Two strains of GAD67-GFP knock-in mice
To evaluate the expression of GFP in GAD67-GFP mice and GAD67-GFP (Δneo) mice (Fig. 1A), Western blot analysis was performed using an anti-GFP antibody (Fig. 1B). The antibody detected a band of GFP of the expected size of 28 kDa in the lysates from both a GAD67-GFP mouse and a GAD67-GFP (Δneo) mouse. The band representing GFP in the lysate from the GAD67-GFP (Δneo) mouse was much denser than that from the GAD67-GFP mouse, which indicates that the cerebral cortex of the GAD67-GFP (Δneo) mouse contained more GFP than the cerebral cortex of the GAD67-GFP mouse. We also compared the GFP contents in the brains of the two strains of knock-in mouse directly by fluorescent microscopy and examined the correlation of the GFP contents with GAD67 immunoreactivities and GABA contents. Figure 1C,D shows green fluorescence observed in the neocortex of a GAD67-GFP mouse and that in a GAD67-GFP (Δneo) mouse, respectively. The sections were obtained by the same fixation procedure. Their digital images were printed without any change in either the brightness or the contrast. Comparison of these images revealed that the section obtained from the GAD67-GFP (Δneo) mouse emitted more intense fluorescence than the section obtained from the GAD67-GFP mouse did, whereas the green fluorescence emitted from the sections of wild-type mouse brain was at a background level. The same sections obtained from the two strains of GAD67-GFP mice and wild-type mice were processed for GAD67 or GABA immunohistochemistry in the same bottle. The GAD67 immunoreactivity of the section obtained from the GAD67-GFP (Δneo) mouse brains and the GAD67-GFP mouse brain seemed to be lower than that from the wild-type mouse (data not shown). Thus, we speculated that the deterioration of the GAD67 immunoreactivity correlated with the increase of GFP expression after deletion of neo cassette. To prove our speculation, we investigated the GABA contents in the forebrain of these mice with a high performance liquid chromatography (HPLC). The results of GABA contents measured by HPLC were shown in Table 1 and in Figure 1E by their ratios or the concentrations of GABA. The forebrain of newborn GAD67-GFP (Δneo) mouse and that of newborn GAD67-GFP mouse (including the cerebral cortex and the basal ganglia) contained GABA at the concentration of 21.7 ± 1.8 nmol/mg (n = 6) and 24.7 ± 2.1 nmol/mg (n = 7), respectively. The GABA contents at postnatal day (P) 0 in the two strains of knock-in mice were significantly lower than that in wild-type mice (t test, P < 0.001). Six weeks later, the GABA contents in the forebrain of GAD67-GFP (Δneo) mouse (19.9 ± 2.0 nmol/mg; n = 6) was still significantly lower than that of wild-type mouse (23.7 ± 2.1 nmol/mg; n = 5; t test, P < 0.05). However, the difference between the GABA content in the forebrains of GAD67-GFP (Δneo) mouse and that of wild-type mouse at 6 weeks old was small. Moreover, the difference between that of GAD67-GFP mouse (26.9 ± 2.1 nmol/mg; n = 6) and that of wild-type mouse (29.0 ± 1.7 nmol/mg; n = 6) at 7 weeks old was no longer significant (t test, P = 0.09). The difference between the GABA contents of the newborn GAD67-GFP (Δneo) mouse and that of the newborn GAD67-GFP mouse was also significant (t test, P < 0.05), but it became barely significant (t test, P = 0.05) 6–7 weeks later. The Western blotting study and the fluorescent microscopic observation revealed that removal of the PGK-neo cassette enhanced GFP expression in the GAD67-GFP (Δneo) mice. In return, as the GFP expression increased, GAD67 expression seemed to be reduced and the difference between the GABA contents in the forebrains of the two strains was significant at birth. However, the GABA contents in the brains of the two strains were increased as they grew. GAD67-GFP (Δneo) and GAD67-GFP mice were seizure-free. They exhibited normal growth, normal reproductive behavior, and no abnormality in the brains at macroscopic level.
Colocalization of GFP with GAD67 and GABA
We investigate the colocalization of GFP with GAD67 and GABA in adult male GAD-GFP mice. Although the GAD67-GFP mouse brains contained less GFP than GAD67-GFP (Δneo) mouse brains did, we used GAD67-GFP mouse brains for the GAD67 and GABA immunohistochemistry, because the GABA content in GAD67-GFP mouse brains was not significantly lower than that in wild-type mice. Thus, the GFP-positive cellular structures appeared in green by GFP fluorescence or by Alexa 488 fluorescence and GAD67- and GABA-immunoreactive structures in red by TSA cyanine 3. The level of green fluorescence depended on the amount of GFP accumulated in the somata, dendrites, axons, and axon terminals. Although both GFP and GAD67 are soluble proteins, their distribution in subcellular structures were different. We examined the difference in the GAD67- and GFP-distributions by confocal microscopic observation in the neocortex (Fig. 2A–F). GFP was present throughout the GFP-positive somata, including in their nuclei, whereas GAD67 was present preferentially in the perikarya of the GAD67-positive somata. The vast majority of the GFP-positive cells had GAD67 immunoreactivity in their perikarya (156 of 196). However, all the GFP-positive cells were not concluded as GAD67-positive. Some of them with GAD67 immunoreactivity close to that in the neuropil (arrows in Fig. 2A,C) were regarded as negative for GAD67. If we included them as GAD67-positive cells, almost all of the GFP-positive cells would be positive for GAD67. Most GAD67-positive cells were identified by GFP fluorescence higher than that in the neuropil. However, some of the GAD67-positive cells were counted as GFP-negative, because they had GFP immunoreactivity close to that in the neuropil. If we included them as GFP-positive cells, basically all the GAD67-positive cells were positive for GFP. Overall, we did not encounter any ectopic GFP expression in apparently GAD67-negative cells, whose red-fluorescence level was lower than that of the neuropil, or any lack of GFP-expression in apparently GAD67-positive neurons in the knock-in mouse. In a double-labeling study of GFP and GABA immunohistochemistry (Fig. 2G–I), most GFP-positive cells were regarded as positive for GABA (141 of 159), but some of them had GABA immunoreactivity similar to that of neuropil and regarded as GABA-negative (arrows in Fig. 2G,I). If we included them as GABA-positive cells, basically all the GFP-positive cells were positive for GABA. All the investigated GABA-positive cells seemed to have a certain amount of GFP immunoreactivity at least similar or higher than that of the neuropil.
Figure 2. A–C: Double labeling of green fluorescence protein (GFP) immunoreactivity (green) and glutamic acid decarboxylase (GAD)67 immunoreactivity (red) in the neocortex. Arrowheads in A and C indicate the cells regarded as positive for GFP but negative for GAD67. D–F: Double labeling of GFP fluorescence and GAD67 immunofluorescence in the ventrolateral thalamic nucleus. GAD67 was distributed preferentially in the axon terminals, whereas GFP was distributed ubiquitously within axons. GFP and GAD67 colocalized in the axon terminals. G–I: Double labeling of GFP immunoreactivity (green) and GABA immunoreactivity (red) in the neocortex. Arrowheads in A and C indicate the cells regarded as positive for GFP but negative for GABA. Scale bars = 10 μm in A (applies to A–C,G–I); 10 μm in D (applies to D–F).
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Distribution of GFP and GAD67 in the axons was investigated in the thalamus where we found many GFP-positive afferent fibers but fewer GAD67-positive cells (see below). GFP was distributed in the axons and axon terminal-like puncta (Fig. 2D,E) as well as in dendrites (Fig. 2A), whereas the GAD67 immunoreactivity was preferentially found in the axon terminal-like puncta (Fig. 2E,F). The double-labeling study of GFP and GAD67 revealed that GFP and GAD67 were preferentially colocalized in the perikarya and in the axon terminal-like puncta (Fig. 2A–F). In the rest of the subcellular structures, GFP and GAD67 would be present in different amounts, depending on the thickness of dendrites and that of axon fibers. GFP expressed under the control of the GAD67 promoter would be a good tracer for detecting somata, dendrites, and axon fibers of GAD67-positive GABAergic neurons but not a good tracer for detecting the intracellular distribution of GAD67 in the knock-in mouse brain.
Temporal regulation of GFP expression
GFP expression in the GAD67-GFP knock-in mouse seemed to be regulated accurately not only spatially but also temporally. Some panels in Figure 4 show negatives of the original fluorescent photomicrographs taken of sections of GAD67-GFP mice. In the E15 embryo head, GFP fluorescence was found not only in the brain but also in the ectodermal placodes of developing whiskers (Fig. 3). In frontal sections through the tooth buds, it was apparent that odontoblasts emitted GFP fluorescence (Fig. 4A–C). All these tissues have been reported to be positive for GABA and GAD67 during embryonic stages (Asada et al., 1997; Maddox and Condie, 2001). In sections through the ganglionic eminence, intense GFP fluorescence was seen to fill the area (Fig. 4D). An in situ hybridization study at E15 revealed a high level of GAD67 mRNA in the ganglionic eminence, especially in the subventricular zone (Fig. 4E). GFP-positive cells were also distributed in the neocortex (Fig. 4D,F) and the olfactory bulb (Fig. 4G). Each GFP-positive cell in the neocortex had a long, thick process and a short tail, which corresponded to the leading and trailing processes (Rakic, 1990) of tangentially migrating GABAergic neurons (Anderson et al., 1997; Tamamaki et al., 1997). The ventricular zone of the neocortex contained a small number of GFP-positive cells (Fig. 4F), which had an appearance typical of migrating cells and were regarded to be GABAergic neurons migrating into the ventricular zone from the intermediate zone (IZ) (Nadarajah et al., 2002). The other ventricular zone cells were negative for GFP and for GAD67 mRNA.
Figure 4. Green fluorescence protein (GFP) fluorescence observed in serial frontal sections obtained from a glutamic acid decarboxylase (GAD)67-GFP knock-in mouse embryo at embryonic day (E) 15, and GAD67 in situ hybridization signal in a frontal section obtained from a wild-type mouse embryo brain at E15. A,B,D,F,G show negatives of original fluorescent light photomicrographs. A: GFP fluorescence in a frontal section through the tooth bud. B: GFP fluorescence in the odontoblast layer (Ob) at higher magnification. C: Counterstained odontoblast layer and enameloblast layer. The section shown in A and B was stained with cresyl violet. D: GFP fluorescence in a frontal section through the lateral ganglionic eminence (LGE). Arrows in D,E indicate the GFP-positive cells in the intermediate zone and marginal zone. The double arrow in D,E indicates the ventricular edge of the neocortex (Cx). E: In situ hybridization signal for GAD67 antisense probe in a frontal section through the lateral ganglionic eminence. F: GFP-positive cells in the neocortex. G: GFP-positive cells in the olfactory bulb. MZ, marginal zone; IZ, intermediate zone; V, ventricle. For other abbreviations , see list. Scale bars = 1 mm in A (applies to A,D,E); 100 μm in B (applies to B,C); 100 μm in G (applies to G,F).
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Figure 3. An embryonic day 15 embryo head. Arrows indicate green fluorescence protein–positive hair roots. Scale bar = 1 mm.
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GFP distribution in the knock-in mice
The double-labeling study and the examination of GFP expression at an embryonic stage indicated that the GFP expression is regulated by GAD67 promoter properly in the knock-in mice and convinced us of the usefulness of the knock-in mice for the studies of GABAergic neurons in various brain regions. Thus, we describe the GFP distribution in the knock-in mouse brain for future use by other researchers.
Although brain sections obtained from GAD67-GFP (Δneo) mice emitted more intense fluorescence than those obtained from GAD67-GFP mice, the distribution pattern of GFP fluorescence in the GAD67-GFP (Δneo) mice was indistinguishable from that in GAD67-GFP mice in all parts of the brain. Therefore, we used GAD67-GFP mice to make a map of GFP distribution in the knock-in mouse brain. Figures 5 and 6 show negatives of the original fluorescent photomicrographs taken of sections of the brains of GAD67-GFP mice. Thus, areas with intense GFP fluorescence appear as dark areas in the figures.
Figure 5. Green fluorescence protein (GFP) fluorescence in a parasagittal section (A) and frontal sections (B–L) obtained from glutamic acid decarboxylase (GAD)67-GFP knock-in mouse brains. All the figures are negatives of the original fluorescent light photomicrographs. For abbreviations , see list. Scale bars = 1 mm in A, C (applies to B–F), G (applies to G–J), K, L.
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Figure 6. Green fluorescence protein (GFP) fluorescence observed in various brain regions of the glutamic acid decarboxylase (GAD)67-GFP knock-in mouse. All the figures are negatives of the original fluorescent light photomicrographs. A: Cortex of the olfactory bulb. B: Neocortex. Arrowheads indicate the GFP-positive somata. The double arrowhead indicates a large GFP-negative hole, and the arrow indicates a small GFP-negative hole. C: Neocortex, white matter, and caudate putamen. D: Hippocampus. E: Caudate putamen. F: Globus pallidus. G: Reticular thalamic nucleus. H: Ventrolateral thalamic nucleus. I: Dorsal lateral geniculate nucleus. J: Superior colliculus. K: Lateral lemniscus. L: Cerebellar cortex. M: Substantia nigra pars reticulata. N: Ventral tegmental nucleus. O: Inferior olive. P: Outer edge of the inner granular layer of the retina. An arrow indicates the area out of focus. Q: Center of the inner granular layer of the retina. R: Inner edge of the inner granular layer of the retina. For other abbreviations , see list. Scale bars = 100 μm in A (applies to A,C,D), B (applies to B,E,F), G (applies to G,H,L), I (applies to I,J), K, M (applies to M–O), P (applies to P–R).
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GFP fluorescence was found throughout the adult GAD67-GFP-mouse brain, from the olfactory bulb to the spinal cord, as shown in the serial frontal sections made from a mouse brain (Fig. 5B–L). The intensity of GFP fluorescence in the knock-in mouse brains varied from site to site. The strongest intensity was seen in the olfactory bulb. When the whole brain was observed under a fluorescence microscope, the fluorescence emitted from the olfactory bulb was most obvious, and that from the cerebellum and cerebral cortex followed. Thus, GFP fluorescence from the olfactory bulb was a good marker to identify knock-in mice. At birth, we could identify such mice by the GFP fluorescence emitted from the olfactory bulb through the meninges, the neurocranium, and the skin. Therefore, phenotyping of the knock-in mouse could be accomplished with a glance through a fluorescence microscope, and there was no need for genotyping of the knock-in DNA sequence. The GFP fluorescence was emitted from the granule cell layer and molecular layer of the olfactory bulb (Fig. 5A,B) (Shipley et al., 1995; Shepherd and Greer, 1998). The distribution was even clearer at higher magnification (Fig. 6A). It is well known that the olfactory glomeruli are the structures with GABAergic neuron processes (Kosaka et al., 1997); the relative intensity of GFP fluorescence was weaker here than in the periglomerular region and, furthermore, weaker than the granule cell layer. Thus, the glomerular layer appeared weak in GFP fluorescence.
In the frontal sections obtained from the caudal level to the olfactory bulb, the anterior part of the caudate putamen and olfactory tubercle emitted GFP fluorescence at moderate intensity. In the olfactory tubercle, the fluorescence of the islands of Calleja was clearly higher than the surrounding structures (Fig. 5C). The GFP fluorescence seen in the olfactory tubercle was continuous with that in the medial septum and that in the diagonal band. The caudate putamen contained medium-sized GFP-positive cells and had higher background fluorescence than the neocortex. The high background of this region was due to small GFP-positive cells and neuropil packed among the fascicles of the internal capsule (Fig. 6E). The globus pallidus emitted more GFP fluorescence among the fascicles of the internal capsule (Fig. 5D). The strong intensity of GFP fluorescence was due to the highly packed GFP-positive cells and GFP-positive neuropil (Fig. 6F).
The neocortex showed less GFP fluorescence than the area described above (Fig. 5C,D). At higher magnification, GFP-positive cell somata, dendrites, and axon terminal-like boutons could be identified in a thin confocal plane (Fig. 6B). The low fluorescence in the matrix suggests that non-GABAergic cells predominate in the neocortex, whereas GABAergic cells predominate in the caudate putamen and globus pallidus. At higher magnification, the non-GABAergic neurons appeared as empty holes, which were often surrounded by GFP-positive puncta. By counting GFP-positive somata (arrowheads in Fig. 6B) and large GFP-negative somata (double arrowhead in Fig. 6B) in areas of the same size as that shown in the Figure 6B, we tried to estimate the proportion of GFP-positive neurons among the neocortical neurons. However, we might have underestimated the number of GFP-positive neurons by regarding the somata with weaker GFP-fluorescence than the fluorescence of the neuropil as GFP-negative neurons. In addition, we might have overestimated or underestimated the GFP-negative neurons (the arrow in Fig. 6B) considered as glial cells. We estimated that the proportion of GFP-positive neurons among the neocortical neurons was 12% (12 ± 1.8%, n = 5), a far lower proportion than that estimated by another method (see below). The white matter contained GFP-positive cells (Fig. 6C), which correspond to the white matter neurons (Clancy et al., 2001), the remnant of the GABAergic neurons found in the IZ at the embryonic stage (Tamamaki et al., 1997, 2002). The hippocampus had weak GFP fluorescence, comparable to that in the neocortex. However, because of the stratified structures, GFP-positive cells in the pyramidal cell layer and the granule cell layer were obvious, especially in the hilus, even at low magnification (Figs. 5E,F, 6D).
The GFP fluorescence in the areas described above was distributed in the somata and in the neuropil (dendrites and axon branches). However, GFP fluorescence in the reticular thalamic nucleus was found predominantly in the large somata (Fig. 6G), whereas GFP fluorescence in the thalamic relay nuclei was found predominantly in the axons and terminal branches with boutons (Fig. 6H). Even if the GFP fluorescence was of similar intensity, the structures emitting the fluorescence might have been different. The rostral part of the thalamus showed fluorescence less than that of the caudate putamen (Fig. 5A,E) and did not show any GAD67 mRNA signal (Fig. 7C). The lack of GAD67-positive GABAergic interneurons in the thalamic relay nuclei is characteristic to rodent thalamus (Ottersen and Storm-Mathisen, 1984). The caudal part of the thalamic relay nuclei emitted a comparable level of GFP fluorescence to the caudate putamen (Fig. 5A,F), and GAD67 mRNA signal was increased in the pretectal area and the lateral geniculate nucleus (Fig. 7C). Figure 7 shows serial sections processed for GAD67 in situ hybridization. In this staining, only the GAD67 mRNA-positive somata appear as dark-colored dots. The distribution of GFP-positive somata coincided well with the distribution of the GAD67 in situ hybridization signal in all parts of the brain. The reticular thalamic nucleus was the area with the most conspicuous GFP-positive and GAD67 mRNA-positive somata in the thalamus. GFP-positive somata were also found in the ventral and dorsal lateral geniculate nuclei (Figs. 5F, 6I) and the pretectal areas (Fig. 5F). In the lateral geniculate nucleus, both the GFP in the somata and neuropil contributed equally to the GFP fluorescence, which was higher than that in other thalamic nuclei (Figs. 5F, 6I). The medial geniculate nucleus contained sparse GFP-positive somata.
Figure 7. In situ hybridization signal for glutamic acid decarboxylase (GAD)67 antisense probe in serial frontal sections obtained from a wild-type mouse brain. Scale bars = 1 mm in A (applies to A–G), H; 100 μm in I.
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In the midbrain, GFP fluorescence was conspicuous in three sites (Fig. 5G). The most conspicuous site was the substantia nigra pars reticulata, the second-most conspicuous site was the superior colliculus, followed by the oculomotor nucleus. GFP fluorescence in the substantia nigra pars reticulata was emitted from many weakly GFP-positive somata and the strongly GFP-positive neuropil (Fig. 6M). Because the intensities of the fluorescence from the somata and neuropil were similar, they were not distinguishable in Figure 6M. The superior colliculus emitted a high level of GFP fluorescence that was conspicuous even at low magnification (Fig. 5A,G,H). At higher magnification, GFP-positive somata were seen to be abundant in the superficial layers of the superior colliculus (Fig. 6J). GFP expression was also conspicuous in the oculomotor nucleus (Fig. 5G) and ventral tegmental nucleus (Fig. 5I). Nuclei related to the auditory system also showed GFP fluorescence at various levels. The medial geniculate nucleus showed GFP fluorescence at a low level, comparable to that of the neocortex (Fig. 5G). The inferior colliculus showed fluorescence at a moderate level (Fig. 5I). Somata displaying a GAD67 mRNA signal were also contained by each nucleus at a level corresponding to the level of fluorescence (Fig. 7F,G). The lateral lemniscus contained both large GFP-positive somata and GFP-positive thick projection axons (Fig. 6K). The cerebellar cortex also emitted GFP fluorescence, but the fluorescent intensity varied from lobule to lobule (Figs. 5A,J, 6L). Ventral lobules in the vermis emitted intense GFP fluorescence. In the confocal image (Fig. 6L), Purkinje cell somata, Golgi cell somata, basket cell somata, and stellate cell somata were seen to be fluorescent (Palay and Chan-Palay, 1974). The GFP fluorescence of the inferior olive was conspicuous because of its intensity and the shape of the nucleus (Fig. 5A,J). At higher magnification, it was obvious that the fluorescence was emitted from the fine axon terminals surrounding cells in the inferior olive (Fig. 6O).
GFP-positive cells were also found in the retina. Figure 6P–R shows confocal images taken by setting a focal plane parallel to the retina and by shifting the focal plane from the outer edge of the inner granular layer (Fig. 6P) to the center of the inner granular layer (Fig. 6Q), and then to inner edge of the inner granular layer (Fig. 6R). The GFP-positive cells at the outer edge of the inner granular layer, which correspond to GABAergic horizontal cells, were aligned in a plane and were difficult to focus on precisely (the area with a faint pattern in Fig. 6P). GFP-positive cells at the inner edge of the inner granular layer, which correspond to GABAergic amacrine cells, were distributed in a layer with a regular, uninterrupted geometric pattern. This pattern of GFP-positive cell arrangement mimicked the GABA-immunoreactive site reported previously in the same layer (Brandon et al., 1979; Famiglietti and Vaughn, 1981; Vaughn et al., 1981; Wu et al., 1981). The similarity between the distribution patterns of GFP-positive cells and those of GABAergic horizontal cells and amacrine cells suggests that GFP expression occurs consistently in the GABAergic neurons in the retina. The center of the inner granular layer emitted intense GFP fluorescence, which was probably due to the GFP contained in the axons and dendrites of horizontal cells and amacrine cells.
Colocalization of GFP with NeuN and neuropeptides
We used the GAD67-GFP (Δneo) mice to investigate the colocalization of GFP with NeuN and several neuropeptides that characterize GABAergic neuron subtypes in the frontal motor cortex. According to the data obtained by anti-NeuN immunohistochemistry, basically all the GFP-positive cells in the neocortex appeared positive for NeuN. The colocalization of GFP with NeuN reconfirmed that the GFP is a good marker of GABAergic neurons in the adult neocortex. We counted the GFP-, NeuN-, CR-, PV-, and SS-positive somata in slits (460 μm wide) set at the frontal motor cortex in 25-μm-thick frontal sections. The slits were divided into zones of layer I, layer II–III, layers V, and layer VI plus white matter, respectively. The motor cortex in mice basically lacks layer IV. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was 19% (291 of 1,496) in a slit (Fig. 8A–C).
Figure 8. Colocalization of green fluorescence protein (GFP) with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in glutamic acid decarboxylase (GAD)67-GFP (Δneo) mice. A–C: Colocalization of GFP with NeuN. All the GFP-positive cells were positive for NeuN and confirmed to be neurons. A–C show GFP fluorescence, the merged image, and NeuN-immunoreactive sites (red) in layers I–II of the frontal motor cortex, respectively. Bars in C indicate the pia mater and the boundary between layers I–II. D–F: Colocalization of GFP with CR. D–F show GFP fluorescence, the merged image, and CR-immunoreactive sites in layers II–III of the frontal motor cortex, respectively. Arrowheads in D, G, and J indicate double-labeled cells. G–I: Colocalization of GFP with PV. G–I show GFP fluorescence, the merged image, and PV-immunoreactive sites in layers II–III of the frontal motor cortex, respectively. J–L: Colocalization of GFP with SS. J–L show GFP fluorescence, the merged image, and SS-immunoreactive sites in layers V–VI of the frontal motor cortex, respectively. Scale bar = 100 μm in A (applies to A–L).
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The distribution and features of CR-positive, PV-positive, and SS-positive cells have been well described in many publications (DeFelipe, 1993; Kubota et al., 1994; Gonchar and Burkhalter, 1997). In brief, CR-positive cells were preferentially found in layers I–II and the deeper part of layer VI. The CR-positive cells had small somata. Other layers contained sparsely distributed CR-positive cells. PV-positive cells were distributed from layers II to VI but not in layer I. PV-positive cells had the largest somata among the GABAergic neuron subtypes. SS-positive cells were found in layers II–VI, but were more abundant in the deeper layers. They had medium-sized somata.
The double-labeling study with GFP revealed that basically almost all of the CR-positive cells were positive for GFP (122 of 131). The intensity of the GFP fluorescence emitted from their small somata varied from moderate to very low (Fig. 8D). The proportion of CR-positive cells among GFP-positive cells was 6.5 ± 4.0% (5 of 89) in layer I, 22.8 ± 1.0% (72 of 341) in layer II–III, 5.2 ± 2.3% (9 of 163) in layer V, and 12.5 ± 5.4% (45 of 358) in layer VI plus white matter. Overall, the proportion of CR-positive cells among GFP-positive cells was 13.9 ± 1.6% (131 of 951) in the neocortex. All the PV-positive cells were positive for GFP (397 of 397). The level of GFP fluorescence emitted from their large somata varied from high to moderate (Fig. 8G). The proportion of PV-positive cells among GFP-positive cells was 0% (0 of 65) in layer I, 44.3 ± 4.7% (198 of 443) in layer II–III, 44.5 ± 6.4% (63 of 146) in layer V, and 41.8 ± 11.3% (136 of 331) in layer VI plus white matter. Overall, the proportion of PV-positive cells among GFP-positive cells was 40.1 ± 7.5% (397 of 985) in the neocortex. Basically almost all of the SS-positive cells were also positive for GFP (187 of 198). The intensity of the GFP fluorescence emitted from their medium somata varied from high to very low (Fig. 8J). The proportion of SS-positive cells among GFP-positive cells was 0% (0 of 52) in layer I, 17.7 ± 3.5% (68 of 381) in layer II–III, 31.4 ± 4.7% (44 of 141) in layer V, and 31.6 ± 5.7% (86 of 275) in layer VI plus white matter. Overall, the proportion of SS-positive cells among GFP-positive cells was 23.4 ± 3.2% (198 of 849) in the neocortex. The proportions identified by colocalization of GFP with CR, PV, and SS in layers were shown in Table 2.
Table 2. GABAergic Neuron Subtypes1
| ||Layer I (%)|| ||Layer II-III (%)|| ||Layer V (%)|| ||Layer VI-WM (%)|| |
|Calretinin||6.5 ± 4.0||(5/89)||22.8 ± 1.0||(72/341)||5.2 ± 2.3||(9/163)||12.5 ± 5.4||(45/358)|
|Parvalbumin||0||(0/65)||44.3 ± 4.7||(198/443)||44.5 ± 6.4||(63/146)||41.8 ± 11.3||(136/331)|
|Somatostatin||0||(0/52)||17.7 ± 3.5||(68/381)||31.4 ± 4.7||(44/141)||31.6 ± 5.7||(86/275)|
|Total||6.5|| ||84.8|| ||81.1|| ||85.9|| |
Thus, the major three subtypes of GABAergic neurons accounted for 6.5% of the GFP-positive cells in layer I, 84.8% in layer II–III, 81.1% in layer V, and 85.9% in layer VI plus white matter. Overall, the major three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells in the neocortex. Unidentified GFP-positive cells accounted for 22.6% of the GFP-positive cells, most notably for 93.5% of the GFP-positive cells in layer I. In accord with unidentified GFP-positive cells, which were GAD67-positive as revealed by in situ hybridization (Fig. 7I), many medium spherical somata emitting intense GFP fluorescence were observed in layer I (Fig. 8A–C).