Abstract

Cortical GABAergic interneurons are divided into various subtypes, with each subtype contributing to rich variety and fine details of inhibition. Despite the functional importance of each interneuron subtype, the molecular mechanisms that contribute to sorting them to their appropriate positions within the cortex remain unclear. Here, we show that the chemokine receptor CXCR4 regulates the regional and layer-specific distribution of interneuron subtypes. We removed Cxcr4 specifically in a subset of interneurons at a specific mouse embryonic developmental stage and analyzed the number of interneurons and their laminar distribution in 9 representative cortical regions comprehensively in adults. We found that the number of Cxcr4-deleted calretinin- and that of neuropeptide Y-expressing interneurons were reduced in most caudomedial and lateral cortical regions, respectively, and also in superficial layers. In addition, Cxcr4-deleted somatostatin-expressing interneurons showed a reduction in the number of superficial layers in certain cortical regions but of deep layers in others. These findings suggest that CXCR4 is required for proper regional and laminar distribution in a wider interneuron subpopulation than previously thought and may regulate the establishment of functional cortical circuitry in certain cortical regions and layers.

Introduction

The cerebral cortex is divided into distinct regions and layers that have different roles for behavior. The proper function of each critically depends on the balance between excitation and inhibition, the latter being regulated by GABAergic interneurons. GABAergic interneurons are divided into various subtypes based on morphologies, electrophysiological properties, connectivity patterns, and biochemical constituents (for review, see Markram et al. 2004). Each interneuron subtype contributes to the rich variety and fine details of cortical inhibition. Indeed, abnormalities in the number and distribution of several interneuron subtypes including somatostatin (SST)- and neuropeptide Y (NPY)-expressing interneurons have been found in epileptic mutant mice (Powell et al. 2003; Cobos et al. 2005) and schizophrenic patients (Ikeda et al. 2004; Morris et al. 2008). Thus, the correct distribution of each interneuron subtype in the appropriate cortical regions and layers appears crucial for cortical functioning.

However, the cellular and molecular mechanisms that regulate the number and distribution of each interneuron subtype within cortical regions remain unclear. Each interneuron subtype is derived from spatiotemporally distinct progenitors within the subpallium (Xu et al. 2004; Butt et al. 2005; Fogarty et al. 2007; Miyoshi et al. 2007; Gelman et al. 2009; Morozov et al. 2009; Sousa et al. 2009), and the fate of those subtypes appears to be already determined when interneurons are generated (Nery et al. 2002; Xu et al. 2004; Yozu et al. 2005; Batista-Brito, Close, et al. 2008; Batista-Brito, Machold et al. 2008; Butt et al. 2008). Postmitotic interneurons migrate from the subpallium to the cortex tangentially and, after entering the cortex, most migrate through the subventricular zone (SVZ)/intermediate zone (IZ) and the marginal zone (MZ) before reaching their final destinations within the cortical plate (CP) (for review, see Kriegstein and Noctor 2004; Metin et al. 2006; Nakajima 2007). In the MZ, they migrate in diverse directions on a tangential plane over long distances by diffusion (Ang et al. 2003; Tanaka et al. 2003, 2006, 2009; Yokota et al. 2007), raising the possibility that their migration in the MZ is involved in their final distribution. Recent molecular studies showed that the chemokine CXCL12 secreted from SVZ cells and the meninges overlying the MZ attracted and promoted the migration of CXCR4-expressing interneurons (Stumm et al. 2003; Tiveron et al. 2006; Li et al. 2008; Liapi et al. 2008; López-Bendito et al. 2008; Tanaka et al. 2009), enabling these interneurons to accumulate and move in the SVZ/IZ and the MZ. In accordance with this idea, Cxcr4-deleted interneurons accumulate in neither the SVZ/IZ nor the MZ during their migration and consequently show obvious but subtle abnormalities in their regional and laminar distributions in the postnatal cortex (Stumm et al. 2003; Tiveron et al. 2006; Li et al. 2008; López-Bendito et al. 2008). These results suggest that CXCR4 is required for interneuron migration in both the SVZ/IZ and the MZ and for their proper final distribution in at least a minor population. However, it remains unclear which migratory pathway, the SVZ/IZ or the MZ, is important for correct interneuron distribution. It is also unknown whether CXCR4 is required for all interneuron subtypes to settle to their correct positions and layers.

To these ends, we designed experiments in which Cxcr4 was specifically removed in a subset of interneurons at a specific developmental stage to prevent interneuron distribution in the MZ but not in the SVZ/IZ during migration and analyzed the number and laminar distribution of Cxcr4-deleted interneuron subtypes in 9 representative cortical regions comprehensively in adults. Our previous studies have shown that interneurons migrate in many directions in the MZ over long distances when observed after E15.5 (Tanaka et al. 2006, 2009). Cxcl12 is strongly expressed in both the SVZ/IZ and the meninges as early as E14.5 (Tiveron et al. 2006). At E15.5 onward, however, Cxcl12 expression becomes weak in the SVZ/IZ, although it remains strong in the meninges (Tiveron et al. 2006). Thus, it seems likely that interneuron distribution in the MZ depends on CXCL12/CXCR4 signaling at E14.5 or later, whereas that in the SVZ/IZ depends on signaling before E15.5. Here, we show that inducible removal of Cxcr4 after E15.75 causes interneuron distribution defects of migrating interneurons in the MZ but not those in the VZ/SVZ/IZ and causes both regional and laminar-distribution defects in certain SST-, NPY-, and calretinin (CR)-expressing interneuron subtypes in adults. These data suggest that interneuron distribution in the MZ during migration is involved in the final distribution of interneurons in adults and that CXCR4 is required for proper final regional and laminar distribution in a wider interneuron subpopulation than previously thought (Li et al. 2008).

Materials and Methods

Animals

To selectively remove Cxcr4 in interneurons after E15.75 and examine their final distribution in the adult cortex, we took advantage of 3 genetically modified alleles: 1) a driver line carrying a modified tamoxifen-inducible form of the site-specific Cre recombinase (CreER) under the control of the Dlx1/2 intragenic enhancer (Batista-Brito, Close, et al. 2008; Batista-Brito, Machold et al. 2008); 2) a CAG-CAT-EGFP reporter line (Kawamoto et al. 2000) that expresses EGFP subsequent to the Cre-mediated removal of a stop cassette flanked by loxP sites; and 3) a Cxcr4-floxed line that cannot express functional CXCR4 subsequent to the Cre-mediated removal of Cxcr4 exon 2 flanked by loxP sites (Tokoyoda et al. 2004) (Fig. 1). In this triple transgenic mouse, removal of Cxcr4 in interneurons was achieved upon the administration of tamoxifen at E15.75 (Fig. 1), enabling us to examine the specific function of CXCR4 in interneurons after this time. To obtain control (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/+) and mutant (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/F) mice, Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/+ males were crossed with Cxcr4F/F females. Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/+ males are on mixed backgrounds between C57b/6 and ICR. Cxcr4F/F females were maintained in a pure C57b/6 background. All controls and mutants were from the same degree of mixed backgrounds. Tamoxifen (Sigma, St Louis, MO) (20 mg/mL dissolved in corn oil [Sigma]) was administered into pregnant mice (0.13 mg/g body weight) on embryonic day (E) 15.75 (3:00–9:00 PM) by oral gavaging with silicon-protected needles (Fine Science Tools Inc., North Vancouver, Canada). A cesarean section was performed at E18.5–19.5. Pups were fostered for adult analysis. Noon on the day of vaginal plug detection was termed as E0.5. E19.5 was defined as postnatal day (P) 0. Polymerase chain reaction primers used for genotyping were as follows: Cre forward, 5′-taaagatatctcacgtactgacggtg-3′; Cre reverse, 5′-tctctgaccagagtcatccttagc-3′; CAT forward, 5′-cagtcagttgctcaatgtacc-3′; CAT reverse, 5′-cagtcagttgctcaatgtacc-3′; Cxcr4-floxed forward, 5′- gcatataacaagtgactggt-3′; and Cxcr4-floxed reverse, 5′-tgatgaagtagatggtgggcaggaa-3′. All experiments were performed in accordance with the Osaka University Guidelines for the Welfare and Use of Laboratory Animals and the guidelines of the Japan Neuroscience Society and Keio University School of Medicine.

Figure 1.

Schematic of inducible and simultaneous GFP-labeling and Cxcr4 removal in a subset of cortical GABAergic interneurons. Tamoxifen was administered at E15.75. The distribution of GFP cells was analyzed at E19.5 or adult in control (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/+) and mutant (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/F) cortices. Rectangles indicate loxP sites. CAT, chloramphenicol acetyltransferase.

Figure 1.

Schematic of inducible and simultaneous GFP-labeling and Cxcr4 removal in a subset of cortical GABAergic interneurons. Tamoxifen was administered at E15.75. The distribution of GFP cells was analyzed at E19.5 or adult in control (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/+) and mutant (Dlx1/2-CreERTg/+; CAG-CAT-EGFPTg/+; Cxcr4F/F) cortices. Rectangles indicate loxP sites. CAT, chloramphenicol acetyltransferase.

Immunohistochemistry

E19.5 brains were dissected out and immersion fixed in 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.1 M, pH 7.4) overnight at 4 °C. Adult (P21–24) mice were deeply anesthetized with sodium pentobarbitone (Nembutal; Abbott, North Chicago, IL; 100–200 mg/kg body weight) and perfused with phosphate-buffered saline (PBS; 0.1 M, pH 7.4) followed by 4% PFA in PB. Brains were dissected and postfixed from 2 h to overnight in the same fixative at 4 °C. Fixed brains were embedded in 4% low-melting point agarose and cut sagittally at 50 μm on a vibrating-blade microtome (VT-1000, Leica Microsystems, Tokyo, Japan). Floating sections were incubated in methanol with 0.3% hydrogen peroxide for 20 min at room temperature (RT), washed with PBS, and incubated in PBS with 0.3% (for E19.5 sections) or 0.5% (for adult sections) Triton X-100 and 5% normal goat or donkey serum (NGS or NDS) for 2 h at RT followed by incubation in goat biotinylated anti-GFP antibody (1:2000; Rockland Immunochemicals, Gilbertsville, PA) in PBS with 0.3% (for E19.5 sections) or 0.5% (for adult sections) Triton X-100 and 1% NGS or NDS overnight at RT. The sections were then washed with PBS, incubated with avidin–biotin peroxidase complex (1:50; Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA) in PBS for 2 h and washed with PBS. Adult sections were additionally incubated overnight at RT with one of the following primary antibodies in PBS with 1% NGS: rabbit anti-parvalbumin (PV) (1:2000; Swant, Bellinzona, Switzerland), rat anti-SST (1:400; Chemicon, Temecula, CA), rabbit anti-CR (1:2000; Swant), or rabbit anti-NPY (1:2000; ImmunoStar, Hudson, WI) and washed with PBS. Then both E19.5 and adult sections were incubated in biotinylated tyramide (1:50; TSA Biotin System; PerkinElmer Life Sciences, Waltham, MA) in amplification diluent for 20 min at RT, washed with PBS, incubated with Alexa488-conjugated streptavidin (1:400; Invitrogen, Eugene, OR) in PBS for 2 h at RT and washed with PBS. Adult sections were further incubated in PBS containing 5% NGS or normal horse serum for 1 h at RT followed by incubation in donkey Alexa594-conjugated anti-rabbit IgG (1:750; Invitrogen) or goat Alexa594-conjugated anti-rat IgG (1:750; Invitrogen) antibody in PBS containing 1% NGS or normal horse serum for 2 h at RT. For nuclear staining, E19.5 sections were incubated in 0.03% 4,6-diamidino-2-phenylindole (DAPI) in PBS for 1 h at RT. Images were captured with a confocal microscope (TCS SP2 AOBS; Leica Microsystems or FV1000; Olympus).

Quantification and Statistics

To quantify the laminar distribution of GFP cells in E19.5 cortices, the number of all GFP cells was counted from a 200-μm-wide profile sampled from intermediate cortical regions along the rostrocaudal axis (e.g., Fig. 2C,D). The cortical wall was subdivided into 7 zones based on the density of DAPI-positive nuclei: MZ, upper CP (u-CP), middle CP (m-CP), lower CP (l-CP), upper-IZ (u-IZ), lower IZ (l-IZ) and SVZ/ventricular zone (VZ). Upper CP was defined as the cell-dense CP zone just beneath the MZ.

Figure 2.

Proportions of Cxcr4-deleted GFP cells in both the MZ and upper CP were reduced at E19.5. (A,B) Distribution of GFP cells in sagittal slices from control (Cxcr4F/+) (A) or mutant (Cxcr4F/F) (B) at E19.5. The number of GFP cells around the MZ was reduced in nearly all cortical regions along the rostrocaudal axis in mutants (arrowheads). (C,D) Enlarged views of the boxed regions shown in A(c) and B(d), respectively. (E) Quantitative analysis of the distribution of GFP cells in middle cortical regions along the rostrocaudal axis (e.g., C,D) in controls (n = 491 cells, 4 brains) (gray circles) and mutants (n = 616 cells, 5 brains) (black circles) (average ± standard error of the mean [SEM]). The abscissa indicates the proportion of GFP cells in each cortical zone. Statistical analysis was done between genotypes in each zone. The E19.5 cortical wall was subdivided into 7 zones: MZ, u-CP, m-CP, l-CP, u-IZ, l-IZ, and SVZ/VZ. Upper CP was defined as the cell-dense CP zone just beneath the MZ. m-CP and l-CP are presumptive layers 5 and 6, respectively. R, rostral; V, ventral. *P = 0.016; Mann–Whitney U-test. Scale bars: (A,B) 400 μm and (C,D) 100 μm.

Figure 2.

Proportions of Cxcr4-deleted GFP cells in both the MZ and upper CP were reduced at E19.5. (A,B) Distribution of GFP cells in sagittal slices from control (Cxcr4F/+) (A) or mutant (Cxcr4F/F) (B) at E19.5. The number of GFP cells around the MZ was reduced in nearly all cortical regions along the rostrocaudal axis in mutants (arrowheads). (C,D) Enlarged views of the boxed regions shown in A(c) and B(d), respectively. (E) Quantitative analysis of the distribution of GFP cells in middle cortical regions along the rostrocaudal axis (e.g., C,D) in controls (n = 491 cells, 4 brains) (gray circles) and mutants (n = 616 cells, 5 brains) (black circles) (average ± standard error of the mean [SEM]). The abscissa indicates the proportion of GFP cells in each cortical zone. Statistical analysis was done between genotypes in each zone. The E19.5 cortical wall was subdivided into 7 zones: MZ, u-CP, m-CP, l-CP, u-IZ, l-IZ, and SVZ/VZ. Upper CP was defined as the cell-dense CP zone just beneath the MZ. m-CP and l-CP are presumptive layers 5 and 6, respectively. R, rostral; V, ventral. *P = 0.016; Mann–Whitney U-test. Scale bars: (A,B) 400 μm and (C,D) 100 μm.

To examine the regional distribution of GFP and subtype-marker (PV, SST, CR, or NPY) double-positive cells in adult cortices, a 450-μm-wide region was chosen for rostral, intermediate, and caudal regions of the cortex at 3 different sagittal planes, located at 1.5–2.0, 2.0–2.5, and 2.5–3.0 mm lateral to the midline (Franklin and Paxinos 2008). The number of double-positive cells was counted from the 9 regions. The 3 sagittal planes were termed as medial (m), intermediate (i), and lateral (l) in a mediolateral order. The levels of the regions along the rostrocaudal axis were depicted as frontal (F), parietal (P), and occipital (O) with F being located most rostrally. Thus, the 9 regions were termed Fm, Pm, Om, Fi, Pi, Oi, Fl, Pl, and Ol (see Fig. 3). Data were collected from 7 to 9 brains for each genotype. Fm, Pm, and Om roughly correspond to the frontal association cortex and the secondary and primary motor cortices; the hindlimb and shoulder regions in the primary somatosensory cortex; and the mediolateral region in the secondary visual cortex and the primary visual cortex (V1), respectively (Fig. 3B) (Franklin and Paxinos 2008). Fi, Pi, Oi, Fl, Pl, and Ol corresponded to area 3 in the frontal cortex, the secondary and primary motor cortices, the dysgranular insular cortex, the primary somatosensory cortex (S1), and the jaw and upper lip regions in S1; the forelimb and shoulder regions in S1, the dysgranular zone in S1, and the barrel field in S1; V1; the granular insular cortex, S1, and the jaw and upper lip regions in S1; the barrel field in S1; and V1, respectively (Fig. 3B) (Franklin and Paxinos 2008).

Figure 3.

Schematics of analyzed cortical regions in adult mice. (A) Dorsal view of telencephalon. Medial (m), intermediate (i), and lateral (l) slices were defined as sagittal slices ranging from 1.5 to 2.0, 2.0 to 2.5, and 2.5 to 3.0 mm from the midline, respectively. Frontal (F), parietal (P), and occipital (O) regions indicate rostral, intermediate, and caudal regions, respectively, in m, i, and l slices. (B) Representative distribution of GFP cells in P21 control m, i, and l slices. Rectangles indicate 9 analyzed cortical regions. The approximate cortical area corresponding to each region is shown as an abbreviation (Franklin and Paxinos 2008). DI, dysgranular insular cx; Fr3, frontal cx, area3; FrA, frontal association cx; GI, granular insular cx; M1, primary motor cx; M2, secondary motor cx; S1BF, primary somatosensory cx, barrel field; S1HL; primary somatosensory cx, hindlimb; S1J, primary somatosensory cx, jaw region; S1ULp, primary somatosensory cx, upper lip region; V1, primary visual cx; V2ML, secondary visual cx, mediolat; D, dorsal; and C, caudal. Scale bar: 1 mm.

Figure 3.

Schematics of analyzed cortical regions in adult mice. (A) Dorsal view of telencephalon. Medial (m), intermediate (i), and lateral (l) slices were defined as sagittal slices ranging from 1.5 to 2.0, 2.0 to 2.5, and 2.5 to 3.0 mm from the midline, respectively. Frontal (F), parietal (P), and occipital (O) regions indicate rostral, intermediate, and caudal regions, respectively, in m, i, and l slices. (B) Representative distribution of GFP cells in P21 control m, i, and l slices. Rectangles indicate 9 analyzed cortical regions. The approximate cortical area corresponding to each region is shown as an abbreviation (Franklin and Paxinos 2008). DI, dysgranular insular cx; Fr3, frontal cx, area3; FrA, frontal association cx; GI, granular insular cx; M1, primary motor cx; M2, secondary motor cx; S1BF, primary somatosensory cx, barrel field; S1HL; primary somatosensory cx, hindlimb; S1J, primary somatosensory cx, jaw region; S1ULp, primary somatosensory cx, upper lip region; V1, primary visual cx; V2ML, secondary visual cx, mediolat; D, dorsal; and C, caudal. Scale bar: 1 mm.

To examine the laminar distribution of double-positive cells in each region, layers 1–6 of the cortex was divided into 10 horizontal bins, and all double-positive cells were counted in each bin in each region. Bins 1, 2, and 10 roughly corresponded to layer 1, the upper end of layer 2/3, and the lower end of layer 6, respectively.

To examine differences between controls and mutants, all data were statistically analyzed using the Mann–Whitney U-test.

Results

MZ-Distribution Defects in Cxcr4-Deleted Interneurons in the Embryonic Cortex

Previous studies have shown that removal of Cxcr4 in interneurons caused a reduction in the number of MZ interneurons (Li et al. 2008; Tanaka et al. 2009). To confirm that a similar reduction occurs by the inducible removal of Cxcr4 after E15.75 (Fig. 1), we examined the distribution of GFP cells in E19.5 cortices (n = 4 brains for controls, 5 for mutants). We found that the proportion of GFP cells in the MZ was significantly less in mutants (Cxcr4F/F) compared with controls (Cxcr4F/+) (P = 0.016, Mann–Whitney U-test) (Fig. 2), whereas no significant difference was observed in either the IZ or the SVZ/VZ (upper-IZ, P = 0.19; lower-IZ, P = 1; SVZ/VZ, P = 0.73) (Fig. 2). Thus, inducible removal of Cxcr4 reduced GFP cells in the MZ but not in either the IZ or the SVZ/VZ.

Regional-Distribution Defects in Cxcr4-Deleted CR- and NPY-Expressing Interneurons in the Adult Cortex

Because GFP cells failed to distribute in the MZ in mutant embryos (Fig. 2), we next examined their final regional and laminar distributions in adults. Interneurons were divided into several distinct subtypes based on the expression of calcium-binding proteins like PV and CR and neuropeptides like SST and NPY (Kubota et al. 1994; Gonchar and Burkhalter 1997; Kawaguchi and Kubota 1997; Kawaguchi and Kondo 2002; Xu et al. 2006; Gonchar et al. 2008). To examine the cell-autonomous role of CXCR4 in the regional and laminar distributions of interneuron subtypes, the number and laminar distribution of all GFP and subtype-marker (PV, SST, CR, or NPY) double-positive (GFP/subtype-marker) cells were analyzed in the representative 9 cortical regions in both control and mutant adults (Fig. 3) (see Materials and Methods). Because cortical size and thickness appeared normal in mutants (data not shown), the cell numbers in given areas of a 450-μm-wide profile of the cortical wall were directly compared with measured cell density in the cortical areas.

We first carried out analysis by focusing on the regional distribution of interneuron subtypes. Although the sum of the number of GFP/subtype-marker cells in each of the 9 cortical regions was not significantly different between controls and mutants (GFP/PV, P = 0.69, Mann–Whitney U-test, n = 7 brains for controls, 8 for mutants; GFP/SST, P = 0.28, n = 7 for controls, 8 for mutants; GFP/CR, P = 0.30, n = 7 for controls, 9 for mutants; GFP/NPY, P = 0.32, n = 8 for controls, 9 for mutants), GFP/CR and GFP/NPY cell numbers were significantly reduced in certain mutant cortical regions: GFP/CR cell number was significantly reduced in Om (Fig. 4A,B) and GFP/NPY cell numbers in both Fl (Fig. 4C,E) and Ol (Fig. 4D,E). No significant reduction was observed in any other region (Supplemental Fig. 1 and data not shown). GFP/PV and GFP/SST cells showed no significant difference in number between controls and mutants in any cortical regions (data not shown). To further characterize regional defects in mutants, the 9 regions were grouped into 3 mediaolateral domains (m consisting of Fm/Pm/Om, i consisting of Fi/Pi/Oi, and l consisting of Fl/Pl/Ol) or rostrocaudal domains (F consisting of Fm/Fi/Fl, P consisting of Pm/Pi/Pl, and O consisting of Om/Oi/Ol) (Fig. 3). The number of GFP/subtype-marker cells was examined in each domain. Although GFP/PV-, GFP/SST,- and GFP/CR-cell numbers showed no significant differences between controls and mutants in any domains, the number of GFP/NPY cells was significantly reduced in mutant l domain (P = 0.046; n = 262 cells, 8 brains for controls; 246 cells, 9 brains for mutants). Collectively, these data suggest that CXCR4 is required cell autonomously for the proper regional distribution of certain CR- and NPY-expressing interneurons.

Figure 4.

The number of Cxcr4-deleted GFP/CR and GFP/NPY cells was reduced in caudomedial and lateral regions, respectively. (A) Distribution of GFP (green)- and CR (magenta)-positive cells in Om in control (Cxcr4F/+) and mutant (Cxcr4F/F) cortices. Arrowheads indicate double-positive cells for GFP and CR. Insets show an enlarged view of the cell indicated by the open arrowhead. (B) Quantitative analysis of the number of GFP/CR cells per slice in Om of control (F/+) (n = 53 cells, 8 brains) and mutant (F/F) (n = 32 cells, 9 brains) cortices (average ± SEM). (C,D) Distribution of GFP (green)- and NPY (magenta)-positive cells in Fl (C) and Ol (D) in control and mutant cortices. Arrowheads indicate double-positive cells for GFP and NPY. Insets show an enlarged view of the cell indicated by the open arrowhead. (E) Quantitative analysis of the number of GFP/NPY cells per slice in each region of control (F/+) (n = 32 cells, 8 brains for Fl; 34 cells, 8 brains for Ol) and mutant (F/F) (n = 19 cells, 9 brains for Fl; 21 cells, 9 brains for Ol) cortices (average ± SEM). *P < 0.05; Mann–Whitney U-test. Scale bars: 150 μm; 10 μm for insets.

Figure 4.

The number of Cxcr4-deleted GFP/CR and GFP/NPY cells was reduced in caudomedial and lateral regions, respectively. (A) Distribution of GFP (green)- and CR (magenta)-positive cells in Om in control (Cxcr4F/+) and mutant (Cxcr4F/F) cortices. Arrowheads indicate double-positive cells for GFP and CR. Insets show an enlarged view of the cell indicated by the open arrowhead. (B) Quantitative analysis of the number of GFP/CR cells per slice in Om of control (F/+) (n = 53 cells, 8 brains) and mutant (F/F) (n = 32 cells, 9 brains) cortices (average ± SEM). (C,D) Distribution of GFP (green)- and NPY (magenta)-positive cells in Fl (C) and Ol (D) in control and mutant cortices. Arrowheads indicate double-positive cells for GFP and NPY. Insets show an enlarged view of the cell indicated by the open arrowhead. (E) Quantitative analysis of the number of GFP/NPY cells per slice in each region of control (F/+) (n = 32 cells, 8 brains for Fl; 34 cells, 8 brains for Ol) and mutant (F/F) (n = 19 cells, 9 brains for Fl; 21 cells, 9 brains for Ol) cortices (average ± SEM). *P < 0.05; Mann–Whitney U-test. Scale bars: 150 μm; 10 μm for insets.

Laminar-Distribution Defects in Cxcr4-Deleted SST-, CR-, and NPY-Expressing Interneurons in the Adult Cortex

Next, we examined the role of CXCR4 in the laminar distribution of interneuron subtypes. Cxcr4-deleted GFP/SST, GFP/CR, and GFP/NPY cells showed certain defects, whereas GFP/PV cells showed no significant differences (data not shown). The number of GFP/SST cells was reduced in specific layers of specific regions (Fig. 5A–E) or a domain (Fig. 5F) in mutants (i.e., the GFP/SST cell number was reduced in deep and superficial layers in Pl [Fig. 5E] and O [Fig. 5F], respectively). In general, the numbers of GFP/CR and GFP/NPY cells were significantly reduced in the upper layer of mutants (Fig. 6A,D). GFP/NPY cell numbers were also reduced in Ol (Fig. 6B) and P (Fig. 6C). Thus, CXCR4 is required cell autonomously for the proper laminar distribution of certain SST-, CR-, and NPY-expressing interneurons.

Figure 5.

The numbers of Cxcr4-deleted GFP/SST cells were reduced in certain layers in specific cortical regions. (A) Distribution of GFP (green)- and SST (magenta)-positive cells in Pi in control (Cxcr4F/+) and mutant (Cxcr4F/F) cortices. Arrowheads indicate double-positive cells for GFP and SST. Insets show an enlarged view of the cell indicated by the open arrowhead. (BF) Quantitative analysis of the number of GFP/SST cells in each bin per slice in certain regions (BE) and O (F) of control (F/+) (n = 103 cells, 8 brains for Pi [B]; 112 cells, 8 brains for Fm [C]; 104 cells, 8 brains for Fl [D]; 94 cells, 8 brains for Pl [E]; 289 cells, 8 brains for O [F]) and mutant (F/+) (n = 83 cells, 9 brains for Pi [B]; 104 cells, 9 brains for Fm [C]; 73 cells, 9 brains for Fl [D]; 81 cells, 9 brains for Pl [E]; 251 cells, 9 brains for O [F]) cortices (average ± SEM). Numbers in ordinates indicate bins for quantification. *P < 0.05; Mann–Whitney U-test. Scale bars: 150 μm; 10 μm for insets.

Figure 5.

The numbers of Cxcr4-deleted GFP/SST cells were reduced in certain layers in specific cortical regions. (A) Distribution of GFP (green)- and SST (magenta)-positive cells in Pi in control (Cxcr4F/+) and mutant (Cxcr4F/F) cortices. Arrowheads indicate double-positive cells for GFP and SST. Insets show an enlarged view of the cell indicated by the open arrowhead. (BF) Quantitative analysis of the number of GFP/SST cells in each bin per slice in certain regions (BE) and O (F) of control (F/+) (n = 103 cells, 8 brains for Pi [B]; 112 cells, 8 brains for Fm [C]; 104 cells, 8 brains for Fl [D]; 94 cells, 8 brains for Pl [E]; 289 cells, 8 brains for O [F]) and mutant (F/+) (n = 83 cells, 9 brains for Pi [B]; 104 cells, 9 brains for Fm [C]; 73 cells, 9 brains for Fl [D]; 81 cells, 9 brains for Pl [E]; 251 cells, 9 brains for O [F]) cortices (average ± SEM). Numbers in ordinates indicate bins for quantification. *P < 0.05; Mann–Whitney U-test. Scale bars: 150 μm; 10 μm for insets.

Figure 6.

The numbers of respective Cxcr4-deleted GFP/CR and GFP/NPY cells were reduced in certain layers in specific cortical regions. (A,D) Quantitative analysis of the number of GFP/CR (A) and GFP/NPY (D) cells in each bin per slice in the sum of all 9 regions of control (F/+) (n = 299 cells, 7 brains for CR [A]; n = 262 cells, 8 brains for NPY [D]) and mutant (F/F) (n = 321 cells, 9 brains for CR [A]; n = 246 cells, 9 brains for NPY [D]) cortices (average ± SEM). (B,C) Quantitative analysis of the number of GFP/NPY cells in each bin per slice in Ol (B) and P (C) of control (n = 34 cells, 8 brains for Ol [B]; n = 76 cells, 8 brains for P [C]) and mutant cortices (n = 21 cells, 9 brains for Ol [B]; n = 70 cells, 9 brains for P [C]) (average ± SEM). Numbers in ordinates indicate bins for quantification. *P < 0.05; Mann–Whitney U-test.

Figure 6.

The numbers of respective Cxcr4-deleted GFP/CR and GFP/NPY cells were reduced in certain layers in specific cortical regions. (A,D) Quantitative analysis of the number of GFP/CR (A) and GFP/NPY (D) cells in each bin per slice in the sum of all 9 regions of control (F/+) (n = 299 cells, 7 brains for CR [A]; n = 262 cells, 8 brains for NPY [D]) and mutant (F/F) (n = 321 cells, 9 brains for CR [A]; n = 246 cells, 9 brains for NPY [D]) cortices (average ± SEM). (B,C) Quantitative analysis of the number of GFP/NPY cells in each bin per slice in Ol (B) and P (C) of control (n = 34 cells, 8 brains for Ol [B]; n = 76 cells, 8 brains for P [C]) and mutant cortices (n = 21 cells, 9 brains for Ol [B]; n = 70 cells, 9 brains for P [C]) (average ± SEM). Numbers in ordinates indicate bins for quantification. *P < 0.05; Mann–Whitney U-test.

Discussion

Our results demonstrate that CXCR4 expression in MZ interneurons is required cell autonomously for the proper regional distribution of certain CR- and NPY-expressing interneurons (Fig. 7A) and laminar distribution of certain SST-, CR-, and NPY-expressing interneurons (Fig. 7B). These findings suggest that CXCR4 expressed in interneurons is required for the proper regional and laminar distribution of a wider subpopulation of interneurons than previously thought (Li et al. 2008).

Figure 7.

Summary of regional and laminar reductions of Cxcr4-deleted interneurons. (A) Regional reduction in the number of Cxcr4-deleted interneurons. Regions surrounded by thick black line and filled with gray indicate regions where the number of GFP/subtype-marker cells was “statistically significantly” reduced in mutants. The domain consisting of 3 regions connected by a thick black line indicates the domain where the number of GFP/subtype-marker cells was statistically significantly reduced in a group of 3 regions in mutants. (B) Laminar reduction in the number of Cxcr4-deleted interneurons. Regions surrounded by thick black lines and domains consisting of those regions indicate the regions and domains where statistically significant laminar reduction in GFP/subtype-marker cells were found in mutants, respectively. Thick lines surrounding entire hemispheres indicate the statistically significant laminar reduction of GFP/subtype-marker cells in the sum of all 9 cortical regions in mutants. Numbers next to each area surrounded by thick lines indicate the bin number where the GFP/subtype-marker-cell number was statistically significantly reduced in mutants. Note both in A and B, failure to detect statistically significant difference does not necessarily mean absence of changes in interneuron distribution.

Figure 7.

Summary of regional and laminar reductions of Cxcr4-deleted interneurons. (A) Regional reduction in the number of Cxcr4-deleted interneurons. Regions surrounded by thick black line and filled with gray indicate regions where the number of GFP/subtype-marker cells was “statistically significantly” reduced in mutants. The domain consisting of 3 regions connected by a thick black line indicates the domain where the number of GFP/subtype-marker cells was statistically significantly reduced in a group of 3 regions in mutants. (B) Laminar reduction in the number of Cxcr4-deleted interneurons. Regions surrounded by thick black lines and domains consisting of those regions indicate the regions and domains where statistically significant laminar reduction in GFP/subtype-marker cells were found in mutants, respectively. Thick lines surrounding entire hemispheres indicate the statistically significant laminar reduction of GFP/subtype-marker cells in the sum of all 9 cortical regions in mutants. Numbers next to each area surrounded by thick lines indicate the bin number where the GFP/subtype-marker-cell number was statistically significantly reduced in mutants. Note both in A and B, failure to detect statistically significant difference does not necessarily mean absence of changes in interneuron distribution.

Genetic Inducible Deletion of Cxcr4 in a Subset of Interneurons

We performed inducible deletion of Cxcr4 for 2 purposes. One was to control the timing of CXCR4 deletion in interneurons to specifically examine the importance of their distribution in the MZ during their migration. Previous studies have shown both systemic (Stumm et al. 2003; Tiveron et al. 2006; Li et al. 2008; López-Bendito et al. 2008) and cell-type specific (Li et al. 2008) removal of Cxcr4 disrupt interneuron distribution not only in the MZ but also in the IZ/SVZ. We found that the proportion of GFP cells in the MZ but in neither the IZ nor the SVZ/VZ was reduced by inducible removal of Cxcr4 after E15.75 (Fig. 2). This reinforces the notion that defects in interneuron distribution in mutant adults (Figs. 4–7) may be due to migration defects in the MZ rather than the IZ/SVZ in embryos.

The second purpose of using an inducible system was to label a small number of interneurons to sensitively detect distribution differences between controls and mutants. Indeed, we found several differences (Fig. 7) that were not reported in a previous study in which a larger subpopulation of interneurons was labeled (Li et al. 2008). Although we found significant differences only in specific cortical regions and layers (Figs. 4–7), neuron numbers tended to increase or decrease in other regions and layers in mutants (Figs. 5 and 6, and data not shown). Statistically significant differences might be detected in those regions and layers by increasing the number of samples examined or applying more sensitive methods than those used in the present study.

Cell-Autonomous Requirement of CXCR4 for Proper Regional and Laminar Distributions in Wider Interneuron Subpopulations

Two recent studies have examined the cell-autonomous roles of CXCR4 in the regional and laminar distributions of interneurons in postnatal cortices (Li et al. 2008; López-Bendito et al. 2008). Li and colleagues selectively removed Cxcr4 in cortical interneurons by combining a Dlx5/6-Cre driver and Cxcr4-floxed lines, labeling the recombined cells by further crossing them with a Z/EG reporter line and finally examined their regional and laminar distributions in P14–15 cortices (Li et al. 2008). They found a reduction in the number of Cxcr4-deleted interneurons in the medial cortex (Li et al. 2008), indicating that CXCR4 is required cell autonomously for the proper regional distribution of interneurons. In this regional distribution analysis, however, they did not determine the subtypes of the Cxcr4-deleted interneurons. Here, we found that CXCR4 is required for certain CR- and NPY-expressing interneurons for their proper regional distributions (Figs. 4 and 7A).

Regarding the laminar distribution, Li and colleagues found that Cxcr4-deleted CR-expressing and nNOS-expressing cells showed ectopic clusters in deep layers and ectopic distribution in superficial layers, respectively (Li et al. 2008). Although they did not report any differences in other interneuron subtypes including SST- and NPY-expressing cells between controls and mutants (Li et al. 2008), we found that CXCR4 is required for certain SST- and NPY-expressing interneurons for their proper laminar distributions (Figs. 5, 6B–D, and 7B). In addition, we found that the number of CR-expressing interneurons was reduced in superficial layers (Figs. 6A and 7B). There are 2 possible explanations for these discrepancies: One is the difference in populations analyzed. Although Li and colleagues examined all SST- and NPY-expressing cells (Li et al. 2008), we analyzed a restricted population (GFP cells labeled by a combination of CreER expression driven by a Dlx1/2 enhancer, a CAG-CAT-EGFP reporter mouse line and tamoxifen administration at E15.75) (Fig. 1) of SST- and NPY-expressing cells or GFP/SST and GFP/NPY cells. Another possibility is the difference in the cortical regions analyzed. Although Li and colleagues examined laminar distributions of interneuron subtypes only in the somatosensory cortex (Li et al. 2008), we analyzed these distributions in 9 representative cortical regions comprehensively (Fig. 3).

López-Bendito and colleagues transplanted Cxcr4-null medial ganglionic eminence (MGE) cells labeled with BrdU together with wild-type MGE cells labeled with both GFP and BrdU into wild-type MGE of mouse embryos and examined their regional and laminar distributions in P14 cortices (López-Bendito et al. 2008). They found that approximately one-third of the entire population of Cxcr4-null cells consistently dispersed less distance tangentially along the cortices than their wild-type counterparts (López-Bendito et al. 2008). In addition, many Cxcr4-null cells were not found in the upper layers of the cortex but instead were abnormally located in the deeper layers (López-Bendito et al. 2008). These results clearly showed and reinforced the notion that CXCR4 is required cell autonomously for normal regional and laminar distribution of some interneurons. However, they did not determine the interneuron subtypes of the examined MGE-derived cells. Because most MGE-derived interneurons differentiate into PV- or SST-expressing interneurons (Xu et al. 2004; Fogarty et al. 2007; Miyoshi et al. 2007), the MGE-derived cells López-Bendito and colleagues examined may be mostly PV- and/or SST-expressing interneurons. These findings support the role of CXCR4 in SST-expressing interneuron for their proper laminar distributions (Fig. 5), although differences in methods of Cxcr4 removal make it difficult to compare their results with ours.

The Role of CXCR4 in the Migration and Distribution of Certain Interneuron Subtypes

Previous studies have shown that MZ interneurons migrate in diverse directions on a tangential plane over long distances by diffusion (Ang et al. 2003; Tanaka et al. 2003, 2006, 2009; Yokota et al. 2007). Physiological roles of the migration for their final distribution have remained unclear, however. We have shown that inducible removal of Cxcr4 in a subset of interneurons led to a failure in their proper distribution in the MZ during their migration (Fig. 2) and in proper final regional and laminar distributions in adults (Figs. 3–7). These results suggest that CXCR4 regulates the final distribution of certain interneuron subtypes by controlling their migration in the MZ during development and that migration in the MZ may have important roles for certain interneuron subtypes to reach their final destinations within the cortex.

We found a reduction in Cxcr4-deleted GFP-cell number only in certain cortical regions and layers (Fig. 7). Previous studies, however, have shown that CXCL12 is expressed in the meninges (Stumm et al. 2003; Tiveron et al. 2006), and the secreted CXCL12 would be uniformly distributed throughout the cortical wall. One possible explanation for why Cxcr4-deleted interneuron number is reduced only in certain regions and layers, whereas the expression of CXCL12 is uniform in the meninges is that migratory interneurons respond differently to CXCL12. It is also possible that CXCR4 expression after E15.75 is crucial for only some interneurons. In support of this hypothesis, interneurons migrating toward the meninges, which are likely to secrete CXCL12, have been observed concurrently with those migrating away from them in the same cultured slice (Tanaka et al. 2003).

Our results demonstrate that the numbers of Cxcr4-deleted CR- and NPY-expressing interneurons but not PV- and SST-expressing interneurons were reduced in the caudal cortex (Figs. 4 and 7A). Recent studies have shown almost all cortical GABAergic interneurons are generated in the subpallium in mice (Fogarty et al. 2007), whereas different interneuron subtypes are generated in different subpallial regions. For example, PV- and SST-expressing interneurons are generated exclusively from Lhx6-expressing precursors in the MGE (Wichterle et al. 2001; Nery et al. 2002; Xu et al. 2004; Butt et al. 2005; Fogarty et al. 2007; Miyoshi et al. 2007), whereas most CR- and NPY-expressing cells are generated from germinal zones of the lateral/caudal ganglionic eminences (CGE) that express Gsh2 (Fogarty et al., 2007), possibly within the CGE (Xu et al. 2004; Butt et al. 2005). Although interneurons derived from the MGE migrate rather diffusely along the rostrocaudal axis to enter the cortex, CGE-derived interneurons preferentially migrate caudally along the caudal migratory stream (Yozu et al. 2005; Kanatani et al. 2008). After entering the cortex, CGE-derived cells appear to be distributed more caudally than MGE-derived cells (Nery et al. 2002). Taken together, these findings suggest that although PV- and SST-expressing interneurons distribute diffusely within the cortex, most CR- and NPY-expressing interneurons derived from the CGE migrate caudally and distribute preferentially in the caudal cortex. Therefore, our findings that the number of Cxcr4-deleted CR- and NPY-expressing interneurons but not PV- and SST-expressing interneurons was reduced in most caudomedial (Figs. 4A,B and 7A) and caudolateral regions (Figs. 4D,E and 7A), respectively, might be due to defects in tangential migration along the caudal migratory stream in CGE-derived CR- and NPY-expressing interneurons. This is consistent with the idea that the loss of CXCR4 function in interneurons reduces their capacity for tangential dispersion (Li et al. 2008; López-Bendito et al. 2008).

In conclusion, we have found that CXCR4 is required cell autonomously for the proper regional distribution of certain CR- and NPY-expressing interneuron populations and the proper laminar distribution of certain SST-, CR-, and NPY-expressing interneuron ones. These findings suggest that CXCR4 is required for proper distribution of a wider subpopulation of interneurons than previously thought (Li et al. 2008). Consequently, our results might explain in part the pathogenesis of the abnormal distribution of cortical SST- and NPY-expressing interneurons found in patients of schizophrenia (Ikeda et al. 2004; Morris et al. 2008) and temporal lobe epilepsy (Robbins et al. 1991).

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

SORST from Japan Science Technology Corporation (F.M.); Grant-in-Aid from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan (F.M., K.N., and D.H.T.); Takeda Science Foundation (F.M.); the Promotion and Mutual Aid Corporation for Private Schools of Japan (K.N.); the Naito Foundation (K.N.); and Global Centers of Excellence program for human metabolomic systems biology assigned to Keio University (K.N., D.H.T.).

We thank Drs Robert Machold and Gord Fishell for providing Dlx1/2-CreER mice. We also thank Ms Noriko Ohshiro and Tomoko Matsumoto for mouse care, Mr Mitsutoshi Yanagida for managing samples and Drs Mikiro Nawa and Sadakazu Aiso for technical support in confocal microscopic analysis. We also thank members in both the Murakami and Nakajima laboratories for meaningful discussions. Conflict of Interest: None declared.

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Author notes

5
Current address: Kobe City Medical Center West Hospital, Kobe 653-0013, Japan