Subtypes of GABAergic Neurons Project Axons in the Neocortex

γ-aminobutyric acid (GABA)ergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS)-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in 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 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 cortex and are in a position to be involved in information processing.


INTRODUCTION
GABAergic neurons regulate information processing and are involved in oscillations in the cerebral cortex (Freund and Buzsáki, 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 longrange 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 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 microtome into 50 µm thick coronal sections, and every sixth section was collected and used for GFP-immunohistochemistry or NADPH-d reaction.

VISUALIZATION OF nNOS-POSITIVE NEURONS AND GFP-POSITIVE NEURONS
The procedure for NADPH-d staining was performed according to the method of Vincent and Kimura (1992). Briefl y, fl oating sections were incubated in a solution of 0.1 M phosphate buffer, 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 1.0 mg/ml β-NADPH at 37°C (pH 7.4) for 60 min. Sections were rinsed three times in 0.1 M phosphate buffer, 5 min per rinse, and mounted on gelatinized slides. Sections were then counterstained with 0.3% Neutral red.
Brain sections of GAD67-Cre knock-in mouse/GFP Cre-reporter mouse were incubated with a primary antibody against GFP, and the immunoreactive site was visualized by DAB reaction as reported previously (Tamamaki et al., 2000). This procedure allowed us to carry out the morphometry of GFP-positive structures in blightfi eld microscopy.

COUNTING NADPH-d POSITIVE FIBERS IN THE WHITE MATTER
To estimate the number of NADPH-d positive fi bers in the white matter, we set 200-µm-site where labeled fi bers could be traced as long as possible in the white matter. Then we counted labeled fi bers in more than fi ve windows. Since thickness of the sections was 50 µm, we could estimate the number of labeled fi bers contained in 1 × 10 4 µm 2 of the cut end of the white matter.

NADPH-d POSITIVE CELLULAR STRUCTURES IN THE CAT NEOCORTEX
To reveal axons of nNOS-positive GABAergic neurons, we employed NADPH-d histochemistry in cat cerebral cortex. NADPH-d staining reveals large non-pyramidal neurons (type I) and small nonpyramidal neurons (type II) with dark-blue precipitate in Golgi-like images (Higo et al., 2007) (Figure 1A). nNOS-IR is co-localized with the precipitate in type I cell but not in the type II cells. These two types of NADPH-d-positive cells have been reported in other experimental species (Monkey: Yan and Garey, 1997;Smiley et al., 2000;Rat: Valtschanoff et al., 1993;Kubota et al., 1994) and also in human brain (Judas et al., 1999).
Somata of type I cells are found in the deep layers and white matter of the neocortex (Figures 1A,B). The number of type I cells is highest at the gray matter and white matter boundary. According to the depth in the white matter, the number of type I cells is reduced, and these are almost completely absent near the lateral ventricle ( Figure 1B). On the other hand, somata of type II cells are found in the neocortical layers II-VI (Higo et al., 2007). NADPH-d positive cells are also found in the basal ganglia. Since the internal capsule is almost free of NADPH-d positive fi bers, the NADPH-d positive fi bers and their terminals in the cerebral cortex are assumed to belong to the type I and II NADPH-d positive cells in the cerebral cortex.
NADPH-d positive fi bers branch and ramify in the gray matter of the neocortex ( Figure 1A). On the other hand, NADPH-d positive thick fi bers did not seldom branch in the white matter ( Figure 1C). In parallel with the number of type I cells in the white

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 the GAD67 gene 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, a Pgk-neo-p(A) cassette fl anked by two Flp recognition target (frt) site, a 7 kb fragment at the 3′ side, and a MC1 promoter-driven diphtheria toxin gene.
Culture of embryonic stem (ES) cells and generation of chimeric mice were performed as described previously (Kitayama et al., 2001). Briefl y, linearized pGAD67CreTV was introduced into C57BL/6 mouse ES cells and then, G418-resistant clones were picked up. Homologous recombined ES clone was identifi ed by Southern blotting. To produce germline chimera, the selected ES cells were microinjected into eight cell-stage embryos of CD-1 mouse strain. The germline chimera of GAD67-Cre mice were crossed with C57BL/6 mice to generate the GAD67-Cre mice line. Because the knock-out of both GAD67 alleles is lethal at birth (Asada et al., 1997), mice heterozygous for the altered GAD67 allele were used for all the observations in this study. Genotypes were identifi ed by Southern blot hybridization or PCR. Tail genomic DNA was digested with Spe-I or Afl -II and hybridized with 5′ probe or 3′ probe, respectively. PCR was performed with specifi c three primers. The sequence of each primer and the approximate length of the amplifi ed DNA fragments are described as follows: Gad1 Cre , g67-2 (5′-TTCCGGAGGTACCACACCTT-3′), g67-5 (5′-TAAGTCGACGCTAGCGAGCGCCTCCCCA-3′), and CreR1 (5′-TTGCCCCTGTTTCACTATCC-3′); wild type, 1.8 kbp; mutant: 1.4 kbp. The GAD67-Cre knock-in mice were mated with a GFP Cre-reporter mouse (Tanahira et al., 2009). Cre-positive GABAergic neurons are revealed by GFP-expression in the offsprings.

ANIMALS AND RECOVERY OF BRAINS
In this experiment, three adult cats (2.5-to 4-kg body weight) and fi ve GAD67-Cre knock-in/GFP Cre-reporter mice were used. Cats were anesthetized with ketamine (40 mg/kg, i.m.) and Nembutal (50 mg/kg, i.p.). The animals were perfused with phosphate-buffered saline at pH 7.4 (PBS), followed by fi xative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) through the left ventricle. Brains were removed, blocked, saturated with a cold solution of 30% phosphate-buffered sucrose overnight, cut serially on a freezing microtome into 50-µm thick coronal sections, and every fi fteenth section was collected and used for NADPH-d reaction.
Mice were anesthetized with Nembutal (50 mg/kg i.p.), and perfused with PBS and the same fi xative used for cats. Brains were removed, saturated with a cold solution of 30% phosphate-buffered sucrose overnight, and cut serially on a freezing matter, the number of NADPH-d positive thick axons also changes: they are highest at the boundary between the gray matter and the white matter and reduced in the deeper white matter. Moreover, large NADPH-d-positive cells originate NADPH-d positive thick axon in the white matter ( Figure 1D). The labeled thick axons occur in fascicles and are extended with following the trajectory of other non-labeled axons at every depth of the white matter. We could not fi nd the thick labeled axons giving rise large branches or turning to ascend to the gray matter. We found fragmented thick axons, many in the fi mbria (8.8 ± 2.1/10 4 µm 2 ; n = 5) (Figure 1E), a small number in the anterior commissure (1.2 ± 1.2 µm 2 ; n = 5), and only one in fi ve windows in the corpus callosum (0.2 ± 0.4 µm 2 ; n = 5) (see Materials and Methods).

GABAergic PROJECTION AXONS IN THE MOUSE WHITE MATTER
Three brains were obtained from the mice at 10-weeks old and sectioned into coronal sections at 50-µm thickness. The GFP-positive cells in the mouse brain were distributed similarly to the GFPpositive cells in the GAD67-GFP knock-in mouse (Tamamaki et al., 2003). However, GFP-positive cells in the GAD67-Cre/GFP-reporter mouse are signifi cantly less in number than that in GAD67-GFP mouse. GFP-IR is similar in every GFP-positive cells because the expression of GFP is driven by chick actin (CA)-promoter in all the GFP-positive cells.
Axon fi bers with GFP-IR fi ll the neocortical grey matter, although individual fi bers are hard to resolve. GFP-positive axon fi bers also occur in the white matter. They can be traced in a few serial sections, but not suffi ciently far to determine whether the axon fi bers belong to GABAergic projection neurons or not. Therefore, we directed our attention to the fi mbria, the anterior commissure, and the corpus callosum. We found a large number of labeled axon segments in the fi mbria (Figure 2A). The density of the labeled axons in the fi mbria is much higher than that of the NADPH-d positive fi bers in the cat fi mbria (Figure 1D). A small number of labeled axons traveled in the anterior commissure (Figure 2B), and a few sparsely in the corpus callosum.
The NADPH-d reaction in mouse brain sections does not reveal nNOS-positive neurons in Golgi-like fashion. Although NADPH-d reactions revealed somata and dendrites of type I-like non-pyramidal neurons in mouse neocortex, NADPH-d positive axons appear as dotted-lines and it was diffi cult to trace their trajectories. The number of labeled axon fragments is countable only in the fi mbria. We found similar number of fragmented NADPH-d-labeled axons in the mouse fi mbria (3.7 ± 1.5/10 4 µm 2 ; n = 6), (Figure 2C) to the case in the cat fi mbria.

DISCUSSION
The present study confi rmed the presence of a subpopulation of GABAergic neurons with long projection axons in the cat and mouse neocortex. Moreover, we added the evidence that the GABAergic neurons with long projection axons include subpopulations larger than the nNOS-positive GABAergic neurons, especially in the archicortex.
NADPH-d-positive somata in the white matter originate thick NADPH-d-positive fi bers in the white matter of the cat neocortex, which emanate with other non-labeled arcuate fi bers, without originating any thick branches, and without bending toward the gray matter of the neocortex. These features may indicate that the total length of NADPH-d-positive fi bers in the white matter is long enough to be called as projection fi bers, while we regarded GABAergic neuron extending an axon longer than 1.5 mm from the soma as the GABAergic projection neuron in the mouse (Tomioka et al., 2005). The NADPH-d-positive fi bers in the grey matter of the neocortex may include fi bers originating from both type I and type II cells.
Formerly, we produced GAD67-GFP knock-in mouse in order to observe GAD67-positive cells and axon fi bers traveling in the central nervous system (Tamamaki et al., 2003). The GFP-positive axon fi bers were clearly revealed by GFP-fl uorescence and could be traced long distances in the brain stem. However, projection axons belonging to the GABAergic neurons in the neocortex seemed to be thin and diffi cult to identify in the GAD67-GFP mouse. In addition, GAD67-promoter activity seemed to vary in each GABAergic neuron subtype, and the intensity of GFP-fl uorescence differed from cell to cell (Tamamaki et al., 2003). With the approach reported here, however, Cre DNA-recombinase deletes fl oxed stop in the GFP Cre-reporter construct, and GFP was expressed depending on the CA-promoter in the construct (Tanahira et al., 2009). Since Creexpression levels depend on the GAD67-promoter, some subtypes of GABAergic neurons with weak GAD67-promoter activity may fail to delete fl oxed stop and may not be revealed by GFP-expression. As the result, we may have underestimated the GABAergic neurons projecting contra-laterally via the corpus callosum (Gonchar et al., 1995;Kimura and Baughman, 1997;Fabri and Manzoni, 2004). We feel necessity of introducing additional techniques to estimate the number of GABAergic neurons projecting contra-laterally.
NADPH-d reaction labeled axon fi bers of nNOS-positive GABAergic neurons in cats and mice, while GFP-immunohistochemistry in GAD67-Cre/GFP Cre-reporter mouse labeled both projection fi bers originating in nNOS-positive GABAergic neurons and those in the other subtypes of GABAergic neurons. However, the distribution pattern of the labeled axon fragments was similar in both preparations. NADPH-d reactive axons and GFP-IR axons were found to be very sparse in the corpus callosum, sparsely in the anterior commissure, and many in the fi mbria. Labeled fi bers in the fi mbria will include afferent-and efferent-fi bers from and to the subcortical nuclei, in addition to the commissural fi bers through the ventral hippocampal commissure. Excluding subcortical afferent-and efferent-fi bers, we speculate that the GABAergic commissural fi bers reciprocally interconnect archi-and paleo-cortex.
When GABAergic projection neurons terminate on GABAergic interneurons, they will inhibit the interneurons and thus disinhibit excitatory principle neurons in the hippocampus (Freund and Antal, 1988;Toth et al., 1997). Although NADPH-d positive fi bers in cat neocortex ramifi ed and seemed to terminate on many types of neurons (i.e. both principle neurons and inhibitory neurons), NADPH-d-positive fi bers originating in the type I cells may innervate GABAergic neurons preferentially. To address this possibility, we plan to investigate the terminal of labeled nNOS-positive axon fi bers in future. One of the important results elucidated in this study is that a large number of GABAergic neurons are also involved in the cortico-fugal, cortico-cortical, and callosal projections. Each subtype of GABAergic projection neuron may contribute to information processing in the neocortex in different ways.