) .
inhibitory synapse strength can be rapidly controlled by changing the number of gabaars in the postsynaptic domain , which is achieved by receptor insertion into and removal from the plasma membrane at extrasynaptic sites and by dynamic movements of gabaars to and from the synapse via lateral diffusion in the plasma membrane ( arancibia - crcamo et  al . , 2009 ; bannai et  al . , 2009 ;
. however , the molecular mechanisms and regulatory signaling pathways that locally control gabaar surface levels and synaptic stability remain unclear .
the stabilization of synaptic gabaars opposite gabaergic presynaptic terminals is crucial for efficient synaptic inhibition , circuit excitability , and animal behavior ( blundell et  al . , 2009 ; crestani et  al .
gabaar clustering is mediated by a complex inhibitory postsynaptic density , the major constituent of which is the hexameric scaffold , gephyrin ( fritschy et  al . , 2008 ) .
however , in the absence of gephyrin , subsets of inhibitory synapses remain ( essrich et  al . , 1998 ; kneussel et  al . , 1999 ) , suggesting the existence of other inhibitory synaptic scaffold molecules .
the inhibitory postsynaptic specialization also contains key adhesion molecules such as neuroligin 2 and slitrk3 ( takahashi et  al . , 2012 ; varoqueaux et  al . ,
2004 ) , in addition to cytoskeletal - associated proteins , which together contribute to controlling the formation and stabilization of gabaergic synapses .
interestingly , several filamentous actin ( f - actin ) regulatory proteins have been associated with the inhibitory postsynaptic density and gephyrin ( luscher et  al . , 2011a ) , suggesting a potential role for the actin cytoskeleton at inhibitory synapses .
however , little is known regarding the regulatory signaling scaffolds that can act locally to coordinate cytoskeletal dynamics to tune gabaar synaptic stability and synaptic inhibition .
the rho family of small gtpases  rho , rac , and cdc42and their regulators play essential roles in modulating actin dynamics and are increasingly implicated in synaptic pathology and neurological dysfunction .
the activation state of small gtpases is determined by guanine nucleotide exchange factors ( gefs ) and gtpase activating proteins ( gaps ) , which together control gtp - gdp exchange and thereby promote gtpase activation and inactivation , respectively ( nobes and hall , 1999 ) .
local regulation of gtpase signaling can be further controlled by subcellular compartmentalization of gefs and gaps determined by protein scaffolds ( kiraly et  al . , 2010 ) .
currently the key gtpases , regulatory gefs and signaling scaffolds acting to regulate gabaar trafficking and inhibitory transmission are poorly understood . in this study , we have identified a git1/pix / rac1/pak signaling complex that is important for maintaining gabaar surface clusters and synaptic inhibition in neurons . using a combination of imaging , biochemical , and electrophysiological approaches ,
we show that the signaling scaffold protein git1 ( g protein - coupled receptor kinase - interacting protein 1 ) , which interacts with the rac1 gef pix , forms complexes with gabaars and is essential for normal gabaar clustering .
furthermore , we demonstrate that downstream rac1 activity is also crucial to maintain inhibitory synapse stability and works in concert with the key rac1 effector and actin - regulator , pak .
we find that rac1 activity stabilizes gabaars at inhibitory synapses while disrupting git1 , rac1 or pak all lead to impaired inhibitory transmission .
thus git1 and pix , in complex with gabaars , play a key role in locally coordinating rac1 and downstream effector activity to regulate gabaar surface stability and inhibitory synapse strength .
git1 is a signaling scaffold that can recruit pix , a gef for the small gtpase rac1 , to locally control the activation of rac1 ( zhang et  al . , 2005 ) .
git1 has been localized to synaptic sites in neurons but its potential association with gabaars and role in regulating signaling in the inhibitory postsynaptic domain remains unstudied .
we hypothesized that git1 and pix might control rac1 signaling at inhibitory synapses and are important for gabaar clustering and inhibitory synaptic transmission .
we initially determined if git1 and pix were localized at inhibitory synapses using immunocytochemistry and confocal laser scanning microscopy ( clsm ) of hippocampal neurons .
neurons labeled with antibodies to git1 , vgat ( vesicular gaba transporter ) to label inhibitory presynaptic terminals , and the 2 gabaar subunit to label synaptic gabaar clusters showed git1 was distributed along dendrites and exhibited colocalization with synaptic gabaar clusters ( figure  1a ; figure  s1a ) .
approximately 55% of git1 localized at inhibitory synapses , whereas 78% of synaptic gabaar clusters colocalized with git1 .
in addition , git1 is known to be at excitatory synapses ( zhang et  al . , 2003 ) , which we confirm by showing that the excitatory synapse marker , homer , demonstrates 60% colocalization with git1 puncta ( figure  1b ) .
git1 and pix are known to form supramolecular signaling platforms and are consistently found in a tight signaling complex in many cell types ( premont et  al .
we therefore imaged neurons labeled with git1 , pix , and 2 gabaar antibodies , showing that 79% of inhibitory synapses colocalized with pix and thereby verifying the localization of both git1 and pix at inhibitory synapses ( figure  1c ) .
interestingly , git1 localizes with synaptic gabaars ( 2 , 3 , 2 subunits , figures s1b and s1c ) but showed little overlap with extrasynaptic gabaars (  subunits , figures s1d and s1e ) .
we also demonstrated that gabaar 3 and 2 subunit antibodies readily coimmunoprecipitate git1 or pix from rat brain lysate as analyzed by western blotting ( figures 1d1f ) , confirming that git1 and pix can form native complexes with synaptic gabaars in  vivo .
git1 did not interact in a complex with extrasynaptic  subunits ( figure  s1f ) .
we also found exogenous flag - git1 to interact with the intracellular domain of the gabaar-3 subunit , by cos7 cell pull - down assays ( figure  s1 g ) , supporting git1 s interaction with gabaars .
coimmunoprecipitation of git1 with the inhibitory postsynaptic scaffold gephyrin confirmed this postsynaptic localization ( figure  1 g ) , although our data suggest that this interaction is indirect , as demonstrated by lack of coimmunoprecipitations in transfected cos7 cells ( figure  s1h ) .
as an alternative approach for further validating gabaar and pix complexes in neuronal dendrites , we performed proximity ligation assays ( plas ) , which provide valuable information about native protein interactions in  situ ( ko et  al . , 2012 ) . using plas ,
we demonstrate that pix complexes with gabaars in neuronal dendrites ( figures 1h and 1i ) .
furthermore , we show that git1 and gephyrin also interact in  situ via plas ( figures s1j and s1k ) , providing additional evidence to support an inhibitory postsynaptic and close association for these proteins in dendrites . to investigate the role of git1 at inhibitory synapses , we utilized rnai to knock down its protein expression in neurons .
rnai caused a significant reduction of git1 expression levels in addition to causing a small reduction in dendrite length as previously shown ( figures s2a  s2d ; menon et  al .
, 2010 ) . to determine the consequences of git1 knockdown on inhibitory synapse and surface gabaar cluster area
, we performed immunocytochemistry and clsm of neurons expressing git1 or control rnai constructs , using an extracellular 2 subunit antibody to label surface gabaars and antibodies to vgat to identify inhibitory synapses . git1 knockdown neurons exhibited a significant decrease in surface gabaar and vgat cluster area compared to control ( figures 2a2c ) , suggesting a possible role for git1 in maintaining the integrity of inhibitory pre- and postsynaptic domains in neuronal dendrites .
importantly , this effect could be rescued by coexpression of rnai - resistant human git1 ( hgit1 ; figures s2e and s2f ) .
git1 knockdown also caused a large decrease in gephyrin cluster area ( figures 2d and 2e ) , suggesting that git1 is important for maintaining both gabaar clusters and the gephyrin scaffold in neurons .
this was further confirmed by surface biotinylation assays , which revealed that surface gabaar levels were reduced in git1 knockdown neurons compared with control ( figures 2f and 2 g ) . considering its role in other cell types
, we hypothesized that  git1 may be important for localizing f - actin regulatory pathways to inhibitory synapses .
therefore , we sought to determine whether surface gabaars were sensitive to short - term disruption of the actin cytoskeleton by treating neurons with latrunculin - a , an inhibitor of actin polymerization ( renner et  al . , 2009 ) .
we found that a 30  min application of 3  m latrunculin - a to neurons caused a significant decrease in surface gabaars ( figures 2h and 2i ) , with no effect on extrasynaptic gabaar populations ( figures s2 g and s2h ) , suggesting that actin polymerization does indeed play an important role in the maintenance of inhibitory synapses .
we then asked whether the inhibitory synapse effects we observed upon knockdown of git1 were due to altered f - actin regulation .
we therefore treated control or git1 rnai - expressing neurons with the f - actin - stabilizing drug , jasplakinolide ( hering et  al . , 2003 ) , prior to surface biotinylation and western blot analysis ( figure  2j ) .
as predicted , git1 rnai caused a significant loss of surface gabaars compared to control , which was restored by treatment with jasplakinolide ( figures 2j and 2k ) .
this suggests that the decrease in surface gabaars observed in git1-deficient neurons is caused by impaired f - actin regulation , and points toward a mechanism involving actin - regulatory proteins .
git1 s primary binding partner pix is one such actin - regulatory protein and is a strong candidate to collaborate with git1 in mediating actin regulation at inhibitory synapses . to test this hypothesis
, we utilized rnai to pix , which caused a significant reduction of pix expression levels ( figures s3a and s3b ) .
pix knockdown in neurons caused a similar effect to that of git1 rnai , reducing both surface gabaars and vgat cluster area ( figures 3a3c ) .
surface biotinylation assays revealed the same phenotype , with pix knockdown neurons exhibiting reduced surface gabaar levels compared with control cells ( figures 3d and 3e ) .
we found git1 or pix knockdown had no effect on ampa receptor clustering or extrasynaptic - containing gabaars ( figures s3c  s3f ) , suggesting that this protein complex is important for synaptic gabaar clustering only .
we then sought to determine whether git1 and pix are important in controlling actin regulation at inhibitory synapses .
we treated control neurons with phalloidin to label f - actin and  found that 80% of inhibitory synapses were positive for f - actin .
git1 and pix knockdown caused a significant decrease in the percentage of inhibitory synapses containing f - actin ( figures 3f and 3 g ) , suggesting that git1 and pix have an important role in controlling f - actin at inhibitory synapses .
the effect of pix knockdown on surface gabaar clusters was rescued by rnai resistant human pix ( hpix ) showing the specificity of the rnai .
in contrast , versions of hpix that no longer have gef activity for rac1 ( hpix - dn ) , or that contain a mutation of the pix sh3 domain ( which is critical for coupling to a downstream effector , pak [ hoelz et  al . , 2006 ] ) , were unable to rescue pix knockdown induced gabaar declustering ( figures 3h and 3i ) .
this suggests that the ability of pix to activate rac1 and interact with pak is essential for its role in maintaining inhibitory synapses and supports the idea that there exists a key  actin regulatory mechanism controlling inhibitory synapse maintenance .
git1 can anchor pix , which directly interacts with the small gtpase rac1 and mediates its activation ( ten klooster et  al . , 2006 ) . because we could not rescue the effects of pix knockdown with a version of pix with impaired gef activity toward rac1 , we hypothesized that rac1 might be responsible for the changes in gabaar clustering in git1 or pix knockdown neurons .
we verified that rac1 was localized to synaptic gabaar clusters in neurons using immunocytochemistry with antibodies to rac1 , the 2 gabaar subunit and vgat , followed by clsm ( figure  4a ) .
we found that 45% of rac1 colocalized with synaptic gabaar clusters along the dendritic shaft ( figure  4b ) , suggesting that rac1-positive signaling complexes may influence synaptic gabaar trafficking or function . to test
whether rac1 activity has a role in maintaining synaptic gabaar clustering , we utilized a dominant - negative rac1 mutant ( nobes and hall , 1999 ) ( rac1-dn ) to block rac1 activation in neurons .
immunostaining and clsm of hippocampal neurons transfected with gfp or rac1-dn revealed a decrease in surface gabaar cluster area and vgat area in neurons expressing rac1-dn compared with control neurons ( figures 4c4e ) .
in addition , surface biotinylation assays revealed that blocking rac1 activation with the rac1-dn caused a decrease in gabaar surface levels ( figures 4f and 4 g ) .
as an alternative approach , we determined if acute short - term inhibition of rac1 would cause similar effects on gabaar surface levels . to achieve this ,
we incubated neurons for 1  hr with the rac1 inhibitor eht 1864 ( eht ; shutes et  al . , 2007 ) , followed by immunocytochemistry and clsm .
similar to the results with rac1-dn , analysis of these neurons showed that acute inhibition of rac1 activity reduced the area of gabaar and gephyrin clusters ( figures 4h4j ) .
moreover , surface biotinylation assays with neurons treated with eht caused a decrease in surface gabaars ( figures 4k and 4l ) , indicating that rac1 contributes to gabaergic synapse stability in neurons .
we then wanted to explore the mechanism downstream of active rac1 leading to the stabilization of gabaar clusters .
pak is a major effector of rac1 that modulates f - actin to stabilize essential cellular structures ( kreis and barnier , 2009 ) .
we hypothesized that rac1 might mediate its action at inhibitory synapses by activating pak , which is supported by our observation that an sh3-domain mutant of pix ( which can no longer couple to pak ) is unable to rescue the effects of pix rnai on gabaar clusters ( figures 3h and 3i ) .
active pak is autophosphorylated ; therefore , we tested whether we could detect phospho - pak at inhibitory synaptic sites by performing immunocytochemistry and clsm of neurons labeled with antibodies to gabaar 2 subunit , the inhibitory presynaptic marker gad6 and phospho - pak ( figure  5a ) .
active pak was found clustered along dendrites and colocalized with both synaptic gabaar clusters and gad6 , confirming its presence at inhibitory synapses . to determine whether pak activity is important for maintaining gabaar and gephyrin cluster stability , we incubated hippocampal neurons with ipa ( deacon et  al . , 2008 ) , a specific pak inhibitor and assessed gabaar 2 , gephyrin and vgat cluster area .
treatment with ipa caused a substantial decrease in gabaar and gephyrin cluster area , with little  effect on vgat cluster area ( figures 5b5e ) .
surface biotinylation assays showed a decrease in surface gabaar expression in neurons treated with ipa compared with vehicle - treated neurons ( figures 5f and 5 g ) , showing that pak activity is required for maintaining surface gabaars in neurons . to further verify a role for pak in controlling gabaar surface stability
, we utilized the autoinhibitory domain ( aid ) of pak fused to gfp , which has widely been used to inhibit pak activity in culture by blocking its autophosphorylation ( kreis and barnier , 2009 ) .
surface biotinylation of neurons transfected with gfp or gfp - pak - aid revealed that gfp - pak - aid expression caused reduced surface gabaar expression compared with control ( figures 5h and 5i ) . to confirm that pak acts downstream of git1 and pix at inhibitory synapses
, we performed rescue experiments with neurons cotransfected with git1 or pix rnai and a constitutively active ( ca ) mutant of pak .
cotransfection with pak - ca effectively prevented the depletion of gabaar clusters observed with git1 or pix rnai alone ( figures 5j and 5k ) , suggesting that pak acts downstream of git1 and pix to control inhibitory synapse maintenance in neurons .
together , these data suggest that a git1/pix / rac1/pak signaling pathway plays an important role in stabilizing gabaar and gephyrin clusters and the maintenance of inhibitory synapses .
our results suggest that components of a signaling pathway involving git1 , pix , rac1 , and pak are critical for stabilizing surface and synaptic gabaars and maintaining gabaergic synapse integrity in neurons .
whole - cell patch - clamp recordings were performed to measure inhibitory synaptic transmission in neurons expressing the git1 rnai , pix rnai or gfp - pak aid constructs .
analysis of spontaneous ipsc ( sipsc ) traces from these cells revealed that git1 or pix knockdown , or inhibition of pak all caused a considerable decrease in sipsc amplitude compared to control neurons ( figures s4a  s4e ) .
these reduced amplitudes can be seen in representative sipsc traces ( figure  s4a ) and the leftward shift to smaller amplitudes in cumulative probability plots ( figures s4b and s4d ) .
analysis of these data showed there was no significant change in the sipsc frequency ( figures s4c and s4e ) . to further explore the impact of inhibiting the git1 signaling pathway , we measured the impact on miniature ipscs ( mipscs ) .
we recorded mipscs from neurons transfected with control or  git1 rnai ( figures 6a6e ) .
analysis of traces from these neurons showed that knockdown of git1 caused a significant decrease in both mipsc amplitude and frequency , which could be rescued with rnai resistant hgit1 , again showing the specificity of the rnai knockdown .
we also recorded mipscs from neurons transfected with pix rnai and pak - aid , analysis of which demonstrated a similar reduction in mipsc amplitude and frequency as that of git1 rnai - expressing neurons ( figures 6a , and 6c6e ) .
the effects of the pix rnai were successfully rescued by coexpression with hpix , and also by pak - ca , suggesting that pak indeed mediates the effects of pix on gabaergic synaptic transmission .
importantly , there was no significant difference between the decay time constants of the events recorded from neurons expressing git1 rnai , pix rnai , or pak - aid compared with control neurons , indicating that the receptor properties are unaltered ( figure  s4f ) .
in addition , pix rnai expression had no significant effect on miniature excitatory postsynaptic currents ( figures s4g  s4i ) , suggesting that inhibition of this pathway under these conditions has specific effects on inhibitory synaptic transmission . to determine the impact of inhibiting this pathway on gabaergic transmission in an intact network
slices were incubated with either ipa or eht to inhibit pak or rac1 , respectively ( figure  6f ) .
analysis of the strength of inhibition with input / output curves of evoked ipscs ( eipscs ) generated by a series of stimulus intensities showed that inhibition of the rac1-pak pathway in brain slices caused a substantial depression of the input / output eipsc curves ( 40%50% reduction for ipa and 30%50% reduction for eht ; figures 6f and 6 g ) .
this indicates that rac1 and pak are indeed critical to maintain gabaergic synaptic transmission in the brain .
together , these electrophysiological experiments reveal that inhibiting the git1/pix / rac1/pak pathway in neurons not only reduces surface gabaar cluster and gephyrin cluster area , but also leads to reduced inhibitory synaptic transmission .
clustering of gabaars at inhibitory synapses is imperative for correct synaptic inhibition in the brain and is tightly controlled by components of the inhibitory postsynaptic density .
reduced gabaar synaptic clustering equates to reduced inhibition in neuronal circuits and subsequent disruption of excitatory / inhibitory ( e / i ) balance , producing defects in information processing at the network level .
our results describe a signaling complex localized to the inhibitory postsynaptic domain that is crucial for correct inhibitory neurotransmission and the maintenance of gabaergic synaptic transmission .
we show surface gabaar clusters are maintained by a git1/pix / rac1/pak signaling complex that modulates f - actin , thereby stabilizing the inhibitory postsynaptic density and synaptic gabaars ( figure  s4j ) .
we demonstrate that git1 interacts with synaptic gabaar subunits and is localized at inhibitory postsynaptic sites , suggesting that it is intimately involved with the inhibitory postsynapse and its function . indeed , the interaction between git1 and gabaars places it in the exact location to scaffold pix at the inhibitory synapse where it could locally activate rac1 signaling ( zhang et  al . , 2003 )
. consistent with this , we also show that pix is located at inhibitory synapses and interacts in a complex with gabaars .
gefs such as pix are essential signaling coordinators that localize and regulate small gtpase signaling at specific sites within the cell ( kiraly et  al . , 2010 ) . therefore , our data showing the presence of pix and rac1 at the inhibitory synaptic site , combined with the effects of pix knockdown and rac1 inhibition on gabaar clustering and inhibitory synaptic transmission , strongly suggest that a rac1 signaling pathway is important for maintaining synaptic inhibition
. our results also point to a scaffolding role for git1 at the inhibitory synapse , potentially as an additional scaffolding protein to increase the stability of gephyrin and gabaar surface clusters .
git1 and pix are also shown to be important for excitatory synapse function , acting via a rac1/pak / actin pathway ( zhang et  al . , 2005 ) , in an activity - dependent manner ( saneyoshi et  al . , 2008 ) .
our findings are supportive of similar scaffolding mechanisms at inhibitory synapses , and suggest that the git1/pix signaling pathway may represent a key coordinator of actin pathways at synapses .
indeed , this is in agreement with the role of this git1/pix signaling module at regions of cell - cell contact in multiple cell types ( hoefen and berk , 2006 ) .
further study will now be required to define how the git1/pix complex may coordinate potential crosstalk between excitatory and inhibitory synapses .
the number of gabaars at the neuronal surface and synaptic sites directly correlates with the strength of inhibitory synaptic transmission ; therefore , the modulation of gabaar synaptic accumulation is a key mechanism for plasticity of inhibitory synapses . here , we show that git1 or pix knockdown causes reduced gabaar clustering and a decrease in the number of gabaars at the neuronal surface , and this effect is mimicked by inhibition of rac1 or pak in neurons . indeed ,
disruption of gabaar clustering by pix knockdown can not be rescued by pix mutants lacking gef activity for rac1 or the ability to couple to pak , implicating pak as a downstream effector .
importantly , the effects of rnai mediated knockdown of either git1 or pix on gabaar clustering is rescued by active pak .
these biochemical and imaging data are supported by electrophysiological data , which show that knockdown of git1 or pix , or inhibition of rac1 or pak , causes reduced gabaergic currents in neurons , suggesting the reduction in gabaar clusters does indeed correlate with reduced inhibition .
in addition to these postsynaptic effects , we also observe reductions in vgat clustering and mipsc frequency , suggestive of additional presynaptic effects of git1/pix knockdown . in our imaging and electrophysiological experiments ,
we analyze git1/pix knockdown in the postsynaptic neuron , suggesting the presynaptic effects we observe are due to destabilization of the presynaptic gabaergic synaptic bouton concurrent with the loss of postsynaptic receptors , scaffolds , and adhesion molecules .
previously , disruption of gephyrin has been shown to reduce gabaar clusters postsynaptically , accompanied by a loss of presynaptic gabaergic innervation ( marchionni et  al .
similarly , loss of the 2 subunit ( as we demonstrate here occurs upon disruption of git1/pix ) causes loss of both postsynaptic clustering and presynaptic innervation ( li et  al . ,
it is becoming clear that crosstalk between the pre- and postsynaptic sites of inhibitory synapses is essential for their plasticity , demonstrated by the observation that inhibitory pre- and postsynaptic structures are highly mobile and can move as a single entity ( dobie and craig , 2011 ) . because our rnai experiments are targeting the postsynaptic cell , our results are consistent with alteration of the git1/pix complex disrupting inhibitory postsynaptic domains , which also causes subsequent disruption of presynaptic innervation .
the git1/pix / rac1/pak pathway we have presented here documents a signaling pathway that links gabaar stability to the actin cytoskeleton via a gtpase signaling cascade . by treating neurons with latrunculin -
a for 30  min , we show that an intact actin cytoskeleton is important for gabaar stability .
this is in contrast with earlier findings showing no effect of latrunculin - a on gabaar clustering ( allison et  al . , 2000 ) ; however , this study differed in both age of neurons and length of treatment ( 24  hr ) .
we conclude that , although actin is not structurally required at inhibitory synapses as in dendritic spine heads , it is emerging that actin is required for the integrity of the inhibitory postsynaptic site ( charrier et  al . , 2006 ) and for postsynaptic scaffold mobility in general ( kerr and blanpied , 2012 ) .
gephyrin also interacts with collybistin ( kins et  al . , 2000 ) , a gef for the small gtpase cdc42 .
the role of collybistin in region - specific inhibitory synapse formation has been investigated ( papadopoulos and soykan , 2011 ) , although it is still unclear whether cdc42 activity is required for gephyrin clustering with several studies suggesting that collybistin functions independently of its gef activity ( reddy - alla et  al . , 2010 ) .
thus , other rho gtpase signaling and scaffolding mechanisms are likely to be present at inhibitory synapses . in agreement with this
, we show that pix and rac1 activity is required for inhibitory synapse function , by maintaining gabaar surface levels at synapses , gephyrin clustering , and gabaergic currents .
git1 knockout ( ko ) mice have been investigated in the context of neuronal function in two independent reports .
reduced dendritic spine density and dendrite length in the hippocampal ca1 region have been reported in one git1 ko model , in addition to impaired performance in learning tasks ( menon et  al . , 2010 ) .
in  a second study , investigators also observed memory and learning impairments , with increased hyperactivity and reduced inhibitory presynaptic input in ca1 ( won et  al . , 2011 ) .
here , we demonstrate the effects of acute git1 knockdown and the short - term effects of inhibiting the git/pix / rac1/pak pathway on gabaar clustering and synaptic inhibition and find reduced clustering of essential inhibitory synapse components including gephyrin . we therefore attribute the differences in our findings and those of won et  al .
, to the use of global ko strategies , which cause git1 knockdown in all cell types throughout development , which is in contrast to the short - term rnai targeting and pharmacological treatment that we employ here
. reduced inhibition due to depletion of gabaars from the neuronal cell surface can alter the e / i balance of neuronal circuits , causing disrupted information processing , which may lead to altered animal behavior ( blundell et  al . , 2009 ; crestani et  al .
, 1999 ; papadopoulos et  al . , 2007 ; tretter et  al . , 2009 ; yizhar et  al . ,
deficits in gabaergic neurotransmission leading to an altered e / i balance have also been implicated in multiple neuropsychiatric disorders including depression ( luscher et  al . , 2011b ) , bipolar disorder ( craddock et  al . ,
2010 ) , schizophrenia ( charych et  al . , 2009 ) , and attention deficit hyperactivity disorder ( adhd ; won et  al .
therefore , identifying the molecular mechanisms that are essential for maintaining inhibitory synaptic transmission in the brain is also critical to understanding the development of these neuropsychiatric disorders , where it may become necessary to readdress pathological alterations in e / i balance .
our findings showing that a rac1 signaling pathway is important for regulating inhibitory synaptic transmission may shed light on the molecular mechanisms underlying mental illness .
indeed , many of the proteins involved in the gtpase signaling pathway we describe here have been directly linked to mental disorders ( allen et  al .
, 2004 ; govek et  al . , 2004 ; won et  al .
. altered pak signaling due to mutations in the pak3 gene has been linked to patients with mental retardation , and pak signaling is additionally implicated in models of fragile - x syndrome and schizophrenia ( chen et  al .
, 2007 ; hayashi - takagi et  al . , 2014 ) , making it an important molecule in the synaptic pathology of psychiatric disorders .
in addition , git1 was recently shown to harbor a single nucleotide polymorphism causing reduced git1 expression that is strongly linked to adhd in humans ( won et  al . , 2011 ) .
our results suggest how a postsynaptic git1 signaling complex plays a key role in controlling synaptic inhibition , by stabilizing gabaars at the inhibitory synaptic site , and may be an important locus for altered animal behavior and psychiatric and cognitive deficits .
we have characterized a git1/pix / rac1/pak signaling pathway that controls gabaar clustering from a molecular and physiological viewpoint .
we found that git1 , pix , rac1 , and pak are  all essential to maintain surface gabaar clusters and inhibitory currents in neurons , therefore making this signaling pathway an important regulator of inhibitory signaling in the brain .
details regarding antibodies , immunocytochemistry and analysis , coimmunoprecipitations , biotinylations , and cdna cloning are included in the supplemental experimental procedures .
all experimental procedures were carried out in accordance with institutional animal welfare guidelines and the uk animals ( scientific procedures ) act 1986 .
cultures of cortical and hippocampal neurons were prepared from e18 sprague - dawley rat embryos as described previously ( pathania et  al . , 2014 ;
twelvetrees et  al . , 2010 ) ; see also the supplemental experimental procedures .
standard voltage - clamp techniques were used for whole - cell recordings of  spontaneous ipsc in cultured neurons ( twelvetrees et  al . , 2010 ) .
electrodes were filled with the following internal solution ( in mm ) : 100 cscl , 30 n - methyl - d - glucamine , 10 hepes , 1 mgcl2 , 5 egta , 2 qx314 , 12 phosphocreatine , 5 mgatp , 0.5 na2gtp , 0.2 leupeptin ( ph 7.27.3 ) , and 270280 mosm . the external solution consisted of the following ( in mm )
: 130 nacl , 26 nahco3 , 3 kcl , 5 mgcl2 , 1.25 nah2po4 , 1 cacl2 , and 10 glucose ( ph 7.4 ) , 300 mosm .
cnqx ( 20  m ) and apv ( 50  m ) were added to block ampa and nmda receptors .
data were analyzed with kaleidagraph ( albeck software ) and mini analysis program ( synaptosoft ) . to measure gabaergic transmission in cortical slices
, we used standard whole - cell recording techniques ( yuen et  al . , 2011 ) .
slices were incubated at room temperature ( 20c 22c ) in artificial csf ( acsf ) bubbled with 95% o2 , 5% co2 , and then slices were treated with various agents for 1  hr before recordings .
the in  situ proximity ligation assay ( pla ) was used according to the manufacturer s instructions ( olink bioscience ) .
neurons were fixed in 4% pfa/30% sucrose , blocked ( 10% horse serum , 0.5% bsa , and 0.2% triton x-100 , 10  min at room temperature ) , and incubated with primary antibodies . for control pla , a single antibody was applied .
cells were washed in 1   pbs and then incubated with secondary antibodies conjugated to oligonucleotides .
ligation and amplification reactions were conducted at 37c , before mounting and visualization with clsm .
e.c.d . performed molecular biology and performed and analyzed some of the biochemistry and confocal imaging experiments .
 summaryeffective inhibitory synaptic transmission requires efficient stabilization of gabaa receptors ( gabaars ) at synapses , which is essential for maintaining the correct excitatory - inhibitory balance in the brain . 
 however , the signaling mechanisms that locally regulate synaptic gabaar membrane dynamics remain poorly understood . using a combination of molecular , imaging , and electrophysiological approaches , 
 we delineate a git1/pix / rac1/pak signaling pathway that modulates f - actin and is important for maintaining surface gabaar levels , inhibitory synapse integrity , and synapse strength . 
 we show that git1 and pix are required for synaptic gabaar surface stability through the activity of the gtpase rac1 and downstream effector pak . manipulating this pathway using rnai , dominant - negative and pharmacological approaches leads to a disruption of gabaar clustering and decrease in the strength of synaptic inhibition . 
 thus , the git1/pix / rac1/pak pathway plays a crucial role in regulating gabaar synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions