The germline chimera of GAD67-Cre mice were crossed with C57BL/6 mice to generate the GAD67-Cre mice line. the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP)-labeled GABAergic neurons in BIX 02189 GAD67-Cre knock-in/GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral BIX 02189 cortex and are in a position to be involved in information processing. Keywords:GABA, cerebral cortex, nicotinamide adenine dinucleotide phosphate diaphorase, nitric oxide synthase == Introduction == GABAergic neurons regulate information processing and are involved in oscillations in the cerebral cortex (Freund and Buzski,1996; Cardin et al.,2009; Sohal et al.,2009). Generally they were thought to be interneurons and act locally. Recently, however, a body of evidence has indicated that GABAergic neurons in the neocortex also project over longer distances. According to our previous report, long-range GABAergic projections originated in layers II, VI and the underlying white matter in mouse neocortex (Tomioka et al.,2005). Several reports have also described the existence of long-range GABAergic projections in the rat’s neocortex (Matsubara and Boyd,1992; McDonald and Burkhalter,1993). Those projections were not limited to the rodent brain but seemed to exist in feline (Higo et al.,2007) and in primate brain (Barone and Kennedy,2000; Tomioka and Rockland,2007). GABAergic neurons with axons projecting in the ipsilateral hemisphere seem to have similar chemical features and often contain somatostatin (SS)-immunoreactivity (IR) (91%), neuropeptide Y (NPY)-IR (82%), and neuronal nitric oxide synthase (nNOS)-IR (71%) (Tomioka et al.,2005; Higo et al.,2007). Considering these observations, the fact that most nNOS-positive neurons SHH are a subpopulation of SS- and NPY-IR neurons, and nNOS-, NPY-, and SS-triple-positive cells are less than 0.5% of GABAergic neurons (Kubota et al.,1994; Gonchar and Burkhalter,1997), it was speculated that the nNOS-positive GABAergic projection neurons compose a very small subpopulation in the neocortical GABAergic neurons. In addition, co-localization of GABA synthesizing enzyme, glutamic acid decarboxylase at 67 K-dalton (GAD67)-IR and a retrograde tracer injected into contralateral hemisphere revealed that another subtype of GABAergic neurons may be involved in the contralateral projection (Gonchar et al.,1995; Kimura and Baughman,1997; Fabri and Manzoni,2004). Parvalbumin (PV)-IR GABAergic neurons in the medial septum have been shown to terminate preferentially on hippocampal GABAergic neurons, and act to regulate the activity of pyramidal neurons in the hippocampus (Freund and Antal,1988). SS-IR GABAergic neurons in the hippocampus project back to the medial septum. This circuit was interpreted as the mechanism to cause theta oscillation (Toth et al.,1997). GABAergic projection neurons in the neocortex may act similarly, by inhibiting local GABAergic interneurons and, in turn, allowing cortical principal cell firing. Although GABAergic projection neurons have been recognized as a subset of neocortical neurons, it is very hard to reveal the projection axons technically. Simple immunohistochemistry of markers like calcium binding proteins or short peptides did not reveal the full axon trajectory from the soma to the terminals. Although nNOS-IR also does not reveal projection axons, NADPH-d reaction reveals nNOS-IR BIX 02189 GABAergic neurons in Golgi-like images including their projection fibers (Yan et al.,1996; Barone and Kennedy,2000; Smiley et al.,2000; Higo et al.,2007). Thus we utilized the NADPH-d reaction to reveal the projection axons of nNOS-IR neurons in the neocortex. In addition, we introduce a new tool, GAD67-Cre knock-in mouse, in which DNA encoding Cre DNA-recombinase in P1 phage was targeted to the locus encoding GAD67. Since the vast majority of GABAergic neurons are expected to be labeled by GFP in the offsprings obtained by mating GAD67-Cre knock-in mouse and GFP Cre-reporter mouse, we expect that all the subtypes of GABAergic projection neurons will be revealed at a glance. == Materials and Methods == All procedures were carried out according to the guidelines for the care and use of animals approved by the Animal Care and Use Committee at Kumamoto University in accordance with the National Institutes of Health (NIH). == Production of GAD67-Cre knock-in mouse == The generation of GAD67-Cre knock-in mice will be described into detail elsewhere (Akashi et al. in preparation). To generate the GAD67-knock-in Cre mice, we designed a targeting vector in which Cre recombinase gene was inserted into immediately after the translational initiation site of theGAD67gene in frame. A knock-in vector pGAD67CreTV contained a 3 kb fragment at the 5 side, a Cre gene placed behind the GAD67 translational start, aPgk-neo-p(A) cassette flanked by two Flp recognition target (frt) site, a 7 kb fragment at the 3 side, and a MC1 promoter-driven diphtheria.