Abstract

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

Introduction

During the development of the cerebral cortex, cortical excitatory neurons are generated in the periphery of the ventricles and mature through several sequential developmental stages, including cell proliferation, cell fate specification, migration, dendrite and axon formation, and synaptogenesis (Price et al. 2006; Rakic 2006). How each transition from one stage to the next is controlled remains largely unknown. For example, cortical excitatory neurons that are generated within the ventricular zone (VZ) migrate toward the cortical surface and reach the marginal zone (MZ), where they start dendrite formation only after they stop migration. It has been suggested that Reelin, an extracellular matrix protein secreted by Cajal–Retzius neurons in the MZ, may act as a “stop” signal for migrating neurons (Dulabon et al. 2000; Tissir and Goffinet 2003). However, how neurons that have stopped migration start dendrite formation is not well understood.

What cellular properties change during and/or after migration? Morphologically, cortical neurons undergoing migration show bipolar shape, with a leading process extending toward the MZ and a trailing process toward the VZ, and neurons that have stopped migration have branched dendrites. Physiologically, it has been shown that cortical neurons undergoing migration are less excitable than those after migration, and that this is mainly due to a low level of expression of sodium channels that are critical for generation of action potentials (Luhmann et al. 2000; Picken Bahrey and Moody 2003; Moore et al. 2009). We previously reported that an increase of spontaneous neural activity/calcium transients impedes migration of cortical excitatory neurons (Bando et al. 2014). In addition, dendritic development partially, but critically, depends on the neuronal activity and intracellular calcium signaling (McAllister 2000; Cline 2001; Wong and Ghosh 2002; Lohmann and Wong 2005; Cancedda et al. 2007; Takemoto-Kimura et al. 2007). It is possible that the increase of spontaneous activity terminates neuronal migration and promotes dendrite formation. However, whether there is a direct linkage between changes in the morphology and those in the electrophysiological properties and/or intracellular calcium dynamics remains unexplored.

In this report, we first show that spontaneous calcium transients occurred more frequently in postmigratory neurons than in migrating neurons. We also observed that an increase in the cellular excitability during migration caused migration arrest together with dendrite formation. Ectopic expression of a prokaryotic voltage-gated sodium channel, NaChBac, a genetic tool to elevate neuronal activity (Ren et al. 2001; Lin et al. 2010), dramatically increased the frequency of spontaneous calcium transients in migrating neurons, and induced dendritic branching even before neurons reached the MZ. Based on these observations, we propose that an increase in the frequency of spontaneous calcium transients, mediated by voltage-gated calcium channels, contributes to the termination of migration and initiation of dendrite formation in the developing cortical excitatory neurons.

Materials and Methods

Animals

Wild-type ICR mice were used in all experiments. The experimental procedures were performed in accordance with the guidelines for animal experimentation of the US National Institutes of Health and Kyoto University, and were approved by the local committee for handling experimental animals in the Graduate School of Science, Kyoto University.

Plasmid Construction

The pCAsalEGFP vector (Bando et al. 2014) was used for expression of enhanced green fluorescent protein (EGFP). Wild-type and mutant NaChBac were prepared as previously described (Shimomura et al. 2011). Turbo red fluorescent protein (RFP) cDNA was purchased from Evrogen (FP232). GCaMP3 cDNA was purchased from Addgene (plasmid 22692). TurboRFP, wild-type and mutant NaChBac, and GCaMP3 were cloned into a pCAGplay vector (Kawaguchi and Hirano 2006). pCAG-Cre and pCALNL vectors were obtained from Drs T. Matsuda (Kyoto University) and C.L. Cepko (Harvard University) TurboRFP, and PSD95-EGFP were cloned into a pCALNL vector (Matsuda and Cepko 2007).

In Utero Electroporation

In utero electroporation was performed as reported previously (Saito and Nakatsuji 2001; Tabata and Nakajima 2001; Bando et al. 2014). In brief, ICR mice at E15 were anesthetized with Somnopentyl (50 mg/kg, Kyoritsu Pharmacy), and their uteruses were exposed. Plasmid vectors were prepared at 0.1 μg/mL (pCAG-Cre), 0.5 mg/mL (pCAG-GCaMP3), or 1.0 mg/mL (others) with 0.04% Trypan Blue (Sigma), and 1–2 μL of DNA solution was injected into the right lateral ventricle. Next, 5 electric pulses of 50 V for 50 ms at 1 Hz were applied to embryos using a CUY21EDIT electroporator (NepaGene).

Immunohistochemistry

Pregnant mice were deeply anesthetized with Somnopentyl (Kyoritsu Pharmacy). Neonatal (P0–P5) mice were deeply anesthetized by hypothermia. Brains of embryonic and neonatal mice were fixed with 4% paraformaldehyde in phosphate buffer at 4 °C overnight, transferred to 30% sucrose in PBS, and incubated at 4 °C overnight. Brains were cryoprotected in OCT compound (Sakura), and coronal sections of 50 μm were cut using a cryostat CM1850 (Leica).

Immunohistochemistry was performed using a free-floating procedure. The sections were incubated with blocking solution (5% normal goat serum [Sigma] and 0.2% Triton X-100 in PBS) for 1 h at room temperature, followed by incubation with primary antibodies in blocking solution at 4 °C overnight. Subsequently, the sections were incubated with secondary antibodies for 1 h at room temperature. The sections were mounted with Vectashield (Vector). The following primary and secondary antibodies were used: anti-Cux1 (rabbit, polyclonal, 1 : 100, Santa Cruz Biotechnology), anti-Ki67 (rabbit, polyclonal, 1 : 600, Millipore), anti-Pax6 (rabbit, polyclonal, 1 : 400, Covance), anti-Tbr2 (rabbit, polyclonal, 1 : 500, Abcam), anti-Nestin (mouse, monoclonal, 1 : 600, BD Pharmingen), anti-GM130 (mouse, monoclonal, 1 : 600 BD Transduction Laboratories), Alexa 568-conjugated anti-rabbit IgG, and anti-mouse IgG (produced in goat, 1 : 1000, Molecular Probes).

Detection of Apoptosis with TUNEL Assay

Apoptosis was detected with an ApopTag® Red In Situ Apoptosis Detection Kit (Chemicon). The experimental procedure was based on the protocol provided by the manufacturer. Briefly, 50-μm-thick coronal cortical sections were incubated with ethanol/acetic acid for 5 min at room temperature. Sections were incubated with terminal deoxynucleotidyl-transferase enzyme and digoxigenin (DIG)-labeled dTTP for 30 min at 37 °C. Subsequently, sections were incubated with rhodamine-conjugated anti-DIG antibody for 30 min at room temperature. Finally, sections were mounted with Vectashield (Vector).

Morphological Analysis

Fluorescent images were acquired using a confocal laser-scanning microscope FV1000 (Olympus). To capture images of migrating neurons in fixed cortical sections, the microscope was equipped with a ×10 dry objective. Confocal images of 50-μm-thick z-stacks were acquired for analysis of neuronal migration (Fig. 4), and z-series of images were projected onto two-dimensional representation. To evaluate neuronal migration, the cortex of 100-μm width was divided into 10 bins from pia to VZ. The fraction of TurboRFP-positive neurons in each bin was calculated by dividing the number of TurboRFP-positive neurons in each bin by that of total TurboRFP-positive neurons in the cortex of 100-μm width. At least 10 images (one image per mouse) for each experimental condition were analyzed.

For analysis of dendritic morphology (Figs 5 and 6) and clustering of PSD95-EGFP (Fig. 6), neurons were sparsely labeled using pCAG-Cre and pCALNL-TurboRFP or PSD95-EGFP plasmids. For acquisition of images to analyze the dendritic morphology, the microscope was equipped with a ×20 dry objective. Fifty-micrometer-thick z-series of images were obtained for morphological analysis. Z-series of images were projected onto a two-dimensional plane. Dendrites were traced, and total length and branch points were analyzed with the NeuronJ software (Meijering et al. 2004). At least 5 neurons per mouse were analyzed.

For acquisition of images to analyze the cell fate, proliferation, and apoptosis, the microscope was equipped with a ×20 dry objective. Images of a single confocal plane were acquired for the analysis.

All results were expressed as mean ± SEM. Statistical analysis was performed with Student's t-test or Dunnett's test.

Electrophysiology

Whole-cell patch-clamp recording was performed as previously described (Ohtsuki et al. 2004; Bando et al. 2014). In brief, 300-μm-thick coronal cortical slices of P0 and P1 mice were cut using a vibratome linear slicer PRO 7 (Dosaka EM) in chilled RDS solution (in mM: 130 NaCl, 4.5 KCl, 2 CaCl2, 33 glucose, and 5 HEPES, titrated to pH 7.3 with NaOH). Slices were allowed to recover for longer than 1 h in Krebs' solution saturated with 95% O2 and 5% CO2 (in mM: 124 NaCl, 26 NaHCO3, 1.8 KCl, 1.24 KH2PO4, 2.5 CaCl2, 1.3 MgCl2, and 10 glucose, pH 7.3) at room temperature (22–24 °C). Oxygenated Krebs' solution was used as the extracellular bath solution. The patch pipettes were filled with the internal solution (in mM: 140 mM d-glucuronic acid, 7 KCl, 155 KOH, 5 EGTA, 10 HEPES, 2 Mg-ATP, and 0.2 Na-GTP, pH 7.3). Glass patch pipettes of 4–7 MΩ were used for recording. The junction potential was offset. Whole-cell recordings were performed at room temperature with a patch-clamp amplifier EPC10 (HEKA). All results were expressed as mean ± SEM. Statistical analysis was performed with Student's t-test.

Calcium Imaging

Calcium imaging was performed as previously described (Bando et al. 2014). GCaMP3 only, or GCaMP3 plus wild-type or mutant NaChBac, was transfected by in utero electroporation at E15. At P0 and P1, 500-μm-thick coronal slices were prepared as described above. Krebs' solution was used as the extracellular solution. Optical recording was performed at room temperature, using a fluorescent microscope BX50WI (Olympus) with a ×20 water-immersion objective and an EM-CCD camera C9100 (Hamamatsu Photonics). The sampling rate was 1 Hz. The mean fluorescent signal of a single cell was calculated, and divided by that at the start using the Aquacosmos software (Hamamatsu Photonics). A fluorescence change to 5% above the baseline within 30 s was regarded as a calcium transient. To elucidate calcium channels involved in the calcium transients, a calcium channel blocker, ω-Agatoxin IVA (AgTX; Peptide Institute, Inc.), 200 nM; ω-conotoxin GVIA (CgTx; Peptide Institute, Inc.), 500 nM; nifedipine (TOCRIS), 5 μM; or Ni2+, 500 nM was applied to the bath, and a slice was incubated for 30 min before recording. All results were expressed as mean ± SEM. Statistical analysis was performed with the χ2 test, Student's t-test or Dunnett's test.

Time-Lapse Imaging

Time-lapse imaging of migrating neurons in cortical slices was performed similarly to previous studies (Tabata and Nakajima 2003; Umeshima and Kengaku 2013). Coronal 300-μm-thick cortical slices of P0 mice were cut as described above. Slices were put on a Millicell-CM membrane (Millipore), mounted with collagen gel, and soaked in culture medium (Neurobasal medium with B27). Slices were recovered for 2 h at 37 °C in an incubator chamber fitted on a laser-scanning confocal microscope (FV1000, Olympus) with gas flow (40% O2, 5% CO2, and 55% air). Images were acquired every 10 min for 6 h using a ×20 objective lens. Migration speed was analyzed using the Fiji software (http://fiji.sc/Fiji).

Results

Different Patterns of Spontaneous Calcium Transients Between Putative Migrating Neurons and Postmigratory Neurons

We first examined the pattern of spontaneous calcium transients in developing cortical neurons. For this, we transfected genetically encoded calcium indicator GCaMP3 (Tian et al. 2009) into newborn layer II/III pyramidal neurons at E15 using in utero electroporation (Saito and Nakatsuji 2001; Tabata and Nakajima 2001), and performed calcium imaging at P1 in an acute slice preparation (Fig. 1A,B). Electroporated neurons, which we could identify by their expression of GCaMP3, were dispersed across cortical layers in P1 slices; some neurons had already arrived at layer II/III, whereas others were still located in deep cortical layers. The latter were considered to be in the course of migration, because they frequently moved toward superficial layers, as assessed by live-cell imaging (Supplementary Fig. 1). The fraction of neurons that showed calcium transients and the frequency of calcium transients were compared between electroporated neurons in layers IV–VI (putative migrating neurons) and layer II/III (postmigratory neurons). The fraction of neurons showing calcium transients was higher for postmigratory neurons than for putative migrating neurons (Fig. 1C, putative migrating, 23/295 neurons, 7.8%; postmigratory, 62/244 neurons, 25.4%, P < 0.001, χ2 test). The frequency of calcium transients was also higher in postmigratory neurons than in putative migrating neurons (Fig. 1D, putative migrating, 1.3 ± 0.2 events/10 min, n = 23 neurons; postmigratory, 2.4 ± 0.2 events/10 min, n = 62, P < 0.001, Student's t-test). Expression of GCaMP3 in cortical neurons did not affect their migration speed in slices, suggesting that GCaMP3 expression did not significantly affect viability or movement (Supplementary Fig. 1). These results suggest that the spontaneous activity of postmigratory neurons is higher than that of migrating neurons. We hypothesized that this increase of spontaneous calcium transients is critical for termination of migration and dendritogenesis, and performed the following experiments to examine this idea.

Figure 1.

The pattern of spontaneous calcium transients in the developing cortical neurons. (A and B) Representative time courses of spontaneous calcium transients during 10-min recordings. Time courses of calcium transients of GCaMP3-expressing neurons in layers IV–VI, presumably during migration (A), and in layer II/III, presumably after migration (B). (C) Fraction of neurons showing calcium transients. ***P < 0.001, χ2 test. (D) Frequency of calcium transients. Mean ± SEM is presented. ***P < 0.001, Student's t-test.

Figure 1.

The pattern of spontaneous calcium transients in the developing cortical neurons. (A and B) Representative time courses of spontaneous calcium transients during 10-min recordings. Time courses of calcium transients of GCaMP3-expressing neurons in layers IV–VI, presumably during migration (A), and in layer II/III, presumably after migration (B). (C) Fraction of neurons showing calcium transients. ***P < 0.001, χ2 test. (D) Frequency of calcium transients. Mean ± SEM is presented. ***P < 0.001, Student's t-test.

NaChBac Expression Elevated the Frequency of Spontaneous Calcium Transients

To elucidate the roles of the calcium transients, we elevated the frequency of spontaneous calcium transients in migrating neurons by utilizing NaChBac, which is a prokaryotic voltage-gated sodium channel that conducts large inward current (Ren et al. 2001). In the mammalian central nervous system, ectopic expression of NaChBac strongly elevates the neural activity and contributes to the survival and maturation of adult-born neurons (Lin et al. 2010; Sim et al. 2013). We therefore used NaChBac as a tool to elevate the neural activity. For this, NaChBac was co-transfected into layer II/III neurons with a fluorescent marker (EGFP or TurboRFP). First, we performed whole-cell patch-clamp recording in a slice preparation to examine whether ectopically expressed NaChBac was functional in migrating neurons. Electrophysiological recordings were obtained from EGFP-expressing neurons with or without NaChBac in the intermediate zone (IZ) at P0 and P1. We recorded voltage-gated current in a voltage-clamp condition. Membrane potential was held at −100 mV, and depolarizing voltage pulses were applied with a 10-mV interval to +20 mV. Only small, fast voltage-dependent inward current was recorded from control neurons expressing only EGFP (Fig. 2AC, control, −106.6 ± 67.8 pA at −20 mV, n = 7 neurons), suggesting endogenous expression of voltage-gated sodium channels in these neurons. In contrast, NaChBac-expressing neurons showed large, slow voltage-dependent inward current in addition to small and fast one (Fig. 2AC, NaChBac, −882.1 ± 264.8 pA at −60 mV, n = 6). It should be noted that neurites prevented us from obtaining good space clamp conditions in NaChBac-expressing neurons with large inward current in particular, which together with series resistance error resulted in an apparent negative shift of voltage-dependence of NaChBac. The spontaneous firing rate recorded in the current-clamp condition was higher in NaChBac-expressing neurons than in control neurons (Fig. 2D,F, control, 0 Hz, n = 7 neurons; NaChBac, 0.024 ± 0.009 Hz, n = 11, P = 0.021, Student's t-test). These results suggest that ectopically expressed NaChBac forms functional channels in migrating neurons. Neither input resistance, nor resting membrane potential, was significantly different between control and NaChBac-expressing neurons (Fig. 2E,G, input resistance, control, 3.6 ± 0.8 GΩ, n = 7 neurons; NaChBac, 4.1 ± 1.0 GΩ, n = 11, P = 0.667, Student's t-test; resting membrane potential, control, −54.3 ± 9.3 mV, n = 7 neurons; NaChBac, −57.5 ± 5.2 mV, n = 11, P = 0.766, Student's t-test).

Figure 2.

Increase of neural activity in layer II/III neurons induced by expression of a prokaryotic voltage-gated sodium channel, NaChBac. (A) Current responses to depolarizing voltage steps, recorded from electroporated neurons in deep CP and IZ at P0 and P1. (B) Initial phases of current responses shown in A are presented. A large, sustained inward current was recorded only from NaChBac-expressing neurons, in response to depolarizing voltage steps from −100 mV (arrowhead). Note small, fast inward current recorded from both control and NaChBac-expressing neurons (arrows), suggesting endogenous expression of voltage-gated sodium channels. (C) Current–voltage relationship of the peak inward current. (D) Representative time courses of membrane potential recorded in a current-clamp mode. (EG) Resting potential (E), spontaneous firing rate (F), and input resistance (G) of neurons expressing EGFP with or without NaChBac. *P < 0.05, Student's t-test. (H) Spontaneous calcium transients in neurons expressing only GCaMP3 (control), GCaMP3, and wild-type NaChBac (NaChBac), E43K or D60K mutants. (I) The number of calcium transients during 10-min recordings. Mean ± SEM is presented. **P < 0.01, Dunnett's test.

Figure 2.

Increase of neural activity in layer II/III neurons induced by expression of a prokaryotic voltage-gated sodium channel, NaChBac. (A) Current responses to depolarizing voltage steps, recorded from electroporated neurons in deep CP and IZ at P0 and P1. (B) Initial phases of current responses shown in A are presented. A large, sustained inward current was recorded only from NaChBac-expressing neurons, in response to depolarizing voltage steps from −100 mV (arrowhead). Note small, fast inward current recorded from both control and NaChBac-expressing neurons (arrows), suggesting endogenous expression of voltage-gated sodium channels. (C) Current–voltage relationship of the peak inward current. (D) Representative time courses of membrane potential recorded in a current-clamp mode. (EG) Resting potential (E), spontaneous firing rate (F), and input resistance (G) of neurons expressing EGFP with or without NaChBac. *P < 0.05, Student's t-test. (H) Spontaneous calcium transients in neurons expressing only GCaMP3 (control), GCaMP3, and wild-type NaChBac (NaChBac), E43K or D60K mutants. (I) The number of calcium transients during 10-min recordings. Mean ± SEM is presented. **P < 0.01, Dunnett's test.

Next, we tested whether NaChBac expression elevated spontaneous calcium transients, which would reflect neuronal excitability. GCaMP3 with wild-type or non-functional mutant NaChBac channels was transfected at E15, and calcium imaging was performed on neurons in the cortical plate (CP) at P0 and P1 (Fig. 2H,I). E43K and D60K are mutants of NaChBac whose voltage-dependency is positively shifted (NaChBac, V1/2 = −37 mV; E43K, V1/2 = +18 mV; D60K, V1/2 = +42 mV) (Shimomura et al. 2011). It is expected that these mutants will not be activated around resting membrane potential, due to their positively shifted membrane potential dependence. As expected, NaChBac expression drastically elevated the fraction of neurons showing spontaneous calcium transients and the frequency of calcium transients, while only a few neurons showed calcium transients at a low frequency in control neurons expressing only GCaMP3 (Fig. 2H,I, control, 3.7%, 4/109 neurons, 1.3 ± 0.3 events/10 min; NaChBac, 82.8%, 135/163 neurons, 9.5 ± 0.5 events/10 min, P = 0.004, Dunnett's test). E43K and D60K mutant channels did not show altered patterns of spontaneous calcium transients (Fig. 2H,I, E43K, 6.9%, 11/160 neurons, 1.4 ± 0.2 events/10 min, P = 0.999; D60K, 8.5%, 10/117 neurons, 1.4 ± 0.3 events/10 min, P = 0.999). These results suggest that NaChBac expression elevates spontaneous calcium transients in migrating neurons through its ion channel function.

To identify the ion channels involved in spontaneous calcium transients in NaChBac-expressing neurons, we performed calcium imaging on cells transfected with GCaMP3 and NaChBac in the presence of specific blockers for each voltage-gated calcium channel at P0 and P1. Spontaneous calcium transients were not affected by AgTX, a blocker of P/Q-type calcium channels (Fig. 3A,B, NaChBac, 8.1 ± 0.6 events/10 min, n = 71 neurons; AgTx, 6.3 ± 0.7 events/10 min, n = 82, P = 0.139, Dunnett's test). The frequency of calcium transients was significantly decreased by application of CgTx, a blocker of N-type calcium channels, or nifedipine, a blocker of L-type calcium channels (Fig. 3A,B, CgTx, 1.3 ± 0.3 events/10 min, n = 59, P < 0.001; nifedipine, 1.7 ± 0.6, n = 62, P < 0.001). Application of Ni2+, a blocker of T-type calcium channels, slightly decreased the frequency of calcium transients (Fig. 3A,B, 5.5 ± 0.7 events/10 min, n = 65, P = 0.017). These results suggest that L-, N-, and T-type voltage-gated calcium channels are involved in generating spontaneous calcium transients in migrating cortical layer II/III neurons.

Figure 3.

Involvement of voltage-gated calcium channels in spontaneous calcium transients. (A) Representative time courses of spontaneous calcium transients during 10-min recordings. Recording was performed from neurons in the IZ and CP at P0 and P1. (B) Frequency of calcium transients of NaChBac-expressing neurons with no calcium channel blocker (NaChBac), AgTX (a blocker of P/Q-type calcium channel), CgTx (a blocker of N-type calcium channel), nifedipine (a blocker of L-type calcium channel), or Ni2+ (a blocker of T-type calcium channel). Mean ± SEM is presented. *P < 0.05, ***P < 0.001, Dunnett's test.

Figure 3.

Involvement of voltage-gated calcium channels in spontaneous calcium transients. (A) Representative time courses of spontaneous calcium transients during 10-min recordings. Recording was performed from neurons in the IZ and CP at P0 and P1. (B) Frequency of calcium transients of NaChBac-expressing neurons with no calcium channel blocker (NaChBac), AgTX (a blocker of P/Q-type calcium channel), CgTx (a blocker of N-type calcium channel), nifedipine (a blocker of L-type calcium channel), or Ni2+ (a blocker of T-type calcium channel). Mean ± SEM is presented. *P < 0.05, ***P < 0.001, Dunnett's test.

NaChBac Expression Caused Migration Defect in the Developing Cerebral Cortex

Next, we examined the effect of NaChBac expression on cortical development. RFP-positive neurons along cortical layers were counted at 3 developmental stages (E17, P0, and P3) after transfection of TurboRFP with or without NaChBac at E15. The distribution of RFP-positive neurons was not significantly changed by NaChBac expression at E17 (Fig. 4A,B and Supplementary Table 1). Neuronal migration was, however, significantly impeded in NaChBac-transfected mice at P0 and P3 (Fig. 4A,B and Supplementary Table 1). Expression of E43K or D60K did not impede neuronal migration at P3, suggesting that the ion channel activity of NaChBac is important for the cell migration, because these mutants were confirmed to have no effects on the neuronal activity in migrating neurons (Fig. 4A,B and Supplementary Table 1). These results strongly suggest that increasing neural activity by NaChBac expression, not just artifactual effects of ectopic expression of prokaryotic protein, impedes neuronal migration during development.

Figure 4.

Migration defect caused by expression of NaChBac in the developing cortical neurons. (A) Representative images of coronal cortical sections at E17, P0, and P3. TurboRFP (control) or TurboRFP with wild-type (NaChBac), E43K or D60K mutant NaChBac was electroporated at E15. Dashed lines indicate the surface of the cortex. SVZ, subventricular zone. Arrows indicate neurons whose migration is impeded. Scale bar, 100 μm. (B) Distribution of TurboRFP-positive neurons. For bin analysis, the cortex of 100-μm width (representative control images are presented) was divided into 10 bins of equal size. Bin 1 is the most superficial part. Mean ± SEM is presented. *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett's test.

Figure 4.

Migration defect caused by expression of NaChBac in the developing cortical neurons. (A) Representative images of coronal cortical sections at E17, P0, and P3. TurboRFP (control) or TurboRFP with wild-type (NaChBac), E43K or D60K mutant NaChBac was electroporated at E15. Dashed lines indicate the surface of the cortex. SVZ, subventricular zone. Arrows indicate neurons whose migration is impeded. Scale bar, 100 μm. (B) Distribution of TurboRFP-positive neurons. For bin analysis, the cortex of 100-μm width (representative control images are presented) was divided into 10 bins of equal size. Bin 1 is the most superficial part. Mean ± SEM is presented. *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett's test.

NaChBac Expression Induced Branch Formation of Leading Process Before Neurons Reached Layer II/III

How did NaChBac expression impede neuronal migration? We hypothesized that the increased spontaneous calcium transients caused by NaChBac expression induced maturation of migrating neurons before they reached layer II/III, because neurons at layer II/III after migration exhibited more calcium transients than those during migration, as shown in Figure 1. To test our hypothesis, we analyzed the morphology of neurons during migration. Migrating cortical excitatory neurons in the CP showed bipolar morphology at P1 (Fig. 5A). However, NaChBac-expressing neurons in the CP had branched protrusions, and the total length of protrusions was longer than that in the control at P1 (Fig. 5AC, control, branch points, 0.2 ± 0.1 and total length, 61.7 ± 3.2 μm, n = 40 neurons from 4 mice; NaChBac, branch points, 4.4 ± 0.5, P < 0.001, Dunnett's test and total length, 130.8 ± 8.1 μm, P < 0.001, Dunnett's test, n = 26 neurons from 3 mice). Most of the E43K- and D60K-expressing neurons were bipolar (Fig. 5AC, E43K, branch points, 0.3 ± 0.1, P = 0.999, Dunnett's test and total length, 65.9 ± 2.8 μm, P = 0.814, n = 32 neurons from 5 mice; D60K, branch points, 0.4 ± 0.2, P = 0.939 and total length, 57.1 ± 2.1 μm, P = 0.791, n = 28 neurons from 5 mice). These results suggest that elevation of the neural activity by NaChBac expression induces leading process branching before neurons arrive at layer II/III.

Figure 5.

Formation of leading process branches in NaChBac-expressing neurons. (A) Morphology of migrating neurons at P1. Arrows indicate branches of the leading process. Scale bar, 50 μm. (B and C) Total length of protrusions (B), and number of branch points (C) of control and NaChBac-expressing neurons. Mean ± SEM is presented. ***P < 0.001, Dunnett's test. (D and E) Immunonstaining of Nestin, a marker for radial glial fibers in control (D) and NaChBac-expressing mice (E). Arrows indicate processes attached to radial glia. Arrowheads indicate processes not attached to radial glia. Scale bar, 50 μm.

Figure 5.

Formation of leading process branches in NaChBac-expressing neurons. (A) Morphology of migrating neurons at P1. Arrows indicate branches of the leading process. Scale bar, 50 μm. (B and C) Total length of protrusions (B), and number of branch points (C) of control and NaChBac-expressing neurons. Mean ± SEM is presented. ***P < 0.001, Dunnett's test. (D and E) Immunonstaining of Nestin, a marker for radial glial fibers in control (D) and NaChBac-expressing mice (E). Arrows indicate processes attached to radial glia. Arrowheads indicate processes not attached to radial glia. Scale bar, 50 μm.

We also stained cortical sections for Nestin, a marker for radial glial fibers, and examined whether EGFP and Nestin signals were overlapping. Under control conditions, migrating neurons appeared to attach the radial glial fibers at P1, because EGFP and Nestin signals were overlapping (Fig. 5D). However, Nestin signals were not co-localized with NaChBac-expressing neurons, suggesting that branches of these neurons did not interact with radial glial fibers (Fig. 5E). Taken together, these results suggest that migration of NaChBac-expressing neurons is finished at P1; these neurons stop migration and mature in deep cortical layers rather than simply undergoing delayed migration.

Branched Leading Process of NaChBac-Expressing Neurons Differentiated to Dendrites

To elucidate the dendritic differentiation of the leading process of NaChBac-expressing neurons, we compared the morphology of TurboRFP-positive neurons between control and NaChBac-electroporated animals in later stages of development. At P3, NaChBac-expressing neurons had many branches similar to dendrites of control neurons in layer II/III (Fig. 6AC, control, branch points, 7.7 ± 0.4 and total length, 239.1 ± 14.3 μm, n = 14 neurons; NaChBac layer II/III, branch points, 9.4 ± 0.8, P = 0.142, Dunnett's test and total length, 245.6 ± 20.6 μm, n = 14 neurons, P = 0.955, Dunnett's test; NaChBac layers IV–VI, branch points, 7.8 ± 0.7, P = 0.997 and total length, 292.4 ± 20.3 μm, n = 13 neurons, P = 0.922). To further test whether these processes of NaChBac-expressing neurons were dendritic arbors, we performed immunostaining against GM130, a marker for Golgi apparatus. It was previously reported that GM130 is localized at the root of primary dendrites (Horton et al. 2005; Ye et al. 2007). In accordance with those reports, Golgi apparatus was localized at roots of primary dendrites of EGFP-positive neurons in control mice (Fig. 6D). In NaChBac-electroporated mice, Golgi apparatus was also localized at roots of the apical process of EGFP-positive neurons both in layer II/III and in layers IV–VI (Fig. 6D). These results suggest that branched apical processes of NaChBac-expressing neurons had dendritic properties. In addition, we electroporated PSD95-EGFP as a postsynaptic marker. In both control and NaChBac-expressing neurons, many puncta of PSD95-EGFP were observed in apical processes at P5 (Fig. 6EG), suggesting that many excitatory synapses were formed in apical processes. Taken together, these results suggest that apical processes of NaChBac-expressing neurons in both layer II/III and layers IV–VI differentiate to dendrites.

Figure 6.

Dendritic differentiation of leading process of NaChBac-expressing neurons. (A) Morphology of migrating neurons at P3. Scale bar, 50 μm. (B and C) Total length of protrusions (B), and number of branch points (C) of control and NaChBac-expressing neurons. Mean ± SEM is presented. (D) Representative images of control and NaChBac-electroporated cortex stained for GM130, a marker for Golgi apparatus. Arrows indicate GM130-positive apical processes. Scale bar, 20 μm. (EG) Clustering of PSD95-EGFP in control neurons in layer II/III (E), NaChBac-expressing neurons in layer II/III (F), and layers IV–VI (G) at P5. Left and center panels show representative images of neurons expressing PSD95-EGFP and TurboRFP. Right panels show higher magnification images of the areas indicated by white squares in the center panels. Arrows indicate PSD95-EGFP-positive puncta. Scale bars, 20 μm (left and center panels) and 5 μm (right panels).

Figure 6.

Dendritic differentiation of leading process of NaChBac-expressing neurons. (A) Morphology of migrating neurons at P3. Scale bar, 50 μm. (B and C) Total length of protrusions (B), and number of branch points (C) of control and NaChBac-expressing neurons. Mean ± SEM is presented. (D) Representative images of control and NaChBac-electroporated cortex stained for GM130, a marker for Golgi apparatus. Arrows indicate GM130-positive apical processes. Scale bar, 20 μm. (EG) Clustering of PSD95-EGFP in control neurons in layer II/III (E), NaChBac-expressing neurons in layer II/III (F), and layers IV–VI (G) at P5. Left and center panels show representative images of neurons expressing PSD95-EGFP and TurboRFP. Right panels show higher magnification images of the areas indicated by white squares in the center panels. Arrows indicate PSD95-EGFP-positive puncta. Scale bars, 20 μm (left and center panels) and 5 μm (right panels).

NaChBac Expression Did Not Affect Proliferation, Neuronal Differentiation, Neuronal Subtype Specification, or Apoptosis

We then analyzed other effects of NaChBac expression on cortical development than migration by immunohistochemical examination of neurons electroporated with EGFP with or without NaChBac at E17. First, to analyze whether neuronal proliferation was affected by NaChBac expression, we performed immunostaining for Ki67, a marker for dividing cells (Fujimoto et al. 2009) (Fig. 7A). The fraction of EGFP/Ki67-double-positive cells was not significantly different, suggesting that cell proliferation was not affected by NaChBac expression (Fig. 7A, control, 10.6 ± 1.4%, n = 11 sections; NaChBac, 10.0 ± 1.4%, n = 10 sections, P = 0.780, Student's t-test).

Figure 7.

Proliferation, neuronal differentiation, apoptosis, and radial glial scaffold were not affected by NaChBac expression. Immunostaining for marker proteins. EGFP with or without NaChBac was electroporated at E15, and sections were prepared and stained at E17. (A) Representative images of coronal cortical sections immunostained for Ki67, a marker for proliferating cells, and the fraction of EGFP/Ki67-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (B) Staining of apoptotic cells with a TUNEL method, and the number of apoptotic cells in a 200-μm width of IZ/SVZ/VZ. (C) Immunostaining for Pax6, a marker for radial glial cells, and the fraction of EGFP/Pax6-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (D) Immunostaining for Tbr2, a marker for intermediate progenitors, and the fraction of EGFP/Tbr2-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (E) The morphology of radial glial fibers expressing only EGFP (control) or EGFP and NaChBac (NaChBac). (F) Immunostaining for Cux1, a marker for layer II/III and IV neurons. Scale bars (AD and F) 50 μm and (E) 100 μm. Mean ± SEM is presented.

Figure 7.

Proliferation, neuronal differentiation, apoptosis, and radial glial scaffold were not affected by NaChBac expression. Immunostaining for marker proteins. EGFP with or without NaChBac was electroporated at E15, and sections were prepared and stained at E17. (A) Representative images of coronal cortical sections immunostained for Ki67, a marker for proliferating cells, and the fraction of EGFP/Ki67-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (B) Staining of apoptotic cells with a TUNEL method, and the number of apoptotic cells in a 200-μm width of IZ/SVZ/VZ. (C) Immunostaining for Pax6, a marker for radial glial cells, and the fraction of EGFP/Pax6-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (D) Immunostaining for Tbr2, a marker for intermediate progenitors, and the fraction of EGFP/Tbr2-double-positive cells in a 200-μm width of IZ/SVZ/VZ. (E) The morphology of radial glial fibers expressing only EGFP (control) or EGFP and NaChBac (NaChBac). (F) Immunostaining for Cux1, a marker for layer II/III and IV neurons. Scale bars (AD and F) 50 μm and (E) 100 μm. Mean ± SEM is presented.

Secondly, we tested whether apoptosis was affected by NaChBac expression using the TUNEL assay. The number of TUNEL-positive cells was not significantly different between control and NaChBac-expressing mice (Fig. 7B, control, 5.7 ± 0.9, n = 10 sections; NaChBac, 5.2 ± 0.8, n = 10, P = 0.684, Student's t-test).

Thirdly, we tested whether neuronal differentiation was affected by NaChBac expression. Radial glia, neural stem cells in the cortex, produce neural progenitor cells, called intermediate progenitors, and intermediate progenitors differentiate into neurons (Noctor et al. 2004; Englund et al. 2005). We performed immunostaining for Pax6, a marker for radial glia, and Tbr2, a marker for intermediate progenitors (Fig. 7C,D). The fraction of EGFP/Pax6-double-positive cells was not significantly different between control and NaChBac expression (Fig. 7C, control, 19.6 ± 2.6%, n = 10 sections; NaChBac, 16.6 ± 1.5%, n = 13 sections, P = 0.336, Student's t-test), nor was the fraction of EGFP/Tbr2-double-positive cells (Fig. 7D, control, 14.8 ± 1.5%, n = 10 sections; NaChBac, 14.5 ± 1.4%, n = 11 sections, P = 0.684, Student's t-test). The morphology of EGFP-positive radial glial fibers was not altered by NaChBac expression (Fig. 7E).

We also tested whether neuronal subtype specification was affected by NaChBac expression. Previous studies identified several genes expressed in a layer-specific manner, and some genes have been reported to be determinants of the neuronal subtype specification in the cerebral cortex (Molyneaux et al. 2007). Cux1 is expressed in layers II/III and IV, but not in the other layers (Nieto et al. 2004). Coronal cortical sections were stained with antibodies against Cux1. NaChBac-expressing neurons that reached layer II/III and mislocated neurons were Cux1-positive (Fig. 7F), suggesting that cell fate specification was not affected by NaChBac expression.

Discussion

In this study, we have demonstrated an increase of spontaneous calcium transients during cortical neuron maturation: the frequency of spontaneous calcium transients was higher in postmigratory neurons in layer II/III than in migrating neurons in deep cortical layers. To test the idea that such an increase of neural activity may contribute to the maturation of neurons, we expressed a genetic tool, NaChBac, in layer II/III excitatory neurons to increase their activity. We found that a precocious increase of cellular excitability during migration caused migration arrest together with dendrite formation. Our findings not only confirm the importance of neural activity/spontaneous calcium transients in the control of migration and/or dendrite formation during neuronal maturation (Komuro and Rakic 1993; Redmond et al. 2002; Wong and Ghosh 2002; Zheng and Poo 2007), but also suggest that an optimum, albeit relatively low, level of calcium transient activity may be needed for neuronal migration in neocortical excitatory neurons. Thus, an appropriate level of neural activity at each step during development is critical for correct localization and maturation of cortical neurons in vivo (Fig. 8).

Figure 8.

Summary of this study. (A) Relationship between the level of spontaneous neural activity and the development of cortical layer II/III neurons. In the control condition, neural activity is low during migration, and it increases after neurons arrive at layer II/III, where postmigratory neurons form dendrites. (B) Effects of NaChBac expression on the development of cortical layer II/III neurons. A premature increase of neural activity (indicated by a red line in the lower panel) stops neuronal migration and promotes dendrite formation before neurons reach layer II/III.

Figure 8.

Summary of this study. (A) Relationship between the level of spontaneous neural activity and the development of cortical layer II/III neurons. In the control condition, neural activity is low during migration, and it increases after neurons arrive at layer II/III, where postmigratory neurons form dendrites. (B) Effects of NaChBac expression on the development of cortical layer II/III neurons. A premature increase of neural activity (indicated by a red line in the lower panel) stops neuronal migration and promotes dendrite formation before neurons reach layer II/III.

Regulation of Neural Activity During Development

We have demonstrated that the frequency of spontaneous calcium transients in a migrating neuron is relatively low, and that it increases significantly after a neuron stops migration and starts dendrite formation. By expressing a genetically encoded calcium indicator (GCaMP3) specifically in late-born excitatory neurons destined to become layer II/III neurons and then following their maturation, we attempted to correlate the developmental change in the pattern of calcium transients with developmental processes, such as migration and dendrite formation, in a specific subset of cortical neurons. A general increase in the level of neuronal activity during cerebral cortical neuron development has been suggested by previous studies using electrophysiological methods. For example, Picken Bahrey and Moody (2003) made patch-clamp recordings from immature cortical neurons in acute slices of prenatal and postnatal mouse cerebral cortex, and reported that E14 and E17 cortical neurons showed no spontaneous action potentials and that the frequency of spontaneous action potentials increased from P0 to P10. Developmental maturation of other electrophysiological properties, such as increased ability to generate single/multiple action potentials, and increased amplitude of sodium channel current, has also been reported (Luhmann et al. 2000; Picken Bahrey and Moody 2003; Moore et al. 2009). The increase in sodium channel current could lead to more frequent generation of action potentials, activation of voltage-gated calcium channels, and calcium influx. Thus, the results of our calcium imaging experiments are consistent with previous findings, in that perinatal cortical neurons exhibited greater neuronal activity as they matured, provided that calcium transients reflect neuronal activity. In addition, we showed that generation of spontaneous calcium transients is partially blocked by application of nifedipine (L-type calcium channel blocker) or CgTx (N-type calcium channel blocker; Fig. 3), suggesting that these voltage-gated calcium channels play a role in the regulation of spontaneous calcium transients during neuronal maturation.

Control of Migration by Neural Activity and Calcium Transients

Neuronal migration is regulated in part by fluctuation in the intracellular calcium concentration. Previous studies showed that a decrease of spontaneous calcium transients terminates neuronal migration in cerebellar granule cells and cortical inhibitory interneurons (Kumada and Komuro 2004; Bortone and Polleux 2009). On the other hand, we previously reported that elevation of spontaneous calcium transients impairs neuronal migration in the cortical excitatory neurons (Bando et al. 2014). Our current results confirm previous reports, suggesting that an increase of spontaneous calcium transients suppresses migration in neocortical excitatory neurons (Heck et al. 2007; Bando et al. 2014). In the control of migration, neuronal activity/calcium signaling may act locally to regulate the motility of leading processes (Lohmann and Wong 2005; Tsai and Meyer 2012), whereas it may exert its effects on dendrite formation through the control of transcription (Redmond et al. 2002; Wong and Ghosh 2002; Nakanishi and Okazawa 2006; Ding et al. 2013).

Dendritic Branch Formation in Migrating Neurons by NaChBac Expression

Previous studies have demonstrated that neuronal activity and its downstream calcium signaling are critically involved in dendrite development (McAllister 2000; Cline 2001; Wong and Ghosh 2002; Lohmann and Wong 2005; Cancedda et al. 2007; Takemoto-Kimura et al. 2007; Schwartz et al. 2009). Those studies showed that attenuation of neuronal activity or calcium signaling during dendrite development in vitro or in vivo impairs dendritic growth, elaboration, and/or remodeling. In the current study, using a complementary approach, we elevated the activity of cortical excitatory neurons in vivo and found that a precocious increase of neuronal activity during neuronal development induces initiation of dendrite formation before neurons arrive at their target cortical layers (Fig. 8). Our results suggest that an increase of neuronal activity/intracellular calcium signaling supports, or is possibly a trigger for, dendritogenesis during the cortical excitatory neuron development in vivo.

We concluded that the leading processes of NaChBac-expressing neurons differentiated to dendritic branches for the following reasons. (1) The morphology of the processes resembled dendrites: Branches emerged from a thick main trunk and extended toward the cortical surface with tapering. (2) Neuronal polarity was not perturbed. Invasion of the Golgi apparatus into a leading process is a characteristic event in the initiation of dendrite formation (Horton et al. 2005; Ye et al. 2007). GM130 (a marker for the Golgi apparatus) was located at the base of the leading process in NaChBac-expressing neurons (Fig. 6D) as in control (normal) cortical neurons (Fig. 6D). (3) PSD95-EGFP (a marker for postsynaptic structure) was eventually localized along the processes, suggesting the maturation of dendrites on which excitatory synapses were formed (Fig. 6EG).

Postmigratory neurons showed large, slow calcium transients (Fig. 1B). NaChBac-expressing neurons also showed similar slow calcium transients (Fig. 2H). Artificial expression of NaChBac in migrating neurons partially mimics calcium transients observed in postmigratory neurons under physiological conditions. It remains possible that artificial elevation of neural activity by NaChBac expression promotes dendrite formation in a calcium-independent manner.

We previously reported that mild elevation of calcium transients by knockdown of KCNK potassium channel impaired neuronal migration without branch formation of a leading process (Bando et al. 2014). In the current study, a strong increase of calcium transients by expression of NaChBac arrested neuronal migration with precocious branch formation of a leading process. The difference in the frequency of calcium transients might have resulted in different morphological properties. Forming dendritic branches before reaching the target cortical layers might have caused migration arrest, because neuronal migration requires coordinated interactions between a leading process and a radial glial fiber. Indeed, many branches of NaChBac-expressing neurons were detached from radial glial fibers (Fig. 5E). Based on these results, we think that branch formation of a leading process and partial detachment from radial glia caused by an increase of neural activity/calcium transients might have impeded neuronal migration. However, we cannot exclude the possibility that increase of activity/calcium transients impedes neuronal migration via a mechanism different from branch formation of the leading process.

In summary, we found that spontaneous neural activity was higher in neurons after migration than during migration. Elevation of neural activity in migrating neurons by expression of NaChBac induced precocious dendritic branching before neurons reached layer II/III. Neurons mature in vivo through sequential developmental processes. Our findings suggest that appropriate control of neuronal activity during each developmental process is critical for the development and maturation of cortical neurons.

Supplementary Material

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

Funding

This work was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (23500388 to Y.T.), Grant-in-Aid for Scientific Research on Innovative Areas “Neural Diversity and Neocortical Organization” from MEXT (23123508 and 25123707 to Y.T.), CREST-JST (Y.T.), and the Global Center of Excellence program A06 to Kyoto University (T.H.).

Notes

We thank Dr Elizabeth Nakajima (Kyoto University) for critical reading of the manuscript, and Drs Takahiko Matsuda (Kyoto University) and Constance L. Cepko (Harvard University) for a generous gift of pCAG-Cre and pCALNL vector. Conflict of Interest: None declared.

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