Postnatal development of GABAergic interneurons in the neocortical subplate of mice

Neuroscience 322 (2016) 78–93

POSTNATAL DEVELOPMENT OF GABAergic INTERNEURONS IN THE NEOCORTICAL SUBPLATE OF MICE G.-J. QU, a J. MA, b Y.-C. YU a* AND Y. FU a* a

Institute of Neurobiology, Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, 131 Dong An Road, Shanghai 200032, China

b

School of Life Sciences, Tsinghua University, 30 Shuang Qing Road, Beijing 100084, China

layers. These findings clarify and extend our understanding of SP interneurons in the developing cerebral cortex and will underpin further study of SP function. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: subplate, interneuron, subtype, postnatal development.

Abstract—The subplate (SP) plays important roles in developmental and functional events in the neocortex, such as thalamocortical and corticofugal projection, cortical oscillation generation and corticocortical connectivity. Although accumulated evidence indicates that SP interneurons are crucial for SP function, the molecular composition of SP interneurons as well as their developmental profile and distribution remain largely unclear. In this study, we systematically investigated dynamic development of SP thickness and chemical marker expression in SP interneurons in distinct cortical regions during the first postnatal month. We found that, although the relative area of the SP in the cerebral cortex significantly declined with postnatal development, the absolute thickness did not change markedly. We also found that somatostatin (SOM), the ionotropic serotonin receptor 3A (5HT3AR), and parvalbumin (PV) reliably identify three distinct non-overlapping subpopulations of SP interneurons. The SOM group, which represents
30% of total SP interneurons, expresses neuronal nitric oxide synthase (nNOS) and calbindin (CB) and colocalizes entirely with neuropeptide Y (NPY). The 5HT3AR group, which accounts for
60% of the total interneuronal population, expresses calretinin (CR) and GABA-A receptor subunit delta (GABAARd). The PV group accounts for
10% of total SP interneurons and coexpressed GABAARd. Moreover, distinct interneuron subtypes show characteristic temporal and spatial distribution in the SP. nNOS+ interneurons in the SP increase from the anterior motor cortex to posterior visual cortex, while CR+ and CB+ interneurons the opposite. Interestedly, the majority of GABAARd+ neurons in SP are non-GABAergic neurons in contrast to other cortical

INTRODUCTION The subplate (SP) is a distinctive and highly dynamic structure in the developing neocortex derived from the embryonic cortical preplate (Chun and Shatz, 1989a; Woo et al., 1991; Wood et al., 1992; Molna´r and Blakemore, 1995a; Valverde et al., 1995a; Marı´ nPadilla, 1998). When the layered neocortex starts to form, new migratory neurons split the cortical preplate into two parts, the upper marginal zone and the lower SP zone (Chun and Shatz, 1989a; Woo et al., 1991; Wood et al., 1992; Molna´r and Blakemore, 1995a; Valverde et al., 1995a; Marı´ n-Padilla, 1998). The SP zone undergoes programed cell death in early development, and evolves differently across species (Kostovic and Rakic, 1980, 1990; Al-Ghoul and Miller, 1989; Chun and Shatz, 1989a; Naegele et al., 1991; Ghosh and Shatz, 1993; Allendoerfer and Shatz, 1994; Valverde et al., 1995b; DeFreitas et al., 2001). In rodents, some SP cells persist into adulthood and form a compact layer after birth, named the SP layer (Aboitiz and Montiel, 2007; Hoerder-Suabedissen and Molna´r, 2015), also known as cortical layer 6b or layer 7 (Aboitiz and Montiel, 2007; Perrenoud et al., 2013). The SP is located at the interface between cortical layer 6 and the white matter (WM) in the neocortex at the postnatal stage (Reep and Goodwin, 1988; Vandevelde et al., 1996; Clancy and Cauller, 1999; Reep, 2000), and contains glutamatergic excitatory neurons and GABAergic interneurons as well as various corticofugal, corticopetal and corticocortical projections (Lavdas et al., 1999; Hevner and Zecevic, 2006; Kanold and Luhmann, 2010; Wang et al., 2010; Perrenoud et al., 2013; Hoerder-Suabedissen and Molna´r, 2015). Given that they are the earliest generated cortical neurons (Bayer and Altman, 1990; Hevner and Zecevic, 2006; Wang et al., 2010), SP neurons have been shown to play a critical role in processes such as the guidance of thalamocortical and corticofugal projection (McConnell et al., 1989; Ghosh et al., 1990; Carlos and O’Leary, 1992;

*Corresponding authors. Tel: +86-21-54237852; fax: +86-2154237647. E-mail addresses: [email protected] (Y.-C. Yu), [email protected]. cn (Y. Fu). Abbreviations: 5HT3AR, serotonin receptor 3A; CB, calbindin; CGE, caudal ganglionic eminence; CR, calretinin; DAPI, 40 ,6-diamidino-2phenylindole; GABA, c-aminobutyric acid; GABAARd, GABA-A receptor subunit delta; L1, layer 1; L2/3, layer 2 and Layer 3; L4, layer 4; L5, layer 5; L6, layer 6; MC, motor cortex; MGE, medial ganglionic eminence; nNOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; Nurr1, nuclear receptor-related 1; PBS, phosphatebuffered saline; PFA, paraformaldehyde; PV, parvalbumin; SC, somatosensory cortex; SOM, somatostatin; SP, subplate; VC, visual cortex; VIP, vasoactive intestinal polypeptide; WM, white matter. http://dx.doi.org/10.1016/j.neuroscience.2016.02.023 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 78

G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

Ghosh and Shatz, 1993; Molna´r and Blakemore, 1995b), the maturation and plasticity of thalamocortical circuits (Kanold et al., 2003; Kanold and Shatz, 2006; Kanold, 2009), establishment of ocular dominance and orientation columns (Grossberg and Seitz, 2003; Kanold et al., 2003; Kanold and Luhmann, 2010), supporting corticocortical connectivity (Kostovic and Rakic, 1990; Friedlander et al., 2009; Kostovic´ et al., 2011), and generating oscillatory activity (Balkowiec and Katz, 2000; Dupont et al., 2006; Heck et al., 2008; Hanganu et al., 2009). Moreover, SP abnormalities have been implicated in various neural disorders, including schizophrenia (Bunney and Bunney, 2000; Eastwood and Harrison, 2006; Sua´rez-Sola´ et al., 2009), Alzheimer’s disease (Sua´rez-Sola´ et al., 2009) and autism spectrum disorders (Bunney and Bunney, 2000; Hutsler and Casanova, 2015). Several studies have clearly demonstrated that interneurons in the SP are crucial for functions such as generating neuronal network oscillation in the cerebral cortex (Voigt et al., 2001; Hanganu et al., 2009), and regulating neuronal migration pattern (Manent et al., 2005; Reiprich et al., 2005; Heck et al., 2007; Luhmann et al., 2009). Therefore, further study of SP interneurons is necessary to promote our understanding of SP functions. Although some interneuron markers (somatostatin (SOM), neuropeptide Y (NPY), NOS et al.) were found in SP interneurons in different species (Antonini and Shatz, 1990; Yan et al., 1996; Finney and Shatz, 1998; Rı´ o et al., 2000; Robertson et al., 2000; Friedlander et al., 2009; Perrenoud et al., 2013), so far the molecular composition of SP interneurons as well as their developmental profile and distribution remain largely unknown. In this study, we characterized the developmental changes in SP thickness in the mouse neocortex. We also characterized the subtypes of SP interneurons and quantitatively analyzed their density in distinct cortical regions during postnatal development. Overall, our research conducts an extensive analysis of the neurochemical properties of SP interneurons in the developing neocortex, and will serve as a quantitative resource for future studies of the development and function of SP interneurons.

EXPERIMENTAL PROCEDURES Animals CD-1 mice, GAD67-GFP (Dneo) knock-in mice (Tamamaki et al., 2003; Jiao et al., 2006; Ma et al., 2014) and serotonin receptor 3A (5HT3AR)-GFP transgenic mice (Vucurovic et al., 2010) were used in this study. GAD67-GFP knock-in mice exhibit specific and efficient GFP-labeling of interneurons in the neocortex. The expression of 5HT3AR in neurons was tightly linked with GFP in 5HT3AR-GFP transgenic mice. The day of birth was noted as postnatal day 1 (P1). All animal procedures were conducted according to the guidelines for the Animal Care and Use Committee of Fudan University. Data acquisition We obtained quantitative data from the motor cortex (MC), somatosensory cortex (SC), and visual cortex

79

(VC) from at least three discrete sections from at least three individual animals at each time point. The cortical regions were determined with a stereotaxic map. The SP was a thin layer between layer 6 and WM in postnatal mice (Hoerder-Suabedissen et al., 2009; Luhmann et al., 2009; Hoerder-Suabedissen and Molna´r, 2012, 2013, 2015), equivalent to layer 6b or layer 7 (Reep and Goodwin, 1988; Reep, 2000; Aboitiz and Montiel, 2007; Chung et al., 2009; Hoerder-Suabedissen and Molna´r, 2013; Perrenoud et al., 2013). The laminar borders of the SP were identified on the basis of cell distribution visualized by 40 ,6-diamidino-2-phenylindole (DAPI) staining as previously reported (Reep, 2000; Torres-Reveron and Friedlander, 2007; Viswanathan et al., 2012). By use of nuclear receptor-related 1 (Nurr1), a special molecular marker labels SP (HoerderSuabedissen et al., 2009; Hoerder-Suabedissen and Molna´r, 2013), we confirmed the precise distinguishing of SP in our experiments (Fig. 1A). The analysis of SP thickness in development Using Photoshop CS5 (Adobe Systems) the SP in coronal brain slices was divided into a series of trapezoids n P (Fig. 1B) and the total area ( ðSiÞ) of the trapezoids n i¼1 P and total length ( ðLi þ liÞ) of upper and lower lines of i¼1

the trapezoids were calculated. L and l indicate the upper and lower lengths of the trapezoids. The average length (Length) P of the SP in a coronal brain slice was n calculated as The average i¼1 ðLi þ liÞ=2 (Fig. 1B). P n thickness of the SP was calculated as i¼1 Si=Length (Fig. 1B). The SP area was divided by the neocortical area to calculate the proportion of the SP in the neocortex in each section. Immunohistochemistry Mice of either sex (P2–P32) were perfused intracardially with cold phosphate-buffered saline (PBS, pH 7.4) and cold 4% paraformaldehyde (PFA) in PBS (pH 7.4). The dissected brains were fixed in 4% PFA overnight, washed in PBS and sectioned coronally (60 lm) using a vibratome (VT1000S, Leica, Nusslosh, Germany). After blocking in PBS containing 5% bovine serum albumin, 0.5% Triton X-100 and 0.05% sodium azide for 1.5–2 h, sections were incubated with the primary antibodies (diluted in PBS containing 1% bovine serum albumin, 0.5% Triton X-100 and 0.05% sodium azide solution) for 36–48 h at 4 °C. After five washes in PBST (0.1% Triton X-100 in PBS) for 10 min each, sections were incubated with the appropriate secondary antibodies overnight at 4 °C and subsequently washed five times in PBS for 10 min each. The following primary antibodies were used: goat antiNurr1 (1:100, R&D Systems AB5380, Minneapolis, Minnesota, U.S.A.), chicken anti-GFP (1:1000, Aves #1020, Tigard, Oregon, U.S.A.), rabbit anti-GABA (1:1000, Sigma #A2052), goat anti-SOM (1:500, Santa Cruz #sc-7819, Dallas, Texas, U.S.A.), mouse antiparvalbumin (PV, 1:400, Millipore #MAB1572, Darmstadt, Germany), rabbit anti-NPY (1:1000, Immunostar #22940, Hudson, Wisconsin, U.S.A.), rabbit

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G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

A

Merge

Nurr1

DAPI

DAPI/Nurr1

L6 MC

SP WM L6

SC

SP WM L6

VC

SP WM

L1

SP

L2

S1

S2 l2

l1

C 70

Thickness (µm)

60

* ***

n

***

*

Length =

L3 S3 l3

MC SC VC

50 40 30 20 10 0

P2

P14

(Li +l i )/2 n i =1

D

***

i =1

Thickness =

P32

6 Percentage of SP area in cortex (%)

B

Si /Length

*** *** *

MC SC VC

5 4

***

3

n.s.

**

2 1 0

P2

P14

P32

Fig. 1. Developmental changes in the cortical subplate layer. (A) Coronal sections of MC, SC and VC from CD-1 mice at P32 were stained with DAPI and Nurr1 to distinguish the subplate. The subplate distinguished from DAPI staining is identical to that labeled by Nurr1. The area in the white rectangle (left figure) is magnified in the right three figures. The broken white lines indicate the borders of subplate. L6, layer 6; SP, subplate; WM, white matter. Scale bars (left) = 200 lm; scale bars (right) = 40 lm. (B) The formula for calculating the thickness of the subplate. S: the area of the trapezoid; L: length of the upper line; l: length of the lower line. (C) Developmental changes in the thickness of the subplate in the postnatal neocortex. The subplate thickness did not change markedly during development. In SC, the subplate at P14 (45.39 ± 1.88 lm) was thicker than at P2 (38.21 ± 1.84 lm) and P32 (37.62 ± 1.94 lm). (D) The relative area of the subplate in the cortex decreased from P2 to P14, but stabilized between P14 and P32 in all three cortical regions. MC, motor cortex; SC, somatosensory cortex; VC, visual cortex; P, postnatal. *p < 0.05; ** p < 0.01; ***p < 0.001; n.s.: not significant.

anti-neuronal nitric oxide synthase (nNOS, 1:1000, Millipore #AB5380, Darmstadt, Germany), mouse anti-calbindin (CB, 1:5000, Swant #300, Bellinzona, Ticino, Switzerland), rabbit anti-GABA-A receptor subunit delta (GABAARd) (1:100, Millipore #AB9752, Darmstadt, Germany), rabbit anti-calretinin (CR, 1:1000, Millipore #AB5054, Darmstadt, Germany), goat anti-CR (1:1000, Millipore #AB1550, Darmstadt, Germany), mouse anti-Reelin (1:1000, Millipore #MAB5364, Darmstadt, Germany), and rabbit anti-vasoactive intestinal peptide (VIP, 1:400, Immunostar #22700,

Hudson, Wisconsin, U.S.A.). The secondary antibodies used were: donkey anti-goat (conjugated to Alexa Fluor 488 and Alexa Fluor 555, 1:500, Invitrogen, Carlsbad, California, U.S.A.), donkey anti-rabbit (Alexa Fluor 488 and Alexa Fluor 555, 1:500, Invitrogen, Carlsbad, California, U.S.A.), donkey anti-mouse (Alexa Fluor 488 and Alexa Fluor 555, 1:500, Invitrogen, Carlsbad, California, U.S.A.), and donkey anti-chicken (Alexa Fluor 488, 1:500, Jackson ImmunoResearch, West Grove, Pennsylvania, U.S.A.). All sections were counterstained with DAPI.

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Slides were then mounted and imaged with an Olympus BX41 epifluorescence microscope with a 10 objective. Photos of immunostained sections were statistically analyzed using Photoshop CS5 (Adobe system, San Jose, California, U.S.A.). Confocal images for publication were obtained by an Olympus FV1000 confocal microscope with 10 and 40 objectives. Digital images were brightness, contrast, and color balanced with Photoshop CS5 (Adobe Systems, San Jose, California, U.S.A.). Cell quantification The SP area in each coronal section was calculated with Photoshop CS5 (Adobe Systems, San Jose, California, U.S.A.). The SP size was calculated as the area multiplied by slice thickness (60 lm). The number of analyzed cells was divided by the SP size to obtain the cell density in each slice. The density of studied cells was compared between different ages and different cortical regions. Owing to the difficulty of detecting all markers in the same cortical slices, the proportions of neuron subtypes were calculated with the mean densities in such cases (Tables 3–5). Statistical analysis All data are presented as mean ± standard error of mean (SEM). Statistical analyses were performed using SigmaPlot (Systat Software, San Jose, California, U.S.A.). Statistical comparisons between two groups were analyzed by the Student’s paired t-test. p-Value <0.05 was considered statistically significant.

Developmental changes in the cortical SP layer Given that developmental changes in the SP layer have not been clearly determined (Robertson et al., 2000), we began by systematically investigating the dynamic development of SP thickness in three distinct cortical subregions—MC, SC, and VC—in CD-1 and GAD67-GFP mice during the first postnatal month. The laminar borders of the SP were identified on the basis of cell distribution with DAPI staining (Fig. 1A, also see Experimental procedures). Briefly, the SP is a compact layer between layer 6 and WM (Fig. 1A, also see Experimental procedures). In order to simplify its profile, we contoured the SP with a series of trapezoids (Fig. 1B). The average P thickness of the SP was calculated with the equation, ni¼1 Si=Length, as Table 1. Summary of subplate thickness (lm)

P2

56.34 ± 3.21 (n = 14 mice) P14 60.05 ± 2.89 (n = 16 mice) P32 60.12 ± 2.85 (n = 25 mice)

Data are mean ± SEM.

MC P2

4.99 ± 0.49 (n = 14 mice) P14 2.53 ± 0.23 (n = 16 mice) P32 2.53 ± 0.15 (n = 25 mice)

SC

VC

5.03 ± 0.42 (n = 10 mice) 2.68 ± 0.11 (n = 17 mice) 2.22 ± 0.18 (n = 10 mice)

3.76 ± 0.37 (n = 16 mice) 1.76 ± 0.08 (n = 16 mice) 1.78 ± 0.12 (n = 12 mice)

Data are mean ± SEM.

described in Fig. 1B and Experimental procedures. Although the thickness of the SP in SC at postnatal day 14 (P14) was slightly thicker than at P2 and P32, overall, SP thickness showed no significant change during development (Fig. 1C, Table 1, n P 10 mice per group). Moreover, we found that the SP was thickest in MC, followed by SC and then VC (Fig. 1C, Table 1). To determine the relative area of the SP in the cerebral cortex, we analyzed the SP area as a proportion of the distinct cortical subregions during postnatal development (Fig. 1D) and found that it decreased dramatically from P2 (4.99 ± 0.49% in MC, n = 14; 5.03 ± 0.42% in SC, n = 10; 3.76 ± 0.37% in VC, n = 16) to P14 (2.53 ± 0.26% in MC, n = 16; 2.68 ± 0.11% in SC, n = 17; 1.76 ± 0.83% in VC, n = 16; p < 0.001). However, the proportion of SP area in the cortical subregions at P32 (2.53 ± 0.15% in MC, n = 25; 2.22 ± 0.18% in SC, n = 10; 1.78 ± 0.12% in VC, n = 12) was not significantly different from that at P14. We also found that the SP occupied the smallest area in VC (Fig. 1D, Table 2).

Three major groups of GABAergic interneurons in the SP

RESULTS

MC

Table 2. The relative subplate area in the cerebral cortex (%)

SC

VC

38.21 ± 1.84 (n = 10 mice) 45.39 ± 1.88 (n = 17 mice) 37.62 ± 1.94 (n = 10 mice)

17.62 ± 0.60 (n = 16 mice) 18.29 ± 0.97 (n = 16 mice) 19.45 ± 0.81 (n = 12 mice)

To determine the subtypes of SP interneurons, we analyzed the expression of three markers, SOM, 5HT3AR and PV, that are universally used to classify cortical interneurons (Rudy et al., 2011; Petersen, 2014). We immunostained SOM and PV in coronal brain sections of GAD67-GFP mice, and GABA in coronal brain sections of 5HT3AR-GFP mice. Our results showed that while SOM and PV were expressed in GAD67-GFP+ neurons, and almost all 5HT3AR+ neurons expressed GABA in the SP (Fig. 2A), SOM, PV and 5HT3AR were not coexpressed (Fig. 2B). These results indicated that SOM+, 5HT3AR+ and PV+ neurons are three nonoverlapping groups of SP interneurons. We found that the density of SP interneurons decreased dramatically during the first two postnatal weeks, but stabilized after P14 (38405.80 ± 1121.76 lm3 in MC, 34937.42 ± 1058.12 lm3 in SC, 51246.97 ± 2209.86 lm3 in VC, at P2; 10333.64 ± 230.92 lm3 in MC, 9700.34 ± 209.55 lm3 in SC, 13257.43 ± 950.05 lm3 in VC, at P14) (Fig. 3A1–3; p < 0.001; n P 3 mice for each group). Furthermore, the density of SP interneurons differed significantly between the three cortical subregions during the first two postnatal weeks, but the difference disappeared after P16 (Fig. 3B1). The densities of 5HT3AR+ and SOM+ interneurons decreased dramatically during the first two postnatal weeks, but stabilized after P14;

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G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

A

SOM

GAD67-GFP

DAPI

Merge L6 SP WM

PV

GAD67-GFP

DAPI

Merge L6 SP WM

GABA

5HT3AR-GFP

DAPI

Merge L6 SP WM

B

SOM

5HT3AR-GFP

DAPI

Merge L6 SP WM

PV

5HT3AR-GFP

DAPI

Merge L6 SP WM

PV

SOM

DAPI

Merge L6 SP WM

Fig. 2. Three major groups of GABAergic interneurons in the subplate. (A) GAD67-GFP+ interneurons expressed SOM (top panel, arrowheads) and PV (middle panel, arrowheads) in the subplate (SC, P32). 5HT3AR-GFP+ interneurons expressed GABA in the subplate (SC, P32, bottom panel, arrowheads). The broken white lines indicate the SP borders. L6, layer 6; SP, subplate; WM, white matter. Scale bars = 40 lm. (B) Neither SOM+ (top panel, empty arrowheads) nor PV+ (middle panel, empty arrowheads) subplate interneurons expressed 5HT3AR (SC, P32). PV+ interneurons did not express SOM in the subplate (SC, P32, bottom panel, empty arrowheads). The broken white lines indicate the SP borders. Scale bars = 40 lm.

whereas PV+ SP interneurons emerged at P16 and increased gradually with development (Fig. 3B2–4). Although the proportions of the three markers varied to some degree at specific time points, we found that 5HT3AR+ interneurons were the most abundant in the

SP overall (Table 3). At P32, neurons expressing SOM, 5HT3AR and PV account for nearly 100% of SP interneurons (Fig. 3C). The proportions of SOM+, 5HT3AR+ and PV+ interneurons were
30%,
60% and
10%, respectively, of the total SP interneurons (Fig. 3C).

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10

Density (1000/mm3)

P32

**

10 8 6 4

** *

**

***

2 0

P32 P24 P20 P16 P14 P12 P10 P8 P6 P4 P2

**

10

3

P32 P24 P20 P16 P14 P12 P10 P8 P6 P4 P2

MC SC VC

P24

10

SOM

14 12

P20

20

0

P32 P24 P20 P16 P14 P12 P10 P8 P6 P4 P2

PV

12

*** ** * *** ** *** *** * * * *** *** ** ** ** * *** * * * * * *** ** * * * * * *

B3

P16 P14 P12 P10 P8 P6

0

5HT3AR

P4

10

B4

** **

30

0

P2

20

30

10

0

40

*** ** *** * * * * * ** * **

40

P32 P24 P20 P16 P14 P12 P10 P8 P6 P4

*** ** *

GAD67-GFP 5HT3AR-GFP SOM PV

50

20

10

B2

Interneuron

30

Density (100/mm3)

20

P2

*** ** *** * ** 50 * 60

40

30

P32 P24 P20 P16 P14 P12 P10 P8 P6 P4 P2

B1

Visual cortex

60

Density (1000/mm )

20

A3

3

Density (1000/mm3)

30

0

Somatosensory cortex

40

Density (1000/mm3)

Density (1000/mm3)

A2

Motor cortex

40

Density (1000/mm )

A1

C PV (~10%)

8

5HT3AR

6

(~60%)

4

SOM (~30%)

2 0

P16

P20

P24

P32

Fig. 3. The neurochemical composition of subplate interneurons during postnatal development. (A) The densities of GAD67-GFP+, 5HT3AR+, SOM+ and PV+ interneurons in the subplate of MC (A1), SC (A2) and VC (A3) from P2 to P32. (B1) The densities of subplate interneurons were compared in MC, SC and VC from P2 to P32. Subplate interneurons were preferentially distributed in VC during the first two postnatal weeks. (B2) The densities of 5HT3AR+ interneurons were compared across MC, SC and VC from P2 to P32. 5HT3AR+ interneurons were preferentially distributed in VC from P2 to P32. (B3–4) The densities of SOM+ (B3) and PV+ interneurons (B4) were compared across MC, SC and VC from P2 to P32. *p < 0.05; **p < 0.01; ***p < 0.001. (C) At P32, neurons expressing SOM, 5HT3AR and PV account for nearly 100% of subplate interneurons. The percentages of 5HT3AR+, SOM+ and PV+ in the subplate interneuron population were about 60%, 30% and 10%, respectively.

The neurochemical composition of SOM+ interneurons during postnatal development Having clarified the three major groups of SP interneurons, we subsequently studied the expression of other markers in each group at representative time points (P2, P8, P14 and P32). We found that NPY, nNOS and CB were exclusively expressed in SOM+ SP neurons (Fig. 4A). Moreover, CB was not coexpressed with nNOS (Fig. 4B), indicating that nNOS+ and CB+ neurons are two distinct subgroups of SOM+ SP interneurons. As shown in Fig. 5A1–3, the density of NPY+/SOM+ SP interneurons decreased greatly from P2 to P32 (n P 3 mice for each group) in MC, SC and VC. The proportion of SOM+ interneurons expressing NPY+ increased markedly during the first 2 weeks after birth, with nearly all SOM+ SP interneurons expressing NPY after P14 (Fig. 5A1–3,

Table 4). The density of nNOS+/SOM+ interneurons significantly decreased from P2 to P32 (Fig. 5A1–3, Table 4), and showed no obvious regional variance barring their significantly lower density in MC versus SC and VC at P32 (Fig. 5B2). The proportion of SOM+ interneurons expressing nNOS increased markedly from P2 to P8, then remained relatively constant (Table 4). The expression of CB in SP interneurons started at P24 and increased gradually (Fig. 5B3). At P32, the density of CB+/SOM+ interneurons was significantly higher in MC than VC, contrary to nNOS+/SOM+ interneurons (Fig. 5B3). To account for regional variations, we averaged the proportions across different cortical regions. We found nearly all SOM+ SP interneurons expressed NPY at P32, while
60% expressed nNOS and
40% expressed CB (Fig. 5C).

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Table 3. The densities of 5HT3AR+, SOM+ and PV+ interneurons in subplate Cortical region

Age

Interneuron (n P 3 mice) 3

5HT3AR (n P 3 mice) 3

SOM (n P 3 mice)

Density (No./mm )

Density (No./mm )

Proportion of interneurons (%)

3

Density (No./mm )

PV (n P 3 mice) Proportion of interneurons (%)

Density (No./mm3)

Proportion of interneurons (%)

P2 P4 P6 P8 P10 P12 P14 P16 P20 P24 P32

38405.80 ± 1121.76 31599.22 ± 2407.53 20446.45 ± 918.55 18024.55 ± 739.12 12894.68 ± 623.02 12791.89 ± 617.81 10333.64 ± 230.92 10067.71 ± 578.55 9709.43 ± 443.26 8987.56 ± 548.87 8641.63 ± 378.78

26813.83 ± 1765.79 19387.94 ± 1405.58 12664.57 ± 857.54 9500.96 ± 761.99 7596.18 ± 223.29 7449.79 ± 194.73 6844.43 ± 598.01 6030.40 ± 325.77 5987.77 ± 542.13 5342.35 ± 619.46 4954.58 ± 162.13

69.82 61.36 61.94 52.71 58.91 58.24 66.23 59.90 61.67 59.44 57.33

9724.30 ± 262.15 9448.42 ± 988.55 5821.47 ± 280.71 4435.03 ± 146.28 3576.90 ± 172.33 3590.04 ± 284.47 2956.72 ± 185.06 3010.17 ± 423.87 2727.32 ± 731.21 2606.05 ± 345.56 2425.32 ± 154.74

25.32 29.90 28.47 24.61 27.74 28.06 28.61 29.90 28.09 29.00 28.07

None None None None None None None 146.06 ± 146.06 191.58 ± 191.58 642.10 ± 252.30 913.34 ± 77.20

None None None None None None None 1.45 1.97 7.14 10.57

SC

P2 P4 P6 P8 P10 P12 P14 P16 P20 P24 P32

34937.42 ± 1058.12 31793.28 ± 1371.31 20360.34 ± 1265.50 17134.28 ± 844.09 12949.75 ± 357.08 11597.64 ± 692.00 9700.34 ± 209.55 9708.69 ± 398.35 9272.29 ± 584.09 9139.61 ± 679.57 8114.29 ± 248.47

23114.09 ± 1023.82 13776.03 ± 610.07 12301.73 ± 399.32 8424.15 ± 597.59 7637.30 ± 136.16 6641.44 ± 539.18 5645.80 ± 432.55 5041.06 ± 265.99 4806.23 ± 491.29 4666.19 ± 534.81 4706.48 ± 348.67

66.16 43.33 60.42 49.17 58.98 57.27 58.20 51.92 51.83 51.05 58.00

10555.62 ± 646.94 8094.72 ± 622.64 5995.94 ± 655.04 4886.69 ± 369.27 4658.92 ± 668.56 4296.85 ± 514.58 3060.32 ± 165.15 3238.82 ± 313.74 3224.87 ± 704.50 2709.38 ± 293.88 2313.69 ± 143.90

30.21 25.46 29.45 28.52 35.98 37.05 31.55 33.36 34.78 29.64 28.51

None None None None None None None 481.09 ± 315.38 627.82 ± 306.49 704.26 ± 61.99 980.03 ± 112.01

None None None None None None None 4.96 6.77 7.71 12.08

VC

P2 P4 P6 P8 P10 P12 P14 P16 P20 P24 P32

51246.97 ± 2209.86 45223.93 ± 1079.18 32242.85 ± 2609.23 24639.48 ± 937.68 19224.27 ± 1640.59 15196.04 ± 819.88 13257.43 ± 950.05 11320.60 ± 871.10 9918.17 ± 405.78 9462.72 ± 483.60 8484.04 ± 288.85

33938.37 ± 1778.90 25040.46 ± 822.36 19095.63 ± 793.95 15138.10 ± 685.78 14595.82 ± 829.62 11907.58 ± 1124.98 11716.46 ± 650.32 8709.86 ± 438.87 7779.50 ± 617.71 7341.12 ± 569.02 6002.06 ± 242.59

66.23 55.37 59.22 61.44 75.92 78.36 88.38 76.94 78.44 77.58 70.75

11556.54 ± 810.94 6745.29 ± 556.13 5940.85 ± 249.23 4483.69 ± 396.76 3978.79 ± 692.15 3624.44 ± 409.94 2478.79 ± 197.36 2709.98 ± 444.88 2194.67 ± 200.71 1888.92 ± 199.11 1731.74 ± 324.72

22.55 14.92 18.43 18.20 20.70 23.85 18.70 23.94 22.13 19.96 20.41

None None None None None None None 279.07 ± 200.85 787.90 ± 248.17 619.86 ± 149.58 1070.48 ± 74.07

None None None None None None None 2.47 7.94 6.55 12.62

Data are mean ± SEM.

G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

MC

85

G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

A

SOM

DAPI

Merge

NPY

L6 SP WM

nNOS

L6 SP WM

CB

L6 SP WM

B

CB

n-NOS

DAPI

Merge L6 SP WM

Fig. 4. Subtypes of SOM+ interneurons in the subplate. (A) SOM+ subplate interneurons were positive for NPY (top panel, arrowheads), nNOS (middle panel, arrowheads) and CB (bottom panel, arrowheads) (SC, P32). The broken white lines indicate the SP borders. Scale bars = 40 lm. (B) CB expression did not colocalize with that of nNOS in subplate interneurons (SC, P32). Empty arrowheads indicate the CB+ subplate interneuron negative for nNOS. The broken white lines indicate the SP borders. Scale bars = 40 lm.

The neurochemical composition of 5HT3AR+ interneurons during postnatal development We next investigated 5HT3AR+ interneurons in the SP. To determine the subtypes of 5HT3AR+ interneurons in the developing SP, we immunostained coronal cortical sections from 5HT3AR-EGFP mice with antibodies against GABAARd and CR (Fig. 6A). Although only a small proportion of 5HT3AR+ interneurons expressed GABAARd and CR prior to P14, the vast majority of GFP+ neurons (5HT3AR+ interneurons) were positive for them at P32 (Fig. 6C1–3, Table 5). We found that CR was not coexpressed with GABAARd in SP interneurons (Fig. 6B), indicating that CR+ and GABAARd+ interneurons are two different subtypes of 5HT3AR+ interneurons in the SP. The density of GABAARd+/5HT3AR+ interneurons increased from P2 to P8 in the three different cortical regions and then declined (Fig. 6C1–3, Table 5). The proportion of GABAARd+/5HT3AR+ interneurons increased significantly in the cortical subregions during postnatal development (Table 5). In contrast, CR+ interneurons were rarely found in the SP from P2 to P12 (Fig. 6D, Table 6). The density of CR+/5HT3AR+ interneurons rose sharply around P14, and were much higher in MC and SC than in VC (Fig. 6D, Table 6). At P32, the

proportions of 5HT3AR+ SP interneurons that expressed GABAARd+ were 39.83 ± 1.42% in MC (n = 3 mice), 49.15 ± 2.10% in SC (n = 4 mice) and 49.76 ± 1.54% in VC (n = 4 mice). The proportions of 5HT3AR+ SP interneurons that expressed CR were 51.84 ± 4.54% in MC (n = 4 mice), 52.27 ± 4.81% in SC (n = 3 mice) and 13.12 ± 0.97% in VC (n = 3 mice). Together, our data indicated that at P32, about half of the 5HT3AR+ interneuron population expressed GABAARd, and the rest expressed CR (Fig. 6E). The neurochemical composition of PV+ interneurons during postnatal development Examination of the interneuron subtype markers revealed that the PV+ SP interneurons only coexpressed GABAARd (Fig. 7), and were present in all cortical subregions after P16 (see Table 3). The expression of GABAARd in non-GABAergic SP neurons Consistent with a previous report (Hoerder-Suabedissen et al., 2013), we found that GABAARd-expressing neurons were densely packed in the SP during postnatal development (Fig. 8A, B). Moreover, the density of

G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

6

*

B1 10

P8

*

* *

6 4 2

C

P2

3

L6

2 P2

SP P14

P8

P8

P14

P32

14 12 10 8 6

***

***

* *

2 1

P2

P8

P14

*

4 2 0

P32

SOM NPY nNOS CB

*

P2

P8

P14

P32

CB

25

3

*

*

B3

nNOS

4

0

Visual cortex

P32

5

8

0

4 0

A3

*

WM

*

6

B2

NPY

***

8

P32

P14

*** *

12

2 P2

Somatosensory cortex

10

4

0

Density (1000/mm3)

* *

Density (1000/mm3)

8

*** *** ***

Density (1000/mm3)

Density (1000/mm3)

10

A2

Motor cortex

Density (100/mm3)

A1

Density (1000/mm )

86

*

20

MC SC VC

15 10 5 0

P2-P20

P24

P32

SOM+ interneuron

nNOS (~60%)

CB (~40%)

NPY (~100%)

Fig. 5. The neurochemical composition of SOM+ interneurons in the subplate during postnatal development. (A) The densities of SOM+, NPY+, nNOS+ and CB+ subplate interneurons in MC (A1), SC (A2) and VC (A3) during postnatal development. Asterisks indicate statistical significance between adjacent time points. *p < 0.05; **p < 0.01; ***p < 0.001. (B1) The density of subplate NPY+ interneurons in VC was significantly lower than in other cortical regions at P2. (B2) The density of subplate nNOS+ interneurons in MC was significantly lower than in other cortical regions at P32. (B3) Subplate interneurons started to express CB at P24, with expression increasing gradually with age. CB+ interneurons were preferentially distributed in MC at P32. *p < 0.05; **p < 0.01; ***p < 0.001. (C) Nearly all SOM+ subplate interneurons expressed NPY at P32, while
60% expressed nNOS and
40% expressed CB.

Table 4. The densities of NPY+, nNOS+ and CB+ interneurons in subplate Cortical Age SOM region (n P 3 mice) Density (No./mm3)

NPY (n P 3 mice) Density (No./mm3)

Proportion Density of SOM (%) (No./mm3)

MC

P2 P8 P14 P32

7548.13 ± 374.29 3556.69 ± 156.09 3275.50 ± 216.76 2423.48 ± 204.02

77.62 80.20 110.78 99.92

3371.96 ± 321.61 2616.31 ± 308.54 1814.25 ± 202.79 1194.72 ± 67.97

34.68 58.99 61.36 49.26

None None None 1744.57 ± 294.67

None None None 71.93

SC

P2 10555.62 ± 646.94 8202.77 ± 1237.94 77.71 P8 4886.69 ± 369.27 3522.46 ± 378.74 72.08 P14 3060.32 ± 165.15 2823.82 ± 234.83 92.27 P32 2313.69 ± 143.90 2597.72 ± 217.22 112.28

3720.21 ± 211.56 3164.00 ± 619.38 1985.69 ± 132.87 1677.51 ± 163.78

35.24 64.75 64.89 72.50

None None None 1062.23 ± 83.48

None None None 45.91

VC

P2 11556.54 ± 810.94 5021.51 ± 497.25 P8 4483.69 ± 396.76 3127.46 ± 141.85 P14 2478.79 ± 197.36 2860.87 ± 322.95 P32 1731.74 ± 324.72 1763.82 ± 206.85

4268.26 ± 346.09 36.93 3056.80 ± 444.43 68.18 2344.62 ± 168.17 94.59 1867.28 ± 273.00 107.83

None None None 628.41 ± 335.21

None None None 36.29

9724.30 ± 262.15 4435.03 ± 146.28 2956.72 ± 185.06 2425.32 ± 154.74

Data are mean ± SEM.

nNOS (n P 3 mice)

43.45 69.75 115.41 101.85

CB (n P 3 mice) Proportion Density of SOM (%) (No./mm3)

Proportion of SOM (%)

87

G.-J. Qu et al. / Neuroscience 322 (2016) 78–93

A

DAPI

5HT3AR-GFP

Merge

GABAARδ

L6 SP WM

CR

L6 SP WM

B

GABAARδ

DAPI

CR

Merge L6 SP WM

30

*** *** ***

25

3

*

20 15 10 5 0

P2

P8

P14

20

40

**

***

15 10 5 0

P32

C3

Somatosensory cortex

*** ***

25 Density (1000/mm )

Density (1000/mm3)

C2

Motor cortex

P2

P8

P14

P32

Density (1000/mm3)

C1

Visual cortex

***

30

***

CR

3

Density (1000/mm )

4

MC SC VC

3

***

**

**

20 10 0

P2

5HT3AR

D

**

P8

P14

GABAARδ

P32 CR

E

**

5HT3AR+ interneuron

*

2

GABAARδ (~50%)

CR (~50%)

1

P24

P32

P20

P16

P12

P14

P10

P6

P8

P2

P4

0

Fig. 6. The neurochemical composition of 5HT3AR+ interneurons in the subplate during postnatal development. (A) Coronal sections of SC from 5HT3AR-GFP transgenic mice were stained for GABAARd (top panel, solid arrowhead; empty arrowheads indicate GABAARd+ interneurons that were negative for GFP in the subplate) and CR (bottom panel, solid arrowheads) at P32. The broken white lines indicate the SP borders. Scale bars = 40 lm. (B) GABAARd expression did not colocalize with that of CR in subplate interneurons (SC, P32). Empty arrowheads indicate the CR+ subplate interneuron negative for GABAARd. The broken white lines indicate the SP borders. Scale bars = 40 lm. (C) The densities of 5HT3AR+, GABAARd+ and CR+ subplate interneurons in MC (C1), SC (C2) and VC (C3) during postnatal development. Asterisks indicate statistical significance between adjacent time points. *p < 0.05; **p < 0.01; ***p < 0.001. (D) The density of CR+ subplate interneurons was compared across MC, SC and VC from P2 to P32. CR+ interneurons were rare in the early postnatal days but the density increased sharply around P14 and was significantly lower in VC compared with MC, SC. Asterisks indicate statistical significance between adjacent time points. *p < 0.05; **p < 0.01. (E) 5HT3AR+ interneurons in the subplate were comprised of GABAARd and CB expressing interneurons. Averaging the proportions across different cortical subregions, GABAARd+ and CR+ interneurons each constituted roughly half of the subplate 5HT3AR+ interneuron population at P32.

GABAARd+ cells in the SP was significantly higher than in the adjacent cortical layer 6 (29399 ± 992 mm3 in the SP, n = 4 mice; 6402 ± 460 mm3 in layer 6, n = 4

mice; VC, P14) (Fig. 8C). These data indicated that GABAARd marker can serve as a specific marker to identify the SP in the neocortex. To determine whether the

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Table 5. The densities of GABAARd+ and CR+ interneurons in subplate Cortical region

Age

5HT3AR (n P 3 mice) 3

GABAARd (n P 3 mice) 3

CR (n P 3 mice)

Density (No./mm )

Density (No./mm )

Proportion of 5HT3AR (%)

Density (No./mm3)

Proportion of 5HT3AR (%)

MC

P2 P8 P14 P32

26813.83 ± 1765.79 9500.96 ± 761.99 6844.43 ± 598.01 4954.58 ± 162.13

2269.05 ± 322.60 3801.35 ± 597.06 3164.43 ± 672.65 1858.49 ± 105.95

8.46 40.01 46.23 37.51

1012.65 ± 273.71 293.48 ± 293.48 1505.25 ± 456.68 2478.33 ± 274.68

3.78 3.09 21.99 50.02

SC

P2 P8 P14 P32

23114.09 ± 1023.82 8424.15 ± 597.59 5645.80 ± 432.55 4706.48 ± 348.67

1792.08 ± 135.59 2688.93 ± 72.87 2732.60 ± 86.40 2179.33 ± 138.62

7.75 31.92 48.40 46.30

916.06 ± 341.08 280.03 ± 124.12 1731.60 ± 334.66 2295.76 ± 64.90

3.96 3.32 30.67 48.78

VC

P2 P8 P14 P32

33938.37 ± 1778.90 15138.10 ± 685.78 11716.46 ± 650.32 6002.06 ± 242.59

2998.94 ± 577.16 6459.80 ± 457.39 3969.01 ± 100.28 3064.12 ± 354.23

8.84 42.67 33.88 51.05

466.81 ± 238.43 205.11 ± 205.11 224.71 ± 102.07 761.32 ± 97.31

1.38 1.35 1.92 12.68

Data are mean ± SEM.

Table 6. The densities of CR+ interneurons (No./mm3)

P2 P4 P6 P8 P10 P12 P14 P16 P20 P24 P32

MC (n P 3 mice)

SC (n P 3 mice)

VC (n P 3 mice)

1012.65 ± 273.71 395.77 ± 171.86 382.58 ± 96.03 293.48 ± 293.48 433.74 ± 76.42 824.32 ± 170.52 1505.25 ± 456.68 3303.68 ± 441.12 3089.12 ± 171.74 2926.08 ± 297.39 2478.33 ± 274.68

916.06 ± 341.08 256.97 ± 127.56 193.24 ± 64.63 280.03 ± 124.12 668.70 ± 84.47 493.51 ± 27.80 1731.60 ± 334.66 2689.18 ± 609.71 2492.14 ± 197.45 2256.08 ± 328.20 2295.76 ± 64.90

466.81 ± 238.43 292.62 ± 146.91 325.71 ± 310.71 205.11 ± 199.11 198.26 ± 89.57 376.54 ± 159.00 224.71 ± 102.07 511.97 ± 301.36 712.46 ± 258.29 595.65 ± 215.66 761.32 ± 97.31

Data are mean ± SEM.

GABAARδ

PV

DAPI

Merge L6

SP WM

Fig. 7. PV+ subplate interneurons only coexpress GABAARd. All PV+ subplate interneurons were found to express GABAARd (SC, P32). PV+ interneurons were positive for GABAARd (solid arrowheads; empty arrowheads indicate GABAARd+ interneurons that were negative for PV in the subplate). The broken white lines indicate the SP borders. Scale bar = 40 lm.

majority of GABAARd+ cells in the SP were interneurons, as in other cortical layers (Ma et al., 2014), we examined the colocalization of GABAARd and GFP in GAD67-GFP mice (Fig. 8A, B). Interestingly, we found that only a small population of GABAARd+ cells in the SP expressed GFP in VC at P14 (13.15 ± 0.45%, n = 4 mice), whereas the majority of GABAARd+ cells in cortical layer 6 were labeled by GFP in VC at P14 (94.12 ± 1.40%, n = 4 mice) (Fig. 8D). These data indicated that, contrary to those in other cortical layers, the majority of GABAARd+ cells in the SP were non-GABAergic neurons.

We also looked for expression of VIP and Reelin in the SP but did not detect them (Fig. 9A, B).

DISCUSSION Postnatal development of the SP layer in the neocortex The anatomical architecture and developmental changes of the SP are markedly different across species. Previous studies showed that a large number of SP neurons

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A

GABAARδ

DAPI

GAD67-GFP

Merge L1 L2/3 L4 L5

L6 SP WM

B

GABAARδ

DAPI

GAD67-GFP

Merge L6 SP WM

35 30 25 20 15 10 5 0

***

D

SP

L6

***

100 Percentage (%)

3

Density (1000/mm )

C

80 60 40 20 0

SP

L6

Fig. 8. The expression of GABAARd in non-GABAergic subplate neurons. (A) Coronal sections of VC from GAD67-GFP knock-in mice were immunostained with GABAARd (P14). The broken white lines indicate the SP borders. Scale bar = 100 lm. (B) Magnification of the subplate region in (A). Interneurons (GFP+) in cortical layer 6 expressed GABAARd (solid arrowheads), although many GABAARd-expressing neurons in the subplate were non-GABAergic (GFP) (empty arrowheads). The broken white lines indicate the SP borders. L1, layer 1; L2/3, layer 2 and Layer 3; L4, layer 4; L5, layer 5; L6, layer 6. Scale bar = 40 lm. (C) The density of GABAARd+ interneurons in the subplate was higher than that in cortical layer 6 (VC, P14). ***p < 0.001. (D) GABAARd+/GFP+ neurons as a percentage of the total GABAergic neurons in the subplate and cortical layer 6. The percentage in the subplate was significantly lower than in cortical layer 6 (VC, P14). ***p < 0.001.

undergo apoptosis and the SP disappears gradually during late embryonic and early postnatal life in cats (Luskin and Shatz, 1985; Chun and Shatz, 1989b), monkeys (Kostovic and Rakic, 1980, 1990; HoerderSuabedissen and Molna´r, 2015), and humans (Kostovic and Rakic, 1980, 1990; Hoerder-Suabedissen and Molna´r, 2015), and the surviving SP neurons disperse into WM (Kostovic and Rakic, 1980, 1990; Chun and Shatz, 1989b; Hoerder-Suabedissen and Molna´r, 2015). In contrast, the SP persists in adult rodents (Woo et al., 1991; Valverde et al., 1995b; Reep, 2000; Robertson et al., 2000; Hoerder-Suabedissen and Molna´r, 2015). However, little has been known about the postnatal development of SP thickness in rodents (Robertson et al., 2000). In this study, we investigated dynamic development of SP thickness in distinct cortical subregions during the first postnatal month. We found that, although the relative area occupied by the SP in the cerebral cortex significantly declined with postnatal development, its absolute thickness did not change markedly.

The developmental profile and subregional distribution of SP interneurons in the cortex during the first postnatal month were systematically analyzed in this study. We demonstrated a dramatic decrease in the density of total SP interneurons in the first two postnatal weeks. Several reasons could underlie such a decrease. The first reason could be that late-born interneurons migrate through the SP into other neocortical layers during the early postnatal period (De Marco Garcı´ a et al., 2011; Miyoshi and Fishell, 2011; Inamura et al., 2012). Alternatively, cell apoptosis was observed in the SP during late embryonic and early postnatal development (Chun and Shatz, 1989a; McConnell et al., 1989; Spreafico et al., 1995; Torres-Reveron and Friedlander, 2007), but whether SP interneurons undergo cell death remains unknown. Furthermore, the SP in rats is remarkably expanded in the medial–lateral and anterior–posterior axes during postnatal development (Robertson et al., 2000). Therefore, we speculate that the decrease in the density of SP GABAergic interneurons in early postnatal

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A

GFP

VIP

DAPI

Merge L6 SP WM

B

GFP

Reelin

DAPI

Merge L6 SP WM

Fig. 9. VIP and Reelin expression do not colocalize in subplate interneurons. Coronal sections of SC from GAD67-EGFP knock-in mice (P32) immunostained with VIP (A) and Reelin (B). The subplate interneurons did not express VIP and Reelin. The broken white lines indicate the SP borders. Scale bars = 40 lm.

Subplate Interneurons 0

20

40 60 Percentage (%)

80

100

SOM

5HT3AR

PV

(~30%)

(~60%)

(~10%)

NPY

CR

GABAARδ

(~30%)

(~30%)

(~40%)

nNOS

CB

(~18%)

(~12%)

Fig. 10. Subpopulations of subplate interneurons in the mouse neocortex at P32. At P32, neurons expressing SOM (60%), 5HT3AR (30%) and PV (10%) together account for nearly 100% of subplate interneurons. SOM+ subplate interneurons completely overlapped with NPY+ interneurons, and consisted of nNOS+ and CB+ interneurons. 5HT3AR+ subplate interneurons were comprised of GABAARd+ and CR+ interneuron subtypes. Nearly all PV+ subplate interneurons expressed GABAARd. The corresponding proportions of each interneuron subtype in the total subplate population are indicated.

development results from a combination of interneuron migration, apoptosis, and a dilution effect. Further researches are required to reveal the exact mechanism underlying the decreasing SP interneuron density in development. Distinct composition of SP interneurons In this study, we clearly described the composition and proportion of neurochemical markers in the SP interneurons, as shown in Fig. 10. SOM, 5HT3AR and PV reliably identified three distinct non-overlapping subtypes of SP interneurons. Moreover, SOM+ and 5HT3AR+ interneurons consisted of more than one interneuron subgroup in the mouse neocortex at P32. The SOM group, which represents
30% of total SP interneurons, colocalizes entirely with NPY and expresses nNOS (
18%) and CB (
12%). The 5HT3AR group, which accounts for
60% of total SP

interneurons, expresses CR (
30%) and GABAARd (
30%). The PV group accounts for
10% of total SP interneurons and coexpressed GABAARd (
10%). The proportions of SOM+, 5HT3AR+ and PV+ interneurons in the SP layer clearly differed from other neocortical layers. It has been reported that SOM+, 5HT3AR+ and PV+ interneurons account for nearly 100% of GABAergic neurons in neocortical layers 1–6, accounting for
30%,
30% and
40%, respectively (Rudy et al., 2011). Moreover, 5HT3AR+ interneurons mainly dominate the superficial layers (layer 1 and layer 2/3), while SOM+ and PV+ interneurons harbor in the deep layers (Lee et al., 2010; Rudy et al., 2011). However, although the SP is the deepest layer in the neocortex, we observed that its proportion of 5HT3AR+ interneurons (
60%) is significantly higher than that of SOM+ (
30%) and PV+ (
10%) neurons. Previous studies have shown that 5HT3AR+, SOM+ and PV+ interneuron groups tend to have distinct physiological characteristics and connectivities in the postnatal cortex (Rudy et al., 2011; Petersen, 2014). Our results strongly suggest that interneuron circuitry in the SP is distinctive as compared with other neocortical layers. Besides, it’s generally accepted that cortical SOM+ and PV+ interneurons originate within the medial ganglionic eminence (MGE) (Xu et al., 2004; Butt et al., 2005; Wonders and Anderson, 2006; Fogarty et al., 2007; Ghanem et al., 2007; Rudy et al., 2011), mainly generated from embryonic day 12.5 (E12.5) to E16.5. While 5HT3AR+ interneurons derive from the caudal ganglionic eminence (CGE) (Inta et al., 2008; Lee et al., 2010; Vucurovic et al., 2010; Rudy et al., 2011), generated between E11.5 and E19.5 (Inta et al., 2008; Lee et al., 2010; Vucurovic et al., 2010). Our results suggest that SP interneurons originate from CGE more than MGE, as 5HT3AR+ neurons make up the majority of SP interneurons. The precise origins and birthdates of SP interneurons still require further study. We found that 5HT3AR+ SP interneurons were comprised of GABAARd+ and CR+ subgroups.

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GABAARd is a marker for neurogliaform cells that regulate cortical microcircuits by GABA-mediated volume transmission (Ola´h et al., 2009), and has been found in the hippocampus and neocortex (Ola´h et al., 2009; Ma et al., 2014). Furthermore, it has been reported that GABAARd is an extrasynaptic receptor (Nusser et al., 1998; Moss and Smart, 2001; Fritschy and Bru¨nig, 2003; Farrant and Nusser, 2005; Glykys and Mody, 2007) that receives diffused GABA (Farrant and Nusser, 2005; Brickley and Mody, 2012). Thus, we speculate that GABAARd+ neurogliaform interneurons modulate neural circuits in the SP by volume transmission. As GABAARd is highly expressed in SP interneurons (
40%, Fig. 10), it implies that GABAARd+ neurogliaform cells have important roles in SP function. Interestingly, we also found dense expression of GABAARd+ in excitatory neurons (non-GABAergic neurons, Fig. 8). Their functions in the SP will be further studied. CR+/5HT3AR+ interneurons have been detected in the cerebral cortex (Vucurovic et al., 2010), but their functions are not clear. Besides, it has been reported that the 5HT3AR+ interneuron population partially coexpresses VIP in the other neocortical layers (Petersen, 2014). However, VIP-positive neurons were not detected at any time point in the SP (Fig. 9A). We found SOM+ SP interneurons coexpress NPY, and consisted of nNOS+ and CB+ subgroups in the mouse neocortex at P32. Studies have shown the existence of long-distance projection interneurons in the SP that express SOM, NPY and nNOS (Tomioka et al., 2005; Tamamaki and Tomioka, 2010). In contrast, SP interneurons in ferrets and cats which express SOM, NPY and CB were found to modulate the local neural circuit (Antonini and Shatz, 1990). Taken together, these results indicate that the two non-overlapping subtypes of SOM+ SP interneurons (nNOS+/SOM+ and CB+/ SOM+ interneurons) play different roles in cortical circuits. Our data revealed that PV+ interneurons coexpress GABAARd in the SP. In the hippocampus, these neurons are involved in gamma oscillation (Ferando and Mody, 2015). Further research is needed to address the function of GABAARd+/PV+ interneurons in the SP.

The expression of neurochemical markers in the developing SP Different neurochemical markers show characteristic temporal and spatial expression patterns in the SP. The expression of SOM and NPY starts at embryonic day 16 in the SP (Rı´ o et al., 2000), while PV+ SP interneurons only emerge after the second postnatal week (Fig. 3A1– 3). CR and CB were expressed mainly after P14 (Fig. 6D) and P24 (Fig. 5B3), respectively. The variance in distribution of 5HT3AR+, nNOS+, CR+ and CB+ SP interneurons in different cortical subregions was remarkable. The density of nNOS+ interneurons in the SP followed a decreasing gradient from anterior MC to posterior VC (Fig. 5B2), whereas CR+ and CB+ interneurons exhibited the opposite trend (Figs. 6D and 5B3). The temporal and spatial distributions of SP interneurons indicate that different interneuron subtypes play varied roles during postnatal development.

In sum, we have systematically classified SP interneurons based on molecular markers, and developed a profile of interneuron subtypes in the postnatal neocortex of mice. Furthermore, our study for the first time comprehensively reports on the distribution of various subtypes of SP interneurons in different cortical regions during postnatal development. These findings will underpin future studies of the subgroups of SP interneurons that should greatly contribute to our understanding of SP functions in development.

AUTHOR CONTRIBUTIONS Y.F., Y.-C.Y. and G.-J.Q. conceived the project. G.-J.Q. executed experiments. J.M. performed part of the immunohistochemistry experiments. G.-J.Q. and Y.F. analyzed the data, interpreted the results and wrote the manuscript. All authors edited the manuscript.

CONFLICT OF INTEREST The authors declare no conflict of interest. Acknowledgments—We thank Dr. Lan Ma for providing GAD67GFP (Dneo) mice and Dr. Zhengang Yang for providing 5HT3ARGFP mice. This work was supported by grants from the Natural Science Foundation of China (31200816) and Shanghai Municipal Health & Family Planning Commission (20144Y0105) to Y. F.; grants from the Ministry of Science and Technology of China (2014CB942800, 2012CB966300), the Natural Science Foundation of China (31471036, 31421091, 91332110, 31271157), the Foundation of the Ministry of Education of China (NCET-120128, 20130071110065), the Foundation of Shanghai Municipal Commission of Health and Family Planning (XYQ2011043), and the Foundation of Shanghai Municipal Education Commission (12ZZ007) to Y.-C.Y.

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(Accepted 10 February 2016) (Available online 15 February 2016)