Research ArticleNEURODEVELOPMENT

Disruption of SynGAP–dopamine D1 receptor complexes alters actin and microtubule dynamics and impairs GABAergic interneuron migration

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Science Signaling  06 Aug 2019:
Vol. 12, Issue 593, eaau9122
DOI: 10.1126/scisignal.aau9122

SynGAP1 in neurodevelopment

Disruptions in dopamine signaling and mutations in SYNGAP1 are individually associated with various neurological disorders, particularly those associated with aberrant neuronal activity in response to the neurotransmitter GABA, including epilepsy, schizophrenia, and autism. Su et al. found that the dopamine receptor D1R and SynGAP (synaptic GTPase–activating protein) formed a complex in response to extracellular dopamine. Experiments with an engineered interfering peptide revealed that this complex promoted dopamine-induced migration of GABAergic interneurons during embryonic and early postnatal development in mice. This physiological role appeared to be specific to GABAergic but not glutamatergic interneurons, although D1R and SynGAP form complexes in both neuron types. These findings may provide insights into various neurodevelopmental and connectivity disorders.

Abstract

Disruption of γ-aminobutyric acid (GABA)–ergic interneuron migration is implicated in various neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. The dopamine D1 receptor (D1R) promotes GABAergic interneuron migration, which is disrupted in various neurological disorders, some of which are also associated with mutations in the gene encoding synaptic Ras–guanosine triphosphatase–activating protein (SynGAP). Here, we explored the mechanisms underlying these associations and their possible connection. In prenatal mouse brain tissue, we found a previously unknown interaction between the D1R and SynGAP. This D1R-SynGAP interaction facilitated D1R localization to the plasma membrane and promoted D1R-mediated downstream signaling pathways, including phosphorylation of protein kinase A and p38 mitogen-activated protein kinase. These effects were blocked by a peptide (TAT-D1Rpep) that disrupted the D1R-SynGAP interaction. Furthermore, disrupting this complex in mice during embryonic development resulted in pronounced and selective deficits in the tangential migration of GABAergic interneurons, possibly due to altered actin and microtubule dynamics. Our results provide insights into the molecular mechanisms regulating interneuron development and suggest that disruption of the D1R-SynGAP interaction may underlie SYNGAP1 mutation–related neurodevelopmental disorders.

INTRODUCTION

Inhibitory γ-aminobutyric acid (GABA)–ergic interneurons, which constitute 20 to 30% of the neuronal population in both adult rodents and humans, modulate the output from excitatory pyramidal neurons in the cerebral cortex (1). During neurodevelopment, interneuron progenitors originate in the ganglionic eminence, where they migrate tangentially in a saltatory fashion along the dorsal-ventral axis to integrate into the developing cortex (2–4). Disruptions in this process have been implicated in various developmental disorders, such as autism spectrum disorder (5, 6) and schizophrenia (7–9). Despite much research into interneuron migration (10–13), many aspects of this complex process remain poorly understood.

The dopamine system affects GABAergic interneurons in a number of ways. For example, dopamine can regulate the life cycle of progenitor cells in the ganglionic eminence (14). The fact that dopamine receptors are expressed in interneurons reinforces the possibility that dopamine can regulate interneuron function (15). Furthermore, activation of the dopamine D1 receptor (D1R) can increase neuronal migration from basal forebrain to the cerebral wall in cultured mouse brain slices (16). However, the molecular pathway through which the D1R regulates GABAergic interneuron migration is not yet understood.

The D1R belongs to a superfamily of single polypeptide, seven-transmembrane domain receptors that exert their biological effects mainly through intracellular G protein–coupled signaling cascades (17). The D1R preferentially couples to Gs protein, which subsequently stimulates adenylyl cyclase and protein kinase A (PKA) (17, 18). D1R signaling can also be regulated through its interaction with other receptors and proteins (19–21). These D1R-associated protein complexes are important for D1R function, and disruption of these interactions may lead to structural and functional deficits.

Synaptic Ras–guanosine triphosphatase–activating protein 1 (SynGAP1) is essential for synaptic development and function and is crucial for cognition and other brain functions (22, 23). SynGAP is highly enriched in dendritic spines at excitatory synapses (24, 25). Moreover, SynGAP interacts with postsynaptic density 95 to regulate N-methyl-d-aspartate (NMDA) receptor–mediated extracellular signal–regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling cascades (26, 27). Haploinsufficiency of SynGAP reduces the innervation of parvalbumin (PV)–positive basket cells, a subpopulation of GABAergic cells found in layer 5 of the somatosensory cortex in postnatal mice (28). However, like that above for the link between D1R signaling and GABAergic interneuron migration, the molecular pathway through which SynGAP regulates GABAergic interneurons, especially during prenatal development, is also largely not understood.

In this study, we investigated the role of the D1R and SynGAP in the regulation of interneuron migration and neurodevelopment. We found that the D1R forms a protein complex with SynGAP and that disruption of this interaction produces neurodevelopmental changes.

RESULTS

Activation of D1R facilitates formation of a D1R-SynGAP complex

Because both D1R and SynGAP have a critical functional role on GABAergic interneuron migration, we thought perhaps they operate in the same pathway. We first investigated whether the D1R forms a protein complex with SynGAP using coimmunoprecipitation assays. Primary antibodies against SynGAP coimmunoprecipitated D1R protein, and reciprocally, a D1R antibody coimmunoprecipitated SynGAP from prenatal [embryonic day 14 (E14)] mouse brain protein extracts (Fig. 1, A and B), confirming the existence of the D1R-SynGAP complex.

Fig. 1 Regulation of D1R-SynGAP complex formation.

(A and B) Reciprocal coimmunoprecipitation (CO-IP) analysis of D1R with a SynGAP antibody (A) and SynGAP with a D1R antibody (B) in brain lysates from prenatal mice. Blots represent three independent experiments. IB, immunoblot. (C and D) Representative blots (C) and densitometric analysis (D) of the coimmunoprecipitation of D1R and SynGAP in lysates from HEK293T cells transfected with GFP-tagged D1R and SynGAP and treated with the D1R agonist SKF81297 (10 μM for 30 min) alone or with the D1R antagonist SCH23390 (1 μM for 30 min before treatment with SKF81297). Data are means ± SEM, presented as the percentage of the control sample, from n = 3 independent experiments. **P < 0.01 as compared to controls and #P < 0.05 as compared to SKF81297, by one-way ANOVA followed by Tukey’s test. (E and F) Western blot of pulldown of SynGAP from mouse brain lysates incubated overnight ex vivo with (E) GST-D1RIL3 or GST-D1RCT, or (F) GST-D1RIL3-1, GST-D1RIL3-2, GST-D1RIL3-3, or GST-D1RIL3-4. Blots represent three independent experiments performed. (G and H) Representative blots (G) and densitometric analysis (H) of the coimmunoprecipitation of SynGAP with D1R from mouse brain lysates incubated ex vivo with TAT, TAT-D1Rpep, or the D1R agonist SKF81297 (each 10 μM for 30 min). Data are means ± SEM, presented as the percentage of the control and TAT sample, from n = 3 independent experiments. ***P < 0.001 as compared to the TAT and SKF81297 group by two-way ANOVA followed by Bonferroni post hoc test.

We then examined the effects of D1R activation on D1R-SynGAP complex formation in cells. Activation of the D1R with a specific D1R agonist SKF81297 (10 μM for 30 min) substantially increased the abundance of D1R-SynGAP protein complex in human embryonic kidney (HEK) 293T cells expressing green fluorescent protein (GFP)–tagged D1R and SynGAP, an effect that was effectively blocked by pretreatment with the D1R antagonist SCH23390 (1 μM for 30 min) (Fig. 1, C and D). These results suggest that activation of the D1R facilitates D1R-SynGAP complex formation.

A short amino acid fragment within the D1R enables it to form a complex with SynGAP

We next sought to identify the regions of the D1R that are essential for binding to SynGAP. We initially used glutathione S-transferase (GST)–fusion proteins encoding fragments of intracellular domains of the D1R to affinity purify SynGAP in mouse brain extracts. Because the third intracellular loop (IL3) and the carboxyl tail (CT) of D1R are important for receptor signaling and trafficking, we hypothesized that they were involved in binding SynGAP. To test our hypothesis, we generated GST fusion proteins encoding the CT of D1R (D1RCT: R338-T446) and the IL3 of D1R (D1RIL3: R219-K272) and studied their ability to interact with SynGAP using affinity purification. GST-D1RIL3, but not GST-D1RCT, was able to precipitate SynGAP (Fig. 1E), implicating D1RIL3 in the D1R-SynGAP interaction.

Using the same strategy, we further dissected D1RIL3 into smaller fragments (fig. S1) and concluded that the D1RIL3-2 fragment (R233-G246) contains the region that binds to SynGAP (Fig. 1F). On the basis of this result, an interfering peptide corresponding to the amino acid sequence of D1RIL3-2 (D1Rpep) was synthesized to specifically disrupt the D1R-SynGAP interaction. To increase the efficiency of D1Rpep delivery into embryonic brains through the blood-brain barrier and placenta, we fused it to the cell membrane transduction domain of the HIV-1 transactivator of transcription (TAT) protein, as previously described (29, 30). Assuming that the D1RIL3-2 region is essential for the D1R to interact with SynGAP, TAT-D1Rpep should disrupt this complex by competing with D1R for SynGAP. As expected, TAT-D1Rpep (YGRKKRRQRRRRAAVHAKNCQTTTG) treatment inhibited the agonist-induced increase in D1R-SynGAP complex in mouse brain, as compared to the control TAT (YGRKKRRQRRR) (Fig. 1, G and H). Thus, we concluded that the D1RIL3-2 region is critical for D1R-SynGAP complex formation and that TAT-D1Rpep is capable of disrupting the D1R-SynGAP complex.

Disruption of the D1R-SynGAP interaction inhibits GABAergic interneuron migration

Crandall et al. (16) have shown that activation of D1R promotes GABAergic neuron tangential migration in cultured mouse brain slices. Another study showed that haploinsufficiency of SynGAP reduces the innervation of PV-positive basket cells, a subpopulation of GABAergic cells found in layer 5 of the somatosensory cortex in postnatal mice (28). Furthermore, other previous studies have indicated that D1Rs are prominently expressed in GABAergic neurons and are important for interneuron migration (31–33). To further address the underlying mechanisms involved, we investigated whether the D1R-SynGAP complex is important for D1R-mediated GABAergic interneuron migration. We disrupted the D1R-SynGAP interaction in vivo by injecting TAT-D1Rpep into pregnant mice (3 nmol/g body weight intraperitoneally, once every day) from E12 to E17 (fig. S2A), the period during which most interneuron tangential migration occurs. TAT fusion peptides have previously been shown to cross both the placenta and blood-brain barrier (34), and we also used an antibody against TAT to confirm that TAT-D1Rpep was delivered into the embryonic brains after injection (fig. S2B). We also confirmed that TAT-D1Rpep injection into pregnant dams can disrupt the D1R-SynGAP complex in embryonic mouse brain by coimmunoprecipitation assays (fig. S2, C and D).

We then measured the distribution of interneurons along their tangential migratory path on E18 and P14 (postnatal day 14) brains using immunofluorescence staining with an antibody to calbindin (CB). Because PV is not steadily expressed during embryonic development, CB was chosen to stain interneurons in this study (35). E18 mice treated with TAT-D1Rpep had a significantly lower proportion of CB cells in the dorsal cortex of bins 2 to 4 and a much higher proportion in ventral regions of bins 5 to 7 when compared to mice treated with TAT peptide or saline (Fig. 2, A and B). A similar but less pronounced effect was seen at P14 (Fig. 2B). These results indicate that migrating interneurons in the TAT-D1Rpep–treated mice failed to reach their proper dorsal target position, suggesting that disruption of the D1R-SynGAP interaction impairs the tangential migration of interneurons.

Fig. 2 Disrupted GABAergic interneuron tangential migration in TAT-D1Rpep–injected embryonic mouse brains.

(A and B) Coronal sections of E18 or P14 brains immunostained with CB [calbindin-28 (GABAergic interneuron marker)] antibody were straightened and divided into seven equidistant bins representing the tangential migratory pathway of interneurons (A). Fluorescently labeled cell numbers were counted in each bin and normalized to the total CB-positive cell number in all bins (B). Scale bars, 200 μm. Data are means ± SEM of n = 7 (saline), 9 (TAT), or 6 (TAT-D1Rpep) embryonic brains from three pregnant mice. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to saline group in the same bin by two-way ANOVA. (C and D) Cell migration in Matrigel explants from E18 embryos injected with TAT or TAT-D1Rpep from E12 to E17. The migrating distance out of the tissue was measured and normalized to the radius of the tissue. Data are means ± SEM of n = 12 explants from three embryonic brains in each group. ***P < 0.001 as compared to TAT group by t test. (E and F) Cortical explants from E14 embryos were included in Matrigel and treated with saline, TAT, or TAT-D1Rpep, and cell migration was monitored (E). The migrating distance out of the tissue was measured and normalized to the radius of the tissue. Scale bar, 200 μm. Data are means ± SEM of n = 12 to 15 explants from three embryonic brains in each group. Statistical significance was assessed by one-way ANOVA, followed by Dunnett’s test.

Furthermore, we cultured medial ganglion eminence (MGE) explants from embryos of TAT-D1Rpep– or TAT-injected mice on a Matrigel matrix and observed cell migration out of the explants. After 24 hours, cells in the TAT-D1Rpep group were delayed in migrating out of the explants, and the distance traveled was significantly shorter (Fig. 2, C and D). Similar results were also obtained from cultured MGE treated with TAT-D1Rpep in vitro (fig. S3, A and B). We confirmed that most of the primary MGE cultured cells are GABAergic interneurons with GAD67, a GABAergic interneuron marker (fig. S4A). We also confirmed the existence of the D1R-SynGAP interaction and the ability of TAT-D1Rpep to decrease this interaction in these neurons using coimmunoprecipitation (fig. S4B). These results further showed that the D1R-SynGAP interaction plays an important role in GABA interneuron tangential migration.

Both D1R and SynGAP have been reported to be expressed in glutamatergic neurons (25, 36). It is therefore logical to investigate whether the D1R-SynGAP interaction also exists in these neurons. We examined the D1R-SynGAP interaction in E14 cortical neurons, as previous studies show that glutamatergic neurons migrate radially into the developing cerebral cortex during early embryonic stages (37). We first confirmed that most and possibly all of the neurons are glutamatergic using vesicular glutamate transporter 1 (vGlut1) antibody that stains the vesicular glutamate transporter, which is a characteristic component of glutamatergic neurons (fig. S5A). We then confirmed the existence of the D1R-SynGAP interaction in these neurons using coimmunoprecipitation. In addition, TAT-D1Rpep is able to decrease this interaction in these neurons (fig. S5B). To test whether disruption of the D1R-SynGAP interaction changes the migration of embryonic glutamate neurons, we examined cell migration in cortical explants from embryonic brains (E14) of mice treated with saline, TAT, or TAT-D1Rpep. There is no difference between the three groups (Fig. 2, E and F), suggesting that D1R-SynGAP may not regulate glutamatergic neuron migration.

Prenatal disruption of the D1R-SynGAP complex leads to more PV- and CB-positive GABAergic interneurons located in the lateral neocortex of adult offspring

To determine whether impaired GABAergic interneuron migration due to disruption of the D1R-SynGAP complex persists until adulthood, we analyzed the location of interneurons across the medial-lateral axis of the neocortex in adult offspring of the TAT- and TAT-D1Rpep–injected pregnant mice (Fig. 3, A and B). Previous studies show that haploinsufficiency of Syngap1, the gene encoding SynGAP, reduces perisomatic innervation by PV-positive basket cells, which are most of the GABAergic interneurons (28). Using immunostaining with anti-PV antibody, we found more PV-positive cells in the lateral neocortex and fewer in the medial region in adult offspring from the TAT-D1Rpep group compared to TAT controls. Similar results were observed for CB-positive interneurons (Fig. 3, C to F). These results confirm that impaired embryonic GABAergic interneuron migration results in abnormal GABAergic interneuron location in the adult mouse brain.

Fig. 3 Abnormal distribution of both PV and CB interneurons in adult offspring from TAT-D1Rpep–injected pregnant mice.

(A) Schematic showing the timeline of peptide injection and immunostaining. (B and C) Five sampling ROIs immunolabeled with either PV or CB antibodies were outlined across the neocortex along the medial-lateral axis. The number of PV- and CB-positive cells was counted and presented as a percentage of total cells in the five ROIs. Scale bar, 200 μm. (D) Distribution of PV- and CB-labeled cells in the whole five bins across the neocortex along the medial-lateral axis. Scale bar, 500 μm. (E and F) Statistical significance was assessed for the distribution of PV-labeled (E) and CB-labeled (F) cells in the neocortex along the medial-lateral axis. Data are means ± SEM of n = 8 ROIs from three different mice per group. *P < 0.05 and **P < 0.01 as compared to TAT group by two-way ANOVA, followed by Bonferroni post hoc test. ROI, region of interest; PV, parvalbumin.

SynGAP enhances D1R-mediated signaling through the D1R-SynGAP interaction

Because protein-protein interactions can be a critical regulator of receptor-mediated downstream functions, we sought to determine the potential functions of the D1R-SynGAP complex on D1R-mediated signaling cascades. We first compared D1R-mediated cyclic adenosine monophosphate (cAMP) accumulation in HEK293T cells coexpressing both D1R and SynGAP to cells expressing D1R alone. HEK293T cells are a common model system to overexpress exogenous proteins by transient transfection. Preincubation with SKF81297 (10 μM for 15 min) in HEK293T cells coexpressing D1R and SynGAP resulted in significantly larger cAMP accumulation, compared to HEK293T cells with D1R alone (Fig. 4A).

Fig. 4 SynGAP modulates D1R-mediated signaling and enhances D1R cell surface membrane expression.

(A) cAMP analysis in HEK293T cells expressing D1R with SynGAP or D1R alone in the presence or absence of SKF81297 (10 μM for 15 min). Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to D1R + pcDNA3 + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (B and C) Representative blots (B) and densitometric analysis (C) of PKA phosphorylation (Thr197) in HEK293T cells expressing D1R with SynGAP and HEK293T cells expressing D1R alone. PKA was used as a loading control. The amount of p-PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 by two-way ANOVA, followed by Bonferroni post hoc test. (D and E) Representative blots (D) and densitometric analysis (E) of p38 phosphorylation in HEK293T cells expressing D1R with SynGAP and HEK293T cells expressing D1R alone. The amount of p-p38 was normalized to that of p38. Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to D1R + pcDNA3 + control group and ***P < 0.001 as compared to D1R + pcDNA3 + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (F) cAMP analysis in primary cultured mouse neurons pretreated with TAT or TAT-D1Rpep in the presence or absence of SKF81297 (10 μM for 15 min). Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (G and H) Representative blots (G) and densitometric analysis (H) of PKA phosphorylation (T197) in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of p-PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (I and J) Representative blots (I) and densitometric analysis (J) of p38 phosphorylation in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of p-p38 was normalized to that of p38. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (K and L) Representative blots (K) and densitometric analysis (L) of cell surface membrane localization of GFP-D1R proteins in HEK293T cells expressing GFP-D1R with SynGAP and HEK293T cells expressing GFP-D1R alone. The amount of biotinylated GFP-D1R was normalized to that of total GFP-D1R. Results for each sample are presented as the percentage of the D1R + pcDNA3 sample. **P < 0.01 as compared to D1R + pcDNA3 group by t test. Data are means ± SEM of n = 3 independent experiments. (M and N) Representative blots (M) and densitometric analysis (N) of cell surface membrane localization of D1R in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of biotinylated D1R was normalized to that of total D1R. Results for each sample are presented as the percentage of the TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT group by t test.

We then examined the abundance of D1R-mediated downstream cAMP pathway signaling molecules, including phosphorylated (activated) PKA (at Thr197) and phosphorylated p38 MAPK in HEK293T cells under similar conditions. We found that HEK293T cells expressing both D1R and SynGAP that were treated with SKF81297 (10 μM for 15 min) had significantly greater abundance of phosphorylated PKA (Fig. 4, B and C) and p38 MAPK (Fig. 4, D and E) than cells expressing D1R alone. These data demonstrate the functional role of SynGAP in regulating D1R-mediated signaling. Furthermore, activation of D1Rs in mouse primary cultured neurons endogenously expressing D1R and SynGAP facilitated cAMP accumulation (Fig. 4F) and increased the abundance of phosphorylated PKA (Fig. 4, G and H) and p38 MAPK (Fig. 4, I and J). These effects were decreased by pretreatment with TAT-D1Rpep as compared to those treated with TAT peptide (10 μM for 1 hour) (Fig. 4, F to J), suggesting that the D1R-SynGAP complex is responsible for the observed up-regulation of D1R-mediated signaling by SynGAP.

Through its interaction with D1R, SynGAP enhances the localization of D1R to the cell surface

We have demonstrated that the interaction between D1R and SynGAP enhances D1R-mediated signaling. Because other studies have reported that protein-receptor interactions can affect cellular localization of the receptor (29, 38), we investigated whether the D1R-SynGAP interaction affects D1R surface localization. Using a biotinylation assay to selectively label D1Rs on the plasma membrane, we detected an increase in the abundance of biotinylated D1Rs in HEK293T cells expressing both D1R and SynGAP compared to those expressing D1R alone (Fig. 4, K and L). As expected, pretreatment with TAT-D1Rpep (10 μM for 2 hours) decreased the amount of biotinylated D1Rs in primary cultured mouse neurons, as compared to pretreatment with TAT alone (Fig. 4, M and N), suggesting that SynGAP can facilitate D1R localization to the cell surface through the D1R-SynGAP interaction.

Disruption of the D1R-SynGAP complex promotes actin depolymerization

Many studies have provided evidence that the actin cytoskeleton is crucial in regulating tangential interneuron migration (39). Microtubules and actin filaments are the principal components of the cytoskeleton (40, 41). Therefore, to further investigate whether cytoskeletal abnormalities mediate the deficits in GABAergic interneuron migration observed after TAT-D1Rpep administration, we measured the abundance of phalloidin [filamentous actin (F-actin)] and cortactin, two proteins implicated in actin polymerization and actin cytoskeleton stabilization. TAT-treated interneurons had significantly more F-actin present at the leading process tips (Fig. 5, A to C). However, after TAT-D1Rpep treatment, there was a marked reduction in F-actin concentration at the leading process tips, and the F-actin present was widely dispersed along the process shaft. Similar results were observed when SynGAP expression was knocked down by small interfering RNA (siRNA) transfection in interneurons (fig. S6A) and the effect of SynGAP siRNA was significantly rescued by the cotransfection of siRNA-resistant SynGAP (Fig. 5, D to F). This indicates that disruption of the D1R-SynGAP interaction reduces actin concentration near the developing neurites of interneurons, which may affect proper interneuron migration.

Fig. 5 Disruption of the D1R-SynGAP complex decreases actin at the leading process tips by increasing cortactin proteolysis and decreasing PKA phosphorylation.

(A to C) TAT- and TAT-D1Rpep–treated interneurons labeled for F-actin in green (phalloidin), cortactin in red (anticortactin), and nuclei in blue (DAPI) (A). The percentage of F-actin–positive tips in all the tips within an entire cell was significantly reduced in TAT-D1Rpep–treated interneurons (B), as well as the number of F-actin–positive tips per cell (C). Data are means ± SEM of n = 15 cells from three independent primary cultures. *P < 0.05 and ***P < 0.001 as compared to TAT group by t test. (D to F) Interneurons transfected with control, SynGAP siRNA, or SynGAP siRNA + siRNA-resistant (siRNA-R) SynGAP were labeled for F-actin in green (phalloidin), cortactin in red (anticortactin), and nuclei in blue (DAPI) (D). The percentage of F-actin–positive tips in all the tips within an entire cell was significantly reduced in SynGAP siRNA–transfected interneurons (E), as well as the number of F-actin–positive tips per cell (F). Data are means ± SEM of n = 15 cells from three independent primary cultures. *P < 0.05 and ***P < 0.001 as compared to control group by one-way ANOVA, followed by Dunnett’s test. (G and H) Representative blots (G) and densitometric analysis (H) of proteolytic cortactin in mouse embryonic brain tissue from TAT- or TAT-D1Rpep–injected groups. The amount of proteolytic cortactin was normalized to that of the loading control α-tubulin. Results for each sample are presented as the percentage of the mean of TAT-injected group. Data are means ± SEM of n = 5 (TAT) or 7 (TAT-D1Rpep) embryonic brains from three pregnant mice. *P < 0.05 compared to TAT group by t test. (I and J) Representative blots (I) and densitometric analysis (J) of PKA phosphorylation (Thr197) in embryonic brain tissue from TAT- or TAT-D1Rpep–injected groups. The amount of phosphorylated PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the mean of TAT-injected group. Data are means ± SEM of n = 5 (TAT) or 7 (TAT-D1Rpep) embryonic brains isolated from three pregnant mice. **P < 0.01 compared to TAT group by t test. (K and L) Representative blots (K) and densitometric analysis (L) of proteolytic cortactin in primary cultured mouse neurons transfected with control siRNA, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP. The amount of proteolytic cortactin was normalized to that of the loading control α-tubulin. Results for each sample are presented as the ratio of SKF81297 to control. Data are means ± SEM of n = 6 independent experiments. **P < 0.01 compared to control siRNA group by one-way ANOVA, followed by Dunnett’s test. (M and N) Representative blots (M) and densitometric analysis (N) of PKA phosphorylation (Thr197) in primary cultured mouse neurons transfected with control siRNA, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP. PKA was used as a loading control. The amount of phosphorylated PKA was normalized to that of PKA. Results for each sample are presented as the ratio of SKF81297 to control. Data are means ± SEM of n = 6 independent experiments. ***P < 0.001 as compared to control siRNA group by one-way ANOVA, followed by Dunnett’s test.

We also assessed the proteolytic protein cortactin as an indicator of actin depolymerization. TAT-D1Rpep–injected groups had significantly more proteolytic cortactin when compared to TAT controls (Fig. 5, G and H), suggesting that the reduced F-actin concentration previously observed could be a result of enhanced cortactin proteolysis. There is evidence that cortactin proteolysis is inhibited upon PKA activation (39). Because SynGAP had enhanced D1R-mediated PKA phosphorylation in HEK293T cells and primary cultured neurons (Fig. 4, B and C), we hypothesize that perturbing the D1R-SynGAP interaction would decrease PKA activity in vivo. As seen in HEK293T cells and primary cultured neurons, there was significantly less phosphorylated PKA in TAT-D1Rpep–injected embryonic mouse brain tissues than in those of TAT controls (Fig. 5, I and J). Similar results were observed when the abundance of SynGAP was knocked down in primary cultured neurons by siRNA: Proteolytic cortactin was increased (Fig. 5, K and L, and fig. S6B), phosphorylated PKA was decreased (Fig. 5, M and N, and fig. S6C) in SynGAP siRNA–transfected neurons, and the effect of SynGAP siRNA was significantly rescued by the cotransfection of siRNA-resistant SynGAP (Fig. 5, K to N). Overall, our data indicate that disruption of the D1R-SynGAP complex decreases actin concentration and polymerization, possibly via altered PKA activity and increased cortactin proteolysis.

Disruption of the D1R-SynGAP complex changes microtubule dynamics

Migrating neurons undergo cytoskeletal reorganization during neurite extension and nuclear translocation (42, 43). Previous studies showed that activation of the D1R induces redistribution of cytoplasmic dynein motor protein (16). Thus, we examined whether D1R-SynGAP disruption would alter the distribution of cytoplasmic dynein motor proteins in primary neuron cultures from the MGE. Primary cultured neurons at 5 days in vitro (DIV 5) were treated with TAT-D1Rpep or TAT peptide and immunostained with anti–β III-tubulin (TuJ1) and anti–cytoplasmic dynein heavy chain (CDHC) antibodies. In TAT-treated neurons, CDHC was present in neurites and within the cell body, whereas in TAT-D1Rpep–treated groups, CDHC was mainly localized within the soma, with only a small portion labeled in the proximal parts of the neurites near the cell body (Fig. 6, A to C). Although TuJ1 remained localized in the neurites, the intensity of its detection was lower neurons treated with TAT-D1Rpep. Similar results were observed when SynGAP abundance was knocked down in interneurons by siRNA (Fig. 6, D to F).

Fig. 6 Disruption of the D1R-SynGAP complex changes neuronal cytoskeleton by decreasing MAP2 phosphorylation.

(A to C) Primary cultured MGE-derived cells were treated with TAT or TAT-D1Rpep, and immunostaining was performed to analyze distribution of the motor protein cytoplasmic dynein heavy chain (CDHC; green) or the cytoskeletal protein neuronal β III-tubulin (TuJ1; red). Nuclei were labeled with the DNA stain DAPI (blue) (A). Quantification of the immunolabeling intensity from soma to leading process tip for CDHC (B) and TuJ1 (C). Images represent 15 neurons from three independent experiments performed. (D to F) Primary cultured MGE-derived cells were transfected with control, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP, and immunostaining was performed to analyze distribution of the motor protein CDHC (green) or the cytoskeletal protein neuronal TuJ1 (red). Nuclei were labeled with the DNA stain DAPI (blue) (D). Quantification of the immunolabeling intensity from soma to leading process tip for CDHC (E) and TuJ1 (F). Images represent 15 neurons from three independent experiments performed. (G and H) Representative blots (G) and densitometric analysis (H) of MAP2 phosphorylation in mouse brain slices treated with TAT or TAT-D1Rpep (10 μM for 30 min before SKF81297 treatment) in the absence or presence of SKF81297 (10 μM for 30 min). The amount of p-MAP2 was normalized to that of MAP2. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. **P < 0.01 as compared to TAT group and ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (I to K) Representative blots (I) and densitometric analysis (J and K) of ERK1/2 phosphorylation in mouse brain slices treated with TAT or TAT-D1Rpep (10 μM for 30 min before SKF81297 treatment) in the absence/presence of SKF81297 (10 μM for 30 min). The amount of p-ERK1(/2) was normalized to that of ERK1(/2). Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test.

Microtubule-associated protein 2 (MAP2) and dynein have opposite effects on neuronal migration through their binding to tubulin with the dynein-tubulin interaction facilitating neuronal migration and the MAP2-tubulin interaction inhibiting this process (44, 45). This MAP2-tubulin effect is dependent on MAP2 phosphorylation (46), which can be mediated through D1R signaling (18, 47). Thus, we hypothesized that the inhibitory effect of TAT-D1Rpep on interneuron migration might be related to an increased interaction between MAP2 and tubulin, secondary to decreased MAP2 phosphorylation. Western blot analysis using anti–pan-phosphorylation antibodies and an antibody to MAP2 showed that MAP2 phosphorylation was significantly decreased in protein extracts from mouse brain slices pretreated with TAT-D1Rpep but not with TAT control peptide (Fig. 6, G and H). Because ERK1 and ERK2 (ERK1/2) regulate MAP2 phosphorylation, we also examined ERK1/2 phosphorylation in mouse brain slices. The D1R-mediated increase in phosphorylation of ERK1/2 was also blocked by pretreatment with TAT-D1Rpep but not with the TAT peptide (Fig. 6, I to K). These data indicate that the D1R-SynGAP interaction might regulate GABAergic interneuron migration through modulation of MAP2 phosphorylation and its effect on tubulin.

DISCUSSION

In this study, we identified a previously unknown interaction between the D1R and SynGAP, which enhances D1R-mediated downstream signaling including cAMP production and phosphorylation of PKA, p38, and ERK1/2. This interaction is involved in the regulation of interneuron tangential migration, possibly through affecting actin and microtubule dynamics (Fig. 7).

Fig. 7 Schematic diagram of interaction between D1R and SynGAP.

SynGAP enhances D1R membrane expression and D1R-mediated signaling pathway, including cAMP accumulation, phosphorylation of PKA, p38, and ERK1/2, through the D1R-SynGAP interaction. Increased phospho-PKA leads to decreased cortactin proteolysis and thus increases actin polymerization. Increased phosphorylated ERK1/2 results in increased MAP2 and thus increases cytoplasmic dynein motor protein-tubulin interaction. Both changes in actin and microtubule dynamics lead to enhanced GABAergic interneuron tangential migration.

Conventionally, upon the binding of an agonist, a G protein–coupled receptor exerts its effects by coupling to G proteins, which subsequently stimulates signal transduction pathways involving cAMP accumulation and phosphatidylinositol-mediated protein kinase C activation (17, 18). However, studies have shown that protein-receptor interactions can also modulate receptor-mediated downstream effects by regulating receptor expression, trafficking, and signaling cascades (19–21). For example, our group has previously shown that an NMDA receptor subunit regulates D1R-mediated cAMP accumulation through its interaction with D1Rs (38). The effects of receptor-interacting proteins on cell membrane trafficking or localization have been observed with proteins [such as the glutamate receptor–interacting protein, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–binding protein, and the N-ethylmaleimide–sensitive fusion protein] that interact with AMPA receptor GluR2 subunits to modulate receptor expression (48). Thus, a possible mechanism by which the D1R-SynGAP interaction affects downstream D1R function is that SynGAP binds to the D1Rs and blocks D1R endocytosis or internalization or promotes its incorporation into the membrane, thus increasing D1R surface abundance and D1R-mediated downstream signaling.

Interneuron tangential migration is a complex process, governed by multiple factors including cytoskeletal proteins, neurotrophins, and transcription factors (10–13). Previous studies have demonstrated the expression of D1Rs in interneurons could regulate interneuron migration (15). Crandall et al. (16) showed that activation of D1Rs promotes interneuron migration, which is consistent with our results here, showing that disruption of D1R function perturbs their migration. We found that this deficit was associated with altered actin dynamics, suggesting that cytoskeletal proteins are possibly also affected by D1R-SynGAP disruption as are neurotrophins and other factors. Furthermore, treatment with the disrupting peptide impaired MAP2 phosphorylation, leading to more tubulin binding. This may represent another cytoskeletal pathway causing migration deficits, because previous studies have shown that phosphorylated MAP2 binds with lower affinity to tubulin, thereby promoting dynein-tubulin interaction and neuronal migration. More research is necessary to completely decipher all the cytoskeletal proteins involved in regulating interneuron migration that are mediated by D1Rs.

We found evidence of a D1R-SynGAP interaction in glutamatergic neurons, but we failed to observe any disrupting peptide-induced changes in glutamatergic neuron migration in embryonic cortical explants, suggesting that the D1R-SynGAP interaction may play a different role in glutamatergic neurons than interneurons. Functional interactions between D1R and NMDA receptors have been investigated by many laboratories, including ours (38, 49), showing that D1R modulates NMDA receptor–mediated long-term potentiation (LTP) through a PKA-dependent pathway (50–52). Our data suggest that SynGAP increases D1R-mediated PKA signaling. Thus, it is logical to predict that the D1R-SynGAP interaction might be involved in D1R-mediated modulation of NMDA receptor functions, such as LTP.

In rodents, the tangential migration of GABAergic interneurons begins from the prenatal stage and is more or less complete by P12, from which point they will continue to mature and establish connections with the neighboring neurons (53). This period of neuron migration during early neurodevelopment is sensitive to disruption, and any alterations in interneurons during this period may have profound consequences. For instance, Belforte et al. (54) created a conditional mutant mouse in which the NR1 subunit of the NMDA receptor was selectively deleted in about half of the cortical and hippocampal interneurons in early postnatal development. Cognitive abnormalities including deficits in spatial memory and social memory emerged in mutants after adolescence. If the conditional knockout was performed after adolescence, then the mutant mice did not exhibit such cognitive deficits. Thus, there appears to be a sensitive period before adolescence during which disruptions to interneuron development can have adverse effects on adult brain function.

The prefrontal cortex (PFC) is highly interconnected with many other brain regions, which is reflected in its functional involvement in a wide range of behaviors (55, 56). For instance, excitatory neurons originating from the PFC innervate GABAergic neurons in the ventral tegmental area (VTA), which can in turn regulate the activity of VTA dopamine neurons and, therefore, the extracellular level of dopamine within the mesolimbic area (such as the nucleus accumbens) (57–59). The extracellular dopamine concentration in the mesolimbic system is known to regulate dopamine-driven behaviors such as voluntary locomotion (60) and sensorimotor gating (61, 62). Lesion studies in rodents have also implicated the PFC in social behaviors (63, 64). Normally, GABAergic interneurons modulate the activity of neighboring pyramidal neurons (65). Disruption of the D1R-SynGAP complex produces interneuron migration deficits, which may therefore have other effects on cognition and behaviors that we did not examine in this study.

In summary, we have identified a previously unknown interaction between the D1R and SynGAP that regulates interneuron tangential migration, actin dynamics, and MAP2 phosphorylation. This new insight about the regulation of interneuron development could be the starting point for further studies and could also represent a previously unknown treatment target for various neurodevelopmental disorders.

MATERIALS AND METHODS

Drugs and peptides

SKF81297 (catalog number: S179, Sigma-Aldrich) and SCH23390 (catalog number: D054, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (catalog number: 276855, Sigma-Aldrich) to a stock solution of 10 mM. TAT-D1Rpep or TAT peptide, which was synthesized by Biomatik, was dissolved in H2O to a stock solution of 10 mM. For injection into pregnant mice, 3 nmol/g body weight of TAT-D1Rpep or TAT peptide was used with 0.9% saline to make a final injection volume of 300 μl.

Coimmunoprecipitation, GST affinity pulldown, and Western blot

Protein affinity purification, coimmunoprecipitation, and Western blot analyses were performed as previously described (29, 30). For coimmunoprecipitation experiments, 500 to 700 μg of solubilized protein extracted from mouse brain tissue or HEK293T cells (American Type Culture Collection) was incubated in the presence of primary antibodies or immunoglobulin G (negative control) (1 to 2 μg) together with protein A/G plus agarose (25 μl per sample; catalog number: sc-2003, Santa Cruz Biotechnology) with gentle shaking for 12 hours at 4°C. Pellets were washed, boiled for 5 min in SDS sample buffer (catalog number: 161–0737, Bio-Rad) and 2-Mercaptoethanol (M7154, Sigma-Aldrich), and subjected to SDS–polyacrylamide gel electrophoresis (PAGE). A total of 50 to 100 μg of protein extracted from tissue was used as a control in each experiment. For protein affinity purification experiments, 500 to 700 μg of protein was incubated with Glutathione Sepharose 4B (30 μl per sample; catalog number: 27-4574-01, Amersham) bound to the indicated GST fusion proteins (50 to 100 μg) at 4°C overnight. Beads were washed, boiled for 5 min in SDS sample buffer, and subjected to SDS-PAGE. After transfer of proteins onto nitrocellulose, membranes were Western-blotted with the primary antibodies specified in the antibody information. The intensity of protein abundance was quantified by densitometry (software: ImageJ from National Institutes of Health and Image Lab from Bio-Rad).

The antibodies used were those against D1R (2 μg per sample; goat, catalog number: sc-1434, Santa Cruz Biotechnology), SynGAP (2 μg per sample; rabbit, catalog number: sc-33598, Santa Cruz Biotechnology), and GFP (2 μg per sample; rabbit, catalog number: sc-8334, Santa Cruz Biotechnology) for immunoprecipitation; D1R (1:1000; rat, catalog number: D2944, Sigma-Aldrich), SynGAP (1:500; rabbit, catalog number: sc-33598, Santa Cruz Biotechnology), and GFP (1:1000; mouse, catalog number: sc-9996, Santa Cruz Biotechnology) for Western blotting; phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; rabbit, catalog number: 9101, Cell Signaling Technology); p44/42 MAPK (ERK1/2) (1:1000; rabbit, catalog number: 9102, Cell Signaling Technology); PKA C-α (1:1000; rabbit, catalog number: 5842, Cell Signaling Technology); phospho PKA α+β (catalytic subunits) (Thr197) (1:1000; rabbit, catalog number: ab5815, Abcam); p38 MAPK (1:1000; rabbit, catalog number: 9212, Cell Signaling Technology); phospho–p38 MAPK (Thr180/Tyr182) (1:1000; rabbit, catalog number: 4511, Cell Signaling Technology); pan-phosphorylation (1:1000; rabbit, catalog number: 61-8300, Invitrogen); MAP2 (1:500; rabbit, catalog number: AB5622, Millipore); and cortactin (1:1000; mouse, catalog number: ab33333, Abcam).

GST fusion protein constructs

GST fusion proteins encoding truncated D1R fragments were amplified by polymerase chain reaction (PCR) from full-length human complementary DNA (cDNA) clones. All constructs were sequenced to confirm the absence of PCR-generated nucleotide errors. GST fusion proteins were prepared from bacterial lysates with Glutathione Sepharose 4B beads as instructed by the manufacturer (Amersham), as previously described. To construct GST fusion proteins encoding truncated D1R, cDNA fragments were amplified by PCR with specific primers. Except where specified, all 5′ and 3′ oligonucleotides incorporated BamHI sites (GGATCC) (catalog number: R0136, New England Biolabs) and XhoI sites (CTCGAG) (catalog number: R0146, New England Biolabs) (20 U per reaction), respectively, to facilitate subcloning into the pGEX-4T3 vector. All constructs were resequenced to confirm appropriate splice fusion and the absence of PCR-generated nucleotide errors.

HEK293T cell culture and DNA transfection

HEK293T cells were maintained at 37°C in Dulbecco’s modified Eagle’s medium (catalog number: 11995-065, Invitrogen) supplemented with 10% fetal bovine serum (catalog number: 10082147, Gibco). Cells were grown to 90% confluency before being transiently transfected with DNA constructs using X-tremeGENE 9 transfection reagent (catalog number: 6365787001, Roche) following the manufacturer’s instruction. Cells were used for various experiments after 24 to 48 hours of transfection. SynGAP siRNA was transfected with Lipofectamine RNAiMAX transfection reagent (catalog number: 13778150, Invitrogen).

SynGAP siRNA construct is a gift from B. J. Hall in Tulane University. It was generated in pSilencer vector and characterized target 19–base pair sequence recognizing the alpha isoform of SynGAP: CCAGCAAGATCCTGATGCA, whereas the control sequence was GCGACATAGCGCACATACT.

cAMP accumulation assay

cAMP accumulation assays were performed in primary cultured mouse neurons or HEK293T cells transiently transfected with D1R and SynGAP or D1R alone using the Parameter cAMP Assay Kit (catalog number: KGE002B, R&D Systems) as directed by the manufacturer (R&D Systems). Briefly, transfected HEK293T cells were pretreated with 10 μM SKF81297 for 15 min, washed in cold phosphate-buffered saline (PBS) [150 mM NaCl, 5.8 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4 (pH 7.4)], and resuspended in cell lysis buffer to a concentration of 1 × 107 cells/ml. Similarly, primary cultured neurons were treated with TAT-D1Rpep or TAT peptide (10 μM for 1 hour) before SKF81297 administration (10 μM for 15 min). Cells were then frozen at −20°C, followed by thawing with gentle mixing, and cell lysis was confirmed using Trypan blue (catalog number: 15250061, Invitrogen) staining. Cellular debris was removed by centrifuging at 600g for 10 min at 4°C, and the supernatant was assayed immediately by the Parameter cAMP Assay.

Surface biotinylation assay

Surface biotinylation assays were performed either with HEK293T cells transfected with GFP-D1R and SynGAP or GFP-D1R alone for 48 hours or with primary cultured mouse neurons treated with TAT-D1Rpep or TAT peptide (10 μM for 2 hours) on DIV 5. Cells were washed twice with ice-cold PBS and incubated for 2 hours at 4°C with EZ-Link Sulfo-NHS-LC-Biotin (1 mg/ml; catalog number: A39257, Pierce) to biotinylate surface proteins. Excess biotin reagent was quenched and removed by washing the cells with ice-cold PBS containing 100 mM glycine. Cells were lysed with lysis buffer [150 mM NaCl, 2.7 mM KCl, 5.8 mM Na2HPO4, 1.5 mM KH2PO4, 1% Triton X-100, 0.1% SDS, and protease inhibitor mixture (catalog number: P8340, Sigma-Aldrich) (pH 7.4)]. The lysates were centrifuged at 12,000g for 5 min to yield the protein extract in the supernatant. Protein concentration was quantified using the bicinchoninic acid (BCA) protein assay (catalog number: PI23224, Pierce). A total of 200 to 500 μg of protein extracts were incubated with Streptavidin Plus UltraLink Resin (15 μl per sample; catalog number: 53116, Pierce) overnight at 4°C to capture biotinylated surface protein. After washing with lysis buffer, bound proteins were eluted by boiling for 5 min with SDS sample buffer and subjected to SDS-PAGE.

Primary culture of MGE-derived neurons

MGE tissues were dissected from E15 embryonic mouse brains, digested with 0.25% trypsin-EDTA (catalog number: 15090-046, Invitrogen) at 37°C for 15 min, followed by Neurobasal medium (catalog number: 21103-049, Gibco) and 10% fetal bovine serum (Invitrogen) to inactivate trypsin activity, and centrifuged at 100g for 5 min. The supernatant solution was replaced with Neurobasal/B27 (catalog number: 17504044, Invitrogen) medium supplemented with GlutaMAX (catalog number: 35050-061, Invitrogen). The pellet was then mechanically dissociated by gentle trituration through a fire-polished Pasteur pipette. The cell suspension was plated at a density of 1 × 105/cm2 onto poly-d-lysine (working concentration is 0.2 mg/ml; catalog number: P1024, Sigma-Aldrich)–coated coverslips (catalog number: 12-545-80, Fisher Scientific) in 24-well culture plates (catalog number: 351147, Falcon), poly-d-lysine–coated 6-well culture plates (catalog number: 351146, Falcon) or 60-mm culture dishes (catalog number: 628160, Greiner). Cultures were incubated at 37°C in a 5% CO2 incubator in Neurobasal/B27+GlutaMAX medium. Half of the medium was changed every 3 days until DIV 10. Cytarabine (Ara-C) (working concentration is 5 μM; catalog number: C1768, Sigma-Aldrich) was added to the medium from DIV 4 onward to prevent glial cell differentiation.

MGE and cortex Matrigel explants

MGE or cortex explants were prepared from E14 CD-1 mice without any treatment or E18 CD-1 mouse embryos after intraperitoneal injection with TAT or TAT-D1Rpep into the pregnant mice from E12 to E17. Small tissue fragments corresponding to the subventricular zone of the MGE or cortex were dissected out, placed in a three-dimensional Matrigel gel matrix (catalog number: 356237, BD Biosciences, San Jose, CA), and cultured for 24 hours in Neurobasal medium supplemented with B27 and GlutaMAX in six-well plates (Falcon) at 37°C in a 5% CO2 incubator, as previously described (66) . For explants from E14 CD-1 mice, saline, TAT or TAT-D1Rpep (10 μM) was added into the culture medium right after the explants were placed in to the plates. After 24 hours, MGE explants were imaged at ×10 magnification on an Olympus FV10i confocal microscope.

Acute mouse brain slices

Acute brain tissues were dissected and prepared from CD-1 mice with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK) and remained in ice-cold artificial cerebrospinal fluid (aCSF) containing 126 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3, and 20 mM glucose, which was bubbled continuously with 95% O2/5% CO2 for 5 min. Freshly cut slices (350-μm-thick) were then placed in an incubating chamber with carbogenated aCSF and recovered from stress at 35°C for 1 hour. They were treated with either TAT-D1Rpep or TAT peptide (10 μM for 1 hour) before treatment with SKF81297 (10 μM for 15 min) and harvested for Western blot analysis or coimmunoprecipitation assays.

Immunocytochemistry

Primary cultures from MGE were fixed in 4% paraformaldehyde (dissolved in PBS; catalog number: P6148, Sigma-Aldrich) for 20 min at room temperature. After washes with PBS, cells were permeabilized for 15 min with PBS containing 0.3% Triton X-100 (catalog number: T8787, Sigma-Aldrich) and incubated in blocking solution [0.3% Triton X-100 and 5% BSA (bovine serum albumin; catalog number: A8806, Sigma-Aldrich) in PBS] for 2 hours at room temperature. The cells were then incubated in primary antibodies and diluted in PBS containing 0.3% Triton X-100 and 1% BSA overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. The coverslips with cells were mounted using ProLong Gold Antifade Mountant (catalog number: P36934, Invitrogen). The primary antibodies used were those against calbindin D-28k (1:200; rabbit, catalog number: AB1778, Millipore), SynGAP (1:200; rabbit, catalog number: ab3344, Abcam), D1R (1:200; rabbit, catalog number: ab20066, Abcam), cortactin (1:200; mouse, catalog number: ab33333, Abcam), β III-tubulin (TuJ1) (1:500; mouse, catalog number: MAB5564, Millipore), and Dynein HC (CDHC) (1:100; rabbit, catalog number: sc-9115, Santa Cruz Biotechnology). Fluorescent secondary antibodies were conjugated to either Alexa Fluor 488 (donkey–anti-rabbit, catalog number: A21206; goat–anti-mouse, catalog number: A11029) or Alexa Fluor 594 (donkey–anti-mouse, catalog number: A21203; goat–anti-rabbit, catalog number: A11037) (1:200; Invitrogen). Alexa Fluor 488 phalloidin (catalog number: A12379, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI) (two drops in 1 ml of PBS; catalog number: R37606, Invitrogen) were used according to the manufacturer’s instructions.

Immunohistochemistry

Both embryonic and postnatal mouse brains were fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose and stored at −80°C before further processing. Frozen coronal sections of 20-μm thickness were cut using a cryostat (Bright Instrument Co. 5030). All sections were initially permeabilized with PBS and 0.3% Triton X-100 (Sigma-Aldrich) and incubated in blocking solution [0.3% Triton X-100 and 5% BSA (Sigma-Aldrich) in PBS] for 2 hours at room temperature to reduce nonspecific background. Then, the sections were incubated with primary antibodies and diluted in PBS containing 0.3% Triton X-100 and 1% BSA overnight at 4°C. After this, the sections were washed in PBS, incubated with secondary antibodies, and diluted in the same solution as primary antibodies for 2 hours at room temperature. Last, the slices were mounted on slides using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). The primary antibodies used were those against calbindin D-28k (1:200; rabbit, catalog number: AB1778, Millipore), parvalbumin (1:200; mouse, catalog number: P3088, Sigma-Aldrich), and HIV-1 TAT (1:500; mouse, catalog number: ab43014, Abcam). Fluorescent secondary antibodies conjugated to either Alexa 488 or Alexa 594 (1:200, Invitrogen) were used to detect the primary antibodies.

Confocal imaging

Confocal images were acquired using a confocal microscope (Olympus FluoView FV1200) at ×20 or ×60 magnification for primary cultured neurons (1.5× zoom) and ×10 for brain slices (Fig. 3A) and MGE explants. All other immunohistochemistry images were captured using a confocal microscope (Zeiss LSM 510 Meta) at ×10 magnification. ImageJ was used for analysis of the distribution of CDHC and TuJ1. The dendritic branches were straightened, and plot profile was acquired. Phalloidin-positive tips in primary cultured neurons were manually counted.

Analysis of immunohistochemistry

As previously described (67), all immunohistochemistry images (except Fig. 3A) were captured using a confocal microscope (Zeiss LSM 510 Meta) at ×10 magnification, converted to gray scale, and normalized to background staining. Sections chosen for analysis were anatomically matched between comparing groups and included samples from rostral, medial, and caudal regions. For the analysis of interneuron distribution across the cortex, a selected curved region (300-μm-wide) from the dorsal cortex to ventral preoptic area was outlined, straightened, and divided into seven equal regions of interest (ROIs) to capture the tangential migratory paths of newborn interneurons (ImageJ). Fluorescently labeled cells within each ROI were counted using the Image-based Tool for Counting Nuclei (ITCN) plugin for ImageJ (ITCN parameters: width, 20 to 25 pixels; minimum distance, 10 to 13 pixels; threshold, 0.3 pixels). For the analysis of interneuron distribution across the neocortex in adult offspring, five ROIs immunolabeled with either PV or CB antibodies were outlined across the neocortex along the medial-lateral axis. The number of PV- and CB-positive cells was counted and presented as a percentage of total cells in the five ROIs.

Statistical analyses

Data were analyzed by t test, one-way analysis of variance (ANOVA) followed by Tukey’s test or Dunnett’s test, or two-way ANOVA followed by Bonferroni post hoc test (GraphPad Prism 5.0, GraphPad Software, CA, USA). All data were expressed as means ± SEM. The significance thresholds of P < 0.05, P < 0.01, or P < 0.001 were used for all analyses.

Study approval

All animal experimental procedures were conducted in accordance with the protocols approved by the Centre for Addiction and Mental Health Animal Care Committee.

SUPPLEMENTARY MATERIALS

stke.sciencemag.org/cgi/content/full/12/593/eaau9122/DC1

Fig. S1. Schematic representation of generated GST fusion proteins encoding truncated D1RIL3 segments.

Fig. S2. TAT fusion peptides are able to cross both the placenta and blood-brain barrier, and TAT-D1Rpep is able to disrupt the D1R-SynGAP interaction.

Fig. S3. Cell migration is delayed in cultured MGE explants treated with TAT-D1Rpep in vitro.

Fig. S4. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured MGE neurons.

Fig. S5. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured glutamatergic neurons.

Fig. S6. SynGAP knockdown decreases the effects of D1R activation on cortactin proteolysis and PKA phosphorylation.

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Acknowledgments: We thank M. B. Kennedy at California Institute of Technology for providing the SynGAP cDNA, B. J. Hall at Tulane University for providing the SynGAP siRNA construct, and A. Wong at University of Toronto for editing the manuscript. Funding: This study was funded with support from Centre for Addiction and Mental Health (to F.L.), a NARSAD Young Investigator Grant from Brain & Behavior Research Foundation (to P.S.), and the Ontario Grant Scholarship (to T.K.Y.L.). Author contributions: F.L. oversaw and supervised the overall project. P.S. and T.K.Y.L. planned and performed biochemistry experiments and analyzed data. P.S., T.K.Y.L., and F.H.F.L. performed immunostaining and analyzed imaging data. P.S. and F.H.F.L. planned and performed revised experiments and analyzed data for revision. F.L., P.S., T.K.Y.L., and F.H.F.L. wrote the manuscript with insightful input from A.R.A. and P.J.F. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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