based on the accumulated information by many neuroanatomists , here we will take the opportunity to focus on the wiring by gabaergic long - range neuronal connections in the brain . we first examine the literature related to gabaergic neurons with cortico - fugal , and subcortical afferent axons ( figure 1 ) . currently reported cortico - cortical , cortico - fugal , and afferent gabaergic projections . bf , basal forebrain ; cb , calbindin ; ms , medial septum ; nkbr , neurokinin - b receptor ; nnos , neuronal no synthetase ; pv , parvalbumin ; ss , somatostatin ; th , tyrosine hydroxylase . gabaergic interneurons are categorized into several subtypes based on characteristic expression profiles of peptides and proteins ( kubota et al . , 1994 ; gonchar and burkhalter , 1997 ) or physiological properties ( miles et al . , 1996 ; markram et al . , 2004 ; puig et al . , for example , medial septal gabaergic neurons are positive for pv and terminate preferentially on hippocampal gabaergic neurons ( freund and antal , 1988 ) . a subset of the hippocampal gabaergic neurons , which often contain calbindin ( cb ) , somatostatin ( ss ) , or neuronal no synthetase ( nnos ) , project axons to the medial septum and innervate the pv - positive gabaergic neurons ( tth and freund , 1992 ; tth et al . , 1993 ; jinno , 2009 ) . when medial septal gabaergic neurons inhibit gabaergic neurons in the hippocampus , so as to cancel inhibition to pyramidal neurons , the pyramidal neurons are allowed to fire ( tth et al . , 1997 ) . the cancellation of inhibition ( disinhibition ) in the hippocampus is terminated by the inhibitory input to the pv neurons in the medial septum . this disinhibiting septo - hippocampal circuit ( and its negative feedback loop ) was found by recording large field potentials in the hippocampus following stimulation of the fimbria ( bilkey and goddard , 1987 ) . the circuit for  disinhibition was analyzed anatomically , and in agreement with the in vivo electrophysiology results , reciprocal projections between gabaergic neurons were found ( freund and antal , 1988 ) . subsequent electrophysiological studies in vitro revealed that the septo - hippocampal circuit generates theta - oscillation in the hippocampus ( heynen and bilkey , 1991 ; tth et al . , 1997 ) . non - pyramidal neurons in the cat hippocampal formation project to the mammillary body ( ino et al . , 1988 ) . tyrosine hydroxylase ( th)-positive gabaergic neurons in the a13 subdivision of the zona incerta project to the neocortex ( lin et al . , 1990 ) . the neocortex , striatum , and basal forebrain are connected by different subtypes of gabaergic projections . a small number of pv - positive gabaergic neurons in the somatosensory area project axons to the striatum ( jinno and kosaka , 2004 ) . neurokinin - b positive gabaergic neurons in the striatum project to neurokinin - b receptor - positive gabaergic neurons in the basal forebrain which project axons to the cerebral cortex ( furuta et al . , 2004 ) . in retrograde labeling studies of excitatory neurons , tracers , such as wga - hrp , fast blue ( fb ) , and fluorogold , were injected into axon terminal areas and detected in the neuronal somata , after retrograde axonal transport . retrograde labeling combined with immunohistochemistry of gad or gaba , or other modified nnos labeling methods correlated with morphology of retrogradely labeled neurons revealed that gabaergic neurons project axons to remote neocortical regions in the ipsilateral hemisphere ( mcdonald and burkhalter , 1993 ; albus and wahle , 1994 ; aroniadou - anderjaska and keller , 1996 ; fabri and manzoni , 1996 ) and in the contralateral hemisphere ( peters et al . , 1990 ; gonchar et al . , 1995 ; fabri and manzoni , 2004 ; tomioka et al . , 2005 ; these cortico - cortical gabaergic projections are also found in the connections between cortical areas ( tomioka and rockland , 2007 ) and between allocortices ( ino et al . , 1990 ; van haeften et al . , 1997 ; jinno , 2009 ) . indeed , microstimulation of the neocortex elicits monosynaptic inhibitory postsynaptic potentials ( ipsps ) that are not preceded by excitation in a small number of neurons in the remote ipsilateral cortex ( ghosh and porter , 1988 ; salin and prince , 1996 ) . to simplify the procedures and to increase the sensitivity of detecting gabaergic projection neurons in the neocortex , we produced a glutamic acid decarboxylase 67 ( gad67 ) green fluorescent protein ( gfp ) knock - in mouse ( tamamaki et al . , 2003 ) , and performed fb retrograde labeling experiments in the mouse ( tomioka et al . , 2005 ) . we plotted gfp- and fb - double - labeled cells in the entire neocortical area and characterized the gabaergic projection neurons by immunohistochemistry ( figure 2 ) . in the study we found fb - single - labeled cells ( excitatory projection neurons ) are accompanied by a small number of gfp- and fb - double - labeled cells ( gabaergic projection neurons ) in most cases of the retrograde labeling ( figure 2d ) . we also found several cortical regions that contained fb- and gfp - double - labeled cells , but few fb - single - labeled cells . this mismatch between the distribution of fb - single - labeled cells and that of fb- and gfp - double - labeled cells implies that gabaergic projections may be distributed more widely over ipsilateral neocortical areas than excitatory projections in the mouse . following fb injection into a visual neocortical area of the gad67-gfp mouse , we found temporal neocortical areas that contained fb- and gfp - double - labeled cells but few fb - single - labeled cells . most of these double - labeled neurons showed immunoreactivity of somatostatin , neuropeptide y ( a ) , and nnos ( b ) . a venn diagram in ( c ) shows the relationship between the immunoreactivity and the gabaergic projection neurons in the neocortex . ( d ) distribution of ss - positive gabaergic projection neurons were revealed by fb injection into the neocortex v1 and represented on the mouse - flattened cortical map and serial frontal sections . each red - filled circle indicates an fb- , gfp- and ss - positive neuron . blue marker and gray large circle ( insets ) shows the fb injection site and cortical region within 1.5 mm from the fb injection site , respectively depending on the projection distance , the retrogradely labeled subtypes of gabaergic neurons vary greatly ( tomioka et al . , 2005 ; tomioka and rockland , 2007 ) . within 1.5  mm of the tracer - injection site , many retrogradely labeled gabaergic neurons exhibit immunoreactivity for pv , ss , or calretinin ( cr ) in all the neocortical layers in mice . at increasing distances , the retrogradely labeled gabaergic finally , in cortical regions more than 1.5  mm from the injection site , almost all of the retrogradely labeled gabaergic neurons express ss - ir in the deepest layer of the neocortex , or in the white matter and layer i. although gabaergic projections in the archicortex and paleocortex originate in the gray matter and layers ii v , respectively ( van haeften et al . , 1997 ; jinno , 2009 ) , the actual distance of these projections may be less than 1.5  mm in rodent . the gabaergic commissural projection , which is longer than 1.5  mm , originates in the deepest layer of the neocortex ( gonchar et al . , 1995 ) , and the laminar distribution of the gabaergic projection neurons mimics that of nnos - positive type 1 neurons in rats and monkeys ( yan et al . , 1996 ; yan and garey , 1997 ) . consistent with this observation , gabaergic projection neurons positive for ss , npy , and nnos ( 91 , 82 , and 71% , respectively ) have been identified . the more that is learned about the features of gabaergic projection neurons , the more they appear to be similar to the nnos - positive type 1 neurons in the neocortex . an intracellular labeling study revealed that the nnos - positive subplate neurons give rise to projecting axon longer than 1.5  mm ( clancy et al . notably , the nnos - positive neurons consist of two different subpopulations in rats and monkeys ( yan and garey , 1997 ; smiley et al . , 2000 ) : one is characterized by cb - ir , small somata , and low nadph - diaphorase activity ( type ii neurons ) and the other by a lack of cb - ir , large somata , and intense nadph - diaphorase activity ( type i neurons ) ( kubota et al . , 1994 ; yan et al . , 1996 ; gonchar and burkhalter , 1997 ; smiley et al . , 2000 ) . yan and garey ( 1997 ) suggested that the vast majority of the nnos - positive neurons in the mouse neocortex are the cb - negative type i neurons . considering these observations , we speculate that the vast majority of cortico - cortical gabaergic projection neurons in the mouse express nnos . nnos - positive gabaergic neurons account for 1% in rat ( gonchar and burkhalter , 1997 ) . taking all these factors into consideration , we speculate that gabaergic projection neurons account for 0.5% of the neocortical gabaergic neurons in mouse . several possible functions of nnos in the central nervous system have been suggested , including the regulation of glutamate release ( kano et al . , 1998 ) and of capillary dilatation ( cauli et al . , 2004 ) , but we do not yet know how the nnos , npy , and ss expressed by gabaergic projection neurons function in the neocortex . it is known that gabaergic projection neurons in the white matter receive neocortical excitatory inputs as a major neuronal input , but these cells do not appear to receive significant thalamic input ( shering and lowenstein , 1994 ; okhotin and kalinichenko , 2003 , tomioka et al . , 2005 ) . to examine the function of gabaergic projection neurons , the primary approach will be to observe the morphology of projection axons , especially their terminal arborization , and to identify their postsynaptic target cells . it is also important to find roles only gabaergic projection neurons can play in the neocortex . an important clue to the function of gabaergic projection neurons may be inferred from the organization of the corpus callosum . the corpus callosum contains the commissural fibers of excitatory neurons and those of gabaergic neurons ( peters et al . , 1990 ; gonchar et al . , 1995 ; fabri and manzoni , 2004 ; tomioka et al . , 2005 ; higo et al . , the latter may be selected from transient gabaergic commissural fibers ( cobas et al . , 1988 ; kimura and baughman , 1997 ) and are stabilized for interhemispheric communication beyond early developmental stages . in the case of sectioning of the corpus callosum , the gamma - oscillations were de - synchronization between the two hemispheres ( figure 3 ) ( engel et al . , 1991 ; uhlhaas and singer , 2010 ) . thus , we may be able to test how excitatory fibers and gabaergic fibers in the corpus callosum contribute to synchronize the gamma - oscillation by specific ablation or blocking of either fiber system . since the mechanism for generating gamma - oscillation is not solely attributable to excitatory neurons , it is possible that the mechanism for synchronizing the gamma - oscillations in multiple cortical areas is also not attributable solely to excitatory neurons . gabaergic projection neurons in the neocortex may make an important contribution to the synchrony of gamma - oscillations . we speculate that gabaergic projection fibers may release gaba and adjust the spike timing of excitatory neurons and pv neurons through gabaa receptor - mediated quick ipsps . callosal connections mediate long - distance synchronization . in the bottom panels are cross - correlograms between responses from different recording sites in the left hemisphere ( lh ) and right hemisphere ( rh ) that indicate the degree of interhemispheric synchronization . when the corpus callosum was intact ( left - hand panel ) , strong interhemispheric synchronization occurred with no phase lag between the lh and rh recording sites . sectioning of the corpus callosum ( right - hand panel ) abolished interhemispheric synchronization while leaving synchronization within hemispheres intact . adapted from uhlhaas and singer ( 2010 ) as discussed above , we speculate that nnos - positive gabaergic projection neurons are involved in important brain functions . to address the function of nnos - positive gabaergic projection neurons in vivo , there are several tools that can silence , activate , or delete the neurons in question , such as , channelrhodopsin-2 variant ( lin et al . , 2009 ) , light - activated electrogenic cl( ) pump ( gradinaru et al . , 2008 ) , and diphtheria toxin receptor ( kuyama et al . , 2010 ) . in any case , we need information of a marker specific to gabaergic projection neurons . in the following , we discuss why nnos may be a useful marker for gabaergic projection neurons in the neocortex . it is widely accepted that during the development of the mammalian neocortex , subplate neurons ( shatz et al . , 1988 ; finney et al . , 1998 ) and marginal zone neurons ( meyer et al . , 1998 ) extend cortico - fugal pioneering axons . it is also accepted that the subplate neurons are transient targets of thalamocortical axons , and although some then undergo apoptosis , some remain in the mature cortex as white - matter neurons ( chun and shatz , 1989a ; kanold and luhmann , 2010 ) . one of the groups is cajal  retzius cells in the marginal zone , which are gaba - negative and originate in the pallial edge ( takiguchi - hayashi et al . , 2004 ; another group is gaba - negative preplate neurons which originate in the ventricular zone of the neocortex . a recent study clarified that the cortico - fugal pioneer fibers originate from the gaba - negative preplate neurons , which are segregated during development into marginal zone neurons and subplate neurons ( espinosa et al . , 2009 ) . a third group is composed of gabaergic neurons originating in the ganglionic eminence migrating tangentially to the neocortex ( anderson et al . , 1997 ; tamamaki et al . early generated gabaergic neurons are distributed broadly in the marginal zone , subplate , and the intermediate zone ( iz ) . in cat neocortex , the subplate gabaergic neurons are often positive for somatostatin , neuropeptide y , and nnos ( chun et al . , 1987 ; chun and shatz , 1989b ) . those in the rat subplate are also positive for nnos ( clancy et al . , 2001 ) . although bayer and altman ( 1991 ) regard the rat layer vii ( vib ) as the subplate and ignore the gabaergic neurons distributed sparsely in the white matter , gabaergic neurons in the rat white matter accumulate in the medial edge of the white matter ( around the cingulate bundle ) ( figure 4 ) . a number of white - matter neurons in the cingulate have axons projecting to the spinal cord ( figure 4 and tamamaki et al . since a part of the white - matter neurons are maintained even after birth and may originate axon branches to innervate several neocortical regions , they can be identified as gabaergic projection neurons in the neocortex . in a similar way , the nnos - positive gabaergic neurons in the marginal zone may be identified as gabaergic projection neurons in layer ii . gabaergic subplate neurons in the intermediate zone ( iz ) also extend their axon cortico - fugally . ( a ) postnatal day 0 rat neocortex . top and second arrows indicate the width of subplate according to bayer and altman ( 1991 ) . second and third arrows indicate the width of white matter which contains many gabaergic neurons . double - arrows indicate the area where we found retrogradely labeled neurons after lipophilic red fluorescent dye , dii injection into the median plane of the spinal cord . ( b ) dii injection into the spinal cord ( c1c3 ) at p0 labeled pyramidal neurons in layer v and subplate neurons in the iz . axons of pyramidal neurons followed those of gabaergic subplate neurons rather than those of gaba - negative subplate neurons ( layer vii ) . ( c ) biotin - dextan - amine ( bda ) injection into the primordium of pons at e18 rat labeled subplate neurons in the iz at p0 . cp , cortical plate ; iz , intermediate zone ; s , subplate containing excitatory subplate neurons . arrow heads in the insets in ( b , c ) indicate the injection site of dii and bda . calibration bars in ( a ) , and in the insets in ( b , c ) are 1  mm . calibration bar in ( b ) is 100 m , that in ( c ) is 50 m . based on the information shown above , we hope to label and manipulate ( including delete ) the gabaergic projection neurons in vivo by an application of genetic tools driven by nnos promoters . however , since in mice all excitatory neurons in the neocortex also express nnos transiently by 2 weeks postnatal days , the process of experimentation will be complex . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
features and functions of long - range gabaergic projection neurons in the developing cerebral cortex have been reported previously , although until now their significance in the adult cerebral cortex has remained uncertain . 
 the septo - hippocampal circuit is one exception  in this system , long - range mature gabaergic projection neurons have been well analyzed and their contribution to the generation of theta - oscillatory behavior in the hippocampus has been documented . to have a clue to the function of the gabaergic projection neurons in the neocortex , we view how the long - range gabaergic projections are integrated in the cortico - cortical , cortico - fugal , and afferent projections in the cerebral cortex . 
 then , we consider the possibility that the gabaergic projection neurons are involved in the generation , modification , and/or synchronization of oscillations in mature neocortical neuron activity . 
 when markers that identify the gabaergic projection neurons are examined in anatomical and developmental studies , it is clear that neuronal no synthetase ( nnos)-immunoreactivity can readily identify gabaergic projection neurons . 
 gabaergic projection neurons account for 0.5% of the neocortical gabaergic neurons . to elucidate the role of the gabaergic projection neurons in the neocortex 
 , it will be necessary to clarify the network constructed by nnos - positive gabaergic projection neurons and their postsynaptic targets . 
 thus , our long - range goals will be to label and manipulate ( including deleting ) the gabaergic projection neurons using genetic tools driven by a nnos promoter . 
 we recognize that this may be a complex endeavor , as most excitatory neurons in the murine neocortex express nnos transiently . 
 nevertheless , additional studies characterizing long - range gabaergic projection neurons will have great value to the overall understanding of mature cortical function .