Smoothened receptor signaling regulates the developmental shift of GABA polarity in rat somatosensory cortex

ABSTRACT Sonic hedgehog (Shh) and its patched–smoothened receptor complex control a variety of functions in the developing central nervous system, such as neural cell proliferation and differentiation. Recently, Shh signaling components have been found to be expressed at the synaptic level in the postnatal brain, suggesting a potential role in the regulation of synaptic transmission. Using in utero electroporation of constitutively active and negative-phenotype forms of the Shh signal transducer smoothened (Smo), we studied the role of Smo signaling in the development and maturation of GABAergic transmission in the somatosensory cortex. Our results show that enhancing Smo activity during development accelerates the shift from depolarizing to hyperpolarizing GABA in a manner dependent on functional expression of potassium–chloride cotransporter type 2 (KCC2, also known as SLC12A5). On the other hand, blocking Smo activity maintains the GABA response in a depolarizing state in mature cortical neurons, resulting in altered chloride homeostasis and increased seizure susceptibility. This study reveals unexpected functions of Smo signaling in the regulation of chloride homeostasis, through control of KCC2 cell-surface stability, and the timing of the GABA excitatory-to-inhibitory shift in brain maturation.


INTRODUCTION
Sonic hedgehog (Shh) is an activity-dependent secreted synaptic molecule (Beug et al., 2011). In the presence of Shh ligand, the receptor patched-1 (Ptch1) relieves its constitutive inhibition of the G-protein-coupled transducer smoothened (Smo), leading to activation of downstream signaling factors (Belgacem and Borodinsky, 2015;Briscoe and Thérond, 2013;Riobo et al., 2006). Shh signaling plays a wide range of functions during embryonic development from neuronal cell proliferation to differentiation (Belgacem et al., 2016;Briscoe and Thérond, 2013;Ruat et al., 2012;Traiffort et al., 2010). Furthermore, in the developing mammalian central nervous system, Shh signaling is involved in axonal elongation (Hammond et al., 2009;Parra and Zou, 2010;Yao et al., 2015) and formation of the cortical connectivity (Harwell et al., 2012;Memi et al., 2018). After birth, Shh signaling components are found in several brain regions, including the cerebral cortex and hippocampus, from an early stage of postnatal development to adulthood (Charytoniuk et al., 2002;Petralia et al., 2011Petralia et al., , 2012Rivell et al., 2019;Traiffort et al., 2010). In the adult hippocampus, the Shh pathway plays an essential role in neurogenesis in the dentate gyrus (Antonelli et al., 2019;Breunig et al., 2008). In addition, Shh receptor Ptch1 and its signal transducer Smo are also expressed at the synaptic junctions of the immature and adult hippocampus (Charytoniuk et al., 2002;Mitchell et al., 2012;Petralia et al., 2011), suggesting roles other than control of neurogenesis (Zuñiga and Stoeckli, 2017). For instance, Shh signaling was reported to exert a modulatory action on neuronal electrical activity in the adult brain (Bezard et al., 2003;Pascual et al., 2005) and, more recently, Shh-Smo signaling has been shown to regulate the formation of glutamatergic and GABAergic terminals in hippocampal neurons (Mitchell et al., 2012). These morphological changes are accompanied by an increase in the frequency of excitatory postsynaptic currents (Feng et al., 2016;Mitchell et al., 2012). Taken together, these findings support the view that the Shh signaling pathway plays a crucial role during early postnatal neuronal circuit construction and synaptic plasticity in the hippocampus and cortex. Thus, its impairment may affect neuronal network formation and lead to brain dysfunction. Likewise, both in human and animal models, accumulating evidence indicates that impairment of the Shh-Smo pathway at postnatal stages may contribute to the emergence of neurodevelopmental disorders, including autism spectrum disorders (ASD) (Al-Ayadhi, 2012;Halepoto et al., 2015) and seizures (Feng et al., 2016;Su et al., 2017).
In the early postnatal life, GABA A receptor function changes from depolarizing to hyperpolarizing in a KCC2 (also known as SLC12A5)-dependent manner (Ben-Ari et al., 1989;Rivera et al., 1999). Alteration in the GABA developmental sequence leads to a compromised balance between excitatory and inhibitory transmission that is associated with severe changes in the neuronal network circuitry (Ben-Ari, 2002;Ben-Ari et al., 2007;Kilb et al., 2013;Sernagor et al., 2010;Wang and Kriegstein, 2009) and, subsequently, with the onset of neurodevelopmental brain disorders (Ben-Ari and Holmes, 2005;Kuzirian and Paradis, 2011;Mueller et al., 2015).
Although both Shh-Smo and GABA signaling are critically involved in similar processes of neuronal network formation and are implicated in etiology of the same neurodevelopmental disorders, their putative interplay remains unclear. Here we postulated that Shh components may contribute to the onset of the GABA polarity shift and suggested that deregulation of the Shh-Smo signaling pathway might then lead to an impaired neuronal network and to the emergence of brain disorders in adulthood. Because loss of Smo function results in an embryonic lethal phenotype (Zhang et al., 2001) and Smo activation in Wnt1-Cre;R26SmoM2 mice induces a hyperplasia of the facial processes at embryonic stages (Jeong et al., 2004), we used in utero electroporation (IUE) to target the somatosensory cortex with constructs encoding either negativephenotype (Smo Δ570-581, referred to as Smo-ΔN) (Kim et al., 2009) or constitutively active (Smo A1, referred to as Smo-CA)  Smo variants. Our results reveal an unexpected function for the Smo signaling pathway in regulation of GABAergic developmental timing in the rat cerebral cortex, notably through the modulation of cell-surface expression and stability of KCC2. We further show that impairment of Smo signaling alters the phosphorylation state of KCC2, thus maintaining a depolarizing action of GABA, which leads to an alteration of GABAergic inhibitory transmission and might contribute to the emergence of brain disorders.

Cortical expression pattern of Smo-CA and Smo-ΔN
Previous studies have shown that Shh signaling pathway components, including Ptch1 and Smo, are present in the postnatal mammalian brain (Harwell et al., 2012;Mitchell et al., 2012;Petralia et al., 2011;Rivell et al., 2019). In the mouse neocortex, Shh signaling regulates the formation of neuronal networks in immature brain, suggesting a potential function in construction and maturation of neural circuits during the postnatal periods. In order to evaluate the role of Shh signaling in the developing brain, we used IUE of plasmids to express GFP alone, as a control, or in combination with constitutively active (Smo-CA) or negative-phenotype (Smo-ΔN) forms of Smo, an essential component for Shh signaling. IUE of rat brains was performed at embryonic day 15 (E15) in order to target mainly pyramidal neurons of layers V and VI (Kriegstein and Noctor, 2004). Because Shh signaling is involved in cell division and growth of cortical progenitors (Araújo et al., 2014;Radonjićet al., 2016), we investigated whether Smo-related constructs might produce alterations in the localization of electroporated neuronal cells in embryonic day 20 (E20) rat embryos. We found no significant difference between the distribution of cells electroporated with Smo-CA or Smo-ΔN and the distribution of GFP-only control cells (Fig. 1A We next performed immunostaining of electroporated tissues at postnatal day 15 (P15) using the neuronal markers NeuN and FoxP2 (deeper layer neuron markers) to assist in the histological identification of cortical layers (Fig. 1C) (Wang et al., 2018), whereas loss of Smo activity leads to increased neuronal apoptosis (Qin et al., 2019). In order to assess whether Smo-CA or Smo-ΔN induces apoptosis in electroporated neurons, we performed immunohistochemical analysis to detect cleaved caspase-3, a marker for cell apoptosis (Logue and Martin, 2008) Fig. 3D).
We show here that, in electroporated somatosensory cortices, the constitutively active form of Smo significantly promotes the expression of the target gene Gli1, whereas the negativephenotype form of Smo acts in the opposite way by reducing Gli1 expression.

Smo controls the developmental GABA excitatory-toinhibitory shift
Cortical neuronal network construction during the postnatal period requires the depolarizing-to-hyperpolarizing shift of GABA to occur with precise timing during maturation (Ben-Ari and Holmes, 2005;Wang and Kriegstein, 2009;Wu and Sun, 2015). Because Shh signaling plays a nodal role in brain development (Álvarez-Buylla and Ihrie, 2014;Delmotte et al., 2020;Yao et al., 2016), we investigated whether the manipulation of Smo activity affects the  onset of the GABA excitatory-to-inhibitory postnatal shift at the network level. We performed field recordings of multiunit activity (MUA) in acute cortical slices from control (GFP), Smo-CA-and Smo-ΔN-expressing rats and measured the effect of bath application of the GABA A receptor (GABA A R) agonist isoguvacine (10 μM) on their spiking activity at P14, P20 and P30. In accordance with the known depolarizing action of GABA in the immature neocortex (Kirmse et al., 2015;Riffault et al., 2018), isoguvacine induced an increase of spiking activity at P14 in cortical slices from control and Smo-ΔN-expressing rodents [median of +33.87% (P=0.019) and +86.12% (P=0.019), respectively; Wilcoxon matched-pairs signed test; Fig. 4A,B]. In contrast, isoguvacine decreased the spiking activity in Smo-CA-expressing animals (−12%; P=0.009, Wilcoxon matched-pairs signed test; Fig. 4A,B). At P20, isoguvacine induced either a decrease or an increase of spiking activity in control (−11.37%; P=0.519, Wilcoxon matched-pairs signed test; Fig. 4B Fig. 4C). In developing neocortex, the depolarizing-to-hyperpolarizing shift of GABA depends primarily on enhancement of the functional expression of KCC2 (Ben-Ari et al., 1989;Rivera et al., 1999). To investigate whether the hyperpolarizing shift of GABA observed in Smo-CA-expressing rats might involve activation of KCC2 we applied the KCC2-selective blocker VU0463271 (VU) on cortical slices obtained from Smo-CA-expressing rats at P14. VU application shifted the isoguvacine response from inhibitory to excitatory when compared with the response of non-treated Smo-CA-expressing slices (−12% without VU versus +57.1% with VU; P=0.0009, Mann-Whitney test; Fig. 4D).
Collectively, our data suggest that increasing Smo activity prematurely shifts GABA polarity from excitatory to inhibitory in a KCC2-dependent manner, whereas inhibiting Smo signaling delays the switch in GABA polarity.  Regulation of the KCC2-dependent developmental shift of GABA relies on a complex mechanism involving a progressive increase in the amount of KCC2 protein and its posttranslational modifications via at least two distinct phosphorylation sites, serine 940 (Ser 940 ) and threonine 1007 (Thr 1007 ) Fig. 6B,C). The membrane stability and transporter activity of KCC2 are dependent on the phosphorylation state of intracellular C-terminal domains. For instance, phosphorylation of the Ser 940 residue increases stability and functionality of KCC2, whereas phosphorylation on the Thr 1007 residue enhances KCC2 endocytosis (Lee et al., 2007;Medina et al., 2014). To determine which steps in KCC2 trafficking may be regulated by Smo-related constructs, we carried out a quantitative immunoblotting assay using antibodies recognizing the Ser 940 (Lee et al., 2011)  The phosphorylation of Ser 940 described above is well known for its ability to stabilize KCC2 in the plasma membrane, whereas dephosphorylation of Ser 940 promotes KCC2 endocytosis (Kahle et al., 2013;Lee et al., 2007). We therefore assessed whether Smorelated constructs are able to regulate KCC2 trafficking to the cell surface. As a tool, we used a KCC2 construct tagged in an external loop with the fluorescent protein pHluorin (KCC2-pH ext ) . This construct allows both measurement of the KCC2-pH ext -dependent shift of [Cl − ] i and visualization of surface expression and/or internalization of KCC2-pH ext. (Friedel et al., 2015(Friedel et al., , 2017. As expected, overexpression of KCC2-pH ext in neurons at 9 DIV resulted in a negative shift of E GABA (median of −53.16 mV in control neurons and −86.25 mV in neurons overexpressing KCC2-pH ext ; P<0.001, Mann-Whitney test; Fig. 7A,B), indicating also that at least some portion of KCC2-pH ext molecules were delivered to the plasma membrane. To corroborate these observations, we performed a live-staining analysis of surface-expressed and internalized KCC2-pH ext . Three different pools of KCC2-pH ext were revealed using a multistep immunolabeling protocol: the total expressed amount of protein (F t ), the amount of KCC2-pH ext cell surface expression (F m ) and the amount of internalized KCC2-pH ext (F i ) (Fig. 7C). As positive and negative controls we used overexpression of KCC2-pH ext mutants T906A/T1007A (A/A-KCC2-pH ext ) and ΔNTD-KCC2-pH ext , which are known for their increased and perturbed surface expression abilities, respectively (Friedel et al., 2017). Live-cell immunolabeling of the control non-mutated KCC2-pH ext revealed a well detectable F m signal in the form of clusters, whereas the posthoc multistep immunolabelings revealed the F i pool of molecules and the total amount of expressed KCC2-pH ext (Fig. 7C) ; Fig. 7D). The relative amounts of F m and the membrane-labeled cluster size in KCC2-pH ext -expressing neurons were lower than those in A/A-KCC2-pH ext -expressing neurons and significantly higher than the values revealed in ΔNTD-KCC2-pH ext -transfected cells (for F m : 0.94 a.u. for KCC2-pH ext versus 1.49 a.u. for A/A-KCC2-pH ext and 0.008 a.u. for ΔNTD-KCC2-pH ext ; P=0.002 and P<0.0001, respectively; Fig. 7E; for single cluster membrane size: 1.02 a.u. for KCC2-pH ext versus 1.09 a.u. for A/A-KCC2-pH ext and 0.55 a.u. for ΔNTD-KCC2-pH ext ; P<0.0001 and P<0.0001, respectively; Mann-Whitney test; Fig. 7G). Co-expression of Smo-CA with KCC2-pH ext did not produce a statistically significant change in F i , F m or single surface-labeled cluster size (Fig. 7E-G Large number of studies have illustrated that KCC2 dysfunction facilitates initiation of epileptic seizures (Chen et al., 2017;Moore et al., 2018). Changes in KCC2 Ser 940 phosphorylation that modify neuronal chloride homeostasis and depolarizing strength of GABA strongly affect seizure susceptibility in KCC2 transgenic mice (Silayeva et al., 2015). Finally, we investigated whether overexpression of Smo mutants may influence the susceptibility to seizures. Rats expressing Smo-ΔN or Smo-CA and age-matched control rats (transfected with GFP alone) at P30 were intraperitoneally injected with subconvulsive doses of pentylenetetrazol (PTZ; 25 mg/kg; Fig. 8A), an inhibitor of the GABA A receptors (Klioueva et al., 2001). Previous studies have shown that the lowest threshold susceptibility to PTZ can be observed during the first 2 postnatal weeks, and the highest at around P30 (Klioueva et al., 2001;Vernadakis and Woodbury, 1969). These results suggest that the vulnerability to PTZ is correlated to the maturation of the GABAergic system during postnatal development. Injections of PTZ were continued every 10 min until each rat had generated a generalized tonic-clonic  These findings reveal an unexpected function of the G-proteincoupled receptor smoothened on chloride homeostasis regulation and, in particular, on the timing of the GABA shift from depolarizing to hyperpolarizing in the postnatal developing brain.
The postnatal developmental switch in GABA polarity is tightly regulated by the actions of several peptides, including neurotrophic factors (Aguado et al., 2003;Kelsch et al., 2001;Ludwig et al., 2011;Riffault et al., 2018), hypothalamic neurohormones (Leonzino et al., 2016;Spoljaric et al., 2017;Tyzio et al., 2006), central and peripheral hormones Sawano et al., 2013). Hence, the precise timing of the GABAergic polarity shift depends on the balance between factors promoting or delaying the onset of GABAergic inhibitory transmission. It is noteworthy that these factors are developmentally regulated and/or operate within specific time windows to control the functional expression of KCC2 and associated chloride homeostasis and, consequently, the postnatal maturation of GABAergic transmission in the central nervous system. Interestingly, the excitatory action of GABA at early stages of development coincide with the predominance of trophic factors responsible for the inhibition of KCC2 expression and function, such as the immature form of BDNF ( proBDNF) (Riffault et al., 2018) and the adipocyte hormone leptin . These two factors are expressed shortly after birth and surge during the first postnatal week in rodents Menshanov et al., 2015). They have been shown to promote low KCC2 protein and mRNA expression levels and, consequently, a high intracellular chloride concentration, thus maintaining a depolarizing action of GABA in neonates. The developmental decreases of leptin and proBDNF observed at the end of the first postnatal week in rodents are concomitant with an increase in oxytocin (Leonzino et al., 2016;Tyzio et al., 2006), mature BDNF (mBDNF) (Maisonpierre et al., 1990) and thyroid hormones (Sawano et al., 2013), which in turn upregulate KCC2, leading to a low intracellular chloride concentration and a hyperpolarizing action of GABA. Although we did not observe any difference in the level of Shh proteins between P15 and P30, a previous study demonstrated that Shh expression paralleled the temporal expression of mBDNF in the cortex of rodents, with a nearly undetectable level at birth and a continuous increased expression at both mRNA and protein levels during the two first postnatal weeks (Rivell et al., 2019). This temporal expression of Shh and its signaling partners during postnatal brain development has been shown to play a role in neuronal network construction through axonal growth and synaptic maturation (Yao et al., 2015). Here, we uncover a new critical function of the Shh transducer Smo in regulation of the GABA polarity switch. Indeed, we showed that activation of the GABA A receptor by isoguvacine in immature (P14) Smo-CA-expressing cortical neurons resulted in an influx of chloride and a decrease in spiking activity, whereas mature (P30) negative-phenotype Smo cortical neurons (Smo-ΔN), maintained a depolarizing and excitatory action of GABA. Thus, in the absence of Smo signaling, the GABA shift is delayed, thereby leading to an altered chloride homeostasis and higher susceptibility to seizures, presumably reflecting an unbalanced excitation to inhibition (E/I) ratio. E/I imbalance has been linked to cognitive disorders such as schizophrenia, ASD, depression and epilepsy (Ben-Ari and Holmes, 2005;Brambilla et al., 2003;Kuzirian and Paradis, 2011;Mueller et al., 2015). Abnormal levels of Shh have also been observed in ASD patients, with increased serum level observed in children with ASD, and decreased mRNA level measured in postmortem adult human brain tissue from autism patients (Choi et al., 2014;Halepoto et al., 2015). In addition, mutations in PTCHD1, a gene encoding a protein that displays secondary structures similar to Ptch1, are found in patients with intellectual disability and ASD (Ung et al., 2018). Consistent with a possible role of Shh components in the regulation of the E/I balance, these observations also suggest that an alteration in the Shh-Ptch-Smo pathway might have an effect on the pathophysiological mechanisms involved in ASD. In line with these observations, the role of Shh signaling in neuronal activity has been recently addressed in an elegant study (Hill et al., 2019) showing that the selective disruption of Shh signaling using a conditional knockout of Smo leads to an increase in neuronal excitability of cortical neurons, thus highlighting the importance of Shh-Smo signaling for the regulation of the E/I equilibrium and the construction of functional cortical networks. The results from this study are consistent with our findings showing that Smo signaling blockade increased neuronal network excitability in rodents expressing the negative-phenotype form of Smo. Furthermore, we found that blockade of Smo activity in Smo-ΔN-expressing rodents led to a decrease in KCC2 function and to a reduced threshold for PTZinduced tonic-clonic seizure. In agreement with a previous study by Chen et al. (2017), these results suggest that downregulation of KCC2 function may contribute to epileptic seizures. The implication of Shh-Smo signaling in epilepsy and seizures has been indirectly addressed in previous studies showing that somatic mutations in Shh pathway genes in humans are associated with hypothalamic hamartoma and drug-resistant epilepsy (Hildebrand et al., 2016;Saitsu et al., 2016). Likewise, Shh expression is increased in hippocampus and neocortex from both human and animal models of temporal lobe epilepsy (TLE) (Fang et al., 2011), thus suggesting a potential relationship between epilepsy and Shh activity. In line with the above studies, Feng et al. (2016) reported that during TLE development, Shh contributes to epileptogenesis through the inhibition of glutamate transporter function leading to abnormal extracellular glutamate levels, whereas the blockade of Shh-Smo signaling reduced the severity of seizures. Taken together, these observations can lead to opposing interpretations regarding the possible interplay between Shh-Smo pathway and epilepsy. These apparent discrepancies could be explained by the treatments utilized (i.e. acute seizure models with PTZ versus spontaneous seizures in the pilocarpine model of chronic TLE), which are able to activate different downstream signaling cascades (Choudhry et al., 2014). Moreover, other studies have found that activation of the Shh-Smo signaling pathway increases the synthesis and secretion of neurotrophic factors like nerve growth factor (NGF) and BDNF (Bond et al., 2013;Chen et al., 2018;Delmotte et al., 2020;Radzikinas et al., 2011), which may have neuroprotective effects in TLE (Bovolenta et al., 2010;Falcicchia et al., 2018;Paradiso et al., 2009). Clearly, additional studies are needed to obtain a deeper knowledge of the cellular and molecular mechanisms linking epilepsy to Shh pathway components.
Importantly, our in vitro experiments revealed that in immature neurons of both primary rat hippocampal cultures and rat neocortical acute slices the E GABA shifted towards a more hyperpolarized level in Smo-CA-expressing neurons, indicating a Smo-CA-initiated decrease of neuronal [Cl − ] i . This effect of Smo was blocked by preincubation with the Gli transcription factor inhibitor GANT61, suggesting that in the current study, Smo acts through Gli-dependent canonical signaling (Belgacem et al., 2016) to modulate the reversal potential of GABA. Our previous research demonstrated that Smo also participates in the maturation of GABAergic networks in the postnatal rat hippocampus through a non-canonical pathway (Delmotte et al., 2020). Non-canonical Shh signaling has been shown to acutely modulate Ca 2+ -mediated spontaneous electrical activity in the embryonic spinal cord through a G αi protein (Belgacem and Borodinsky, 2011). In this context, the G αi -dependent non-canonical signaling acts as a negative regulator of canonical Shh signaling. The activation of Smo leads to an increase of Ca 2+ spike frequency, leading to protein kinase A activation and subsequent inhibition of the activator forms of Gli transcription factors (Gli1 and Gli2 A ) concomitant with an enhancement of the repressor forms of Gli (Gli3 R ) in embryonic spinal neurons (Belgacem and Borodinsky, 2015). Here, we show an enhancement of Gli activity upon Smo activation, indicating that the non-canonical Smo signaling pathway is not involved Borodinsky, 2011, 2015). This highlights the diversity of signaling pathways recruited by the Shh signal transducer Smo among cell types, central nervous structures and developmental stages.
In immature neurons the increased [Cl − ] i and corresponding depolarizing values of E GABA are determined primarily by effective intrusion of Cl − by sodium-potassium-chloride cotransporter type 1 (NKCC1, also known as SLC12A2) and absence of effective Cl − extrusion by KCC2. The blockage of NKCC1 (Yamada et al., 2004) or activation of KCC2 Inoue et al., 2012;Khirug et al., 2005) both lead to a hyperpolarizing shift of E GABA . In this study, we revealed a novel Smo-dependent pathway of KCC2 upregulation during neuronal development. Whether the Smo pathway also regulates NKCC1 activity, as well as other transporters and channels controlling Cl − homeostasis, in immature neurons should be a subject of future studies.
The Cl − extrusion activity of KCC2 in developing neurons depends on protein expression level (Rivera et al., 1999) and posttranslational modifications that regulate the surface targeting and clustering of KCC2 (Côme et al., 2019;Medina et al., 2014). Thus, KCC2 cycles between synaptic sites and endocytic compartments, depending on the phosphorylation state of different amino acid residues (Côme et al., 2019;Kahle and Delpire, 2016;Kahle et al., 2014). However, this membrane turnover also requires regulation by the cytosolic N-and C-termini of KCC2 (Friedel et al., 2017). For instance, N-terminal truncation of KCC2 prevents its export to the cell surface, whereas C-terminal truncation leads to a decreased surface expression of KCC2 (Friedel et al., 2017). The observed loss of KCC2 surface expression in the C-terminal mutants is explained by an increase in the internalization rate without affecting cell-surface delivery. Furthermore, KCC2 function can also be regulated through lateral diffusion at the plasma membrane (Chamma et al., 2013). This suggests that KCC2 membrane dispersion may, in turn, mediate membrane destabilization and endocytosis, resulting in dysregulation of intracellular chloride homeostasis (Côme et al., 2019). In agreement with this, we found that inhibition of the Smo signaling pathway in mature neurons induces a decrease in KCC2 cluster size and enhances the intracellular concentration of chloride, which sets E GABA at more positive values than the resting potential and reduces cell-surface expression levels of KCC2. We further show that these effects occur through an increased rate of KCC2 turnover.
In conclusion, these data uncover an unexpected role for the Shh transducer Smo during early postnatal development through the control of chloride homeostasis. Thus, Smo signaling can tune GABA inhibitory transmission by controlling phosphorylation of KCC2 that affects KCC2 stability in the cell membrane and modulates neuronal chloride homeostasis. Finally, these results suggest that Smo signaling is able to set the cursor of the GABAergic inhibitory switch during the critical maturational period where developmental pathogenesis takes place.

Animal procedures
All animal procedures were carried out according to guidelines set by the INSERM animal welfare committee through the local committee (CEEA n°14) and the European Communities Council Directives (2010/ 63/UE). Male and female Wistar rats were purchased from Janvier Labs (https://www.janvier-labs.com/fiche_produit/rat_wistar/). Animals were raised and mated at INMED A2 animal facility and were housed under a 12 h light/dark cycle at 22-24°C with access to food and water ad libitum.

In utero electroporation
In utero injections and electroporations were performed in embryos from timed pregnant rats (embryonic day 15) that received buprenorphine (Buprecare at 0.03 mg/kg) and were anaesthetized with sevoflurane (4.5%) 30 min later. Briefly, the uterine horns were exposed, and a lateral ventricle of each embryo was injected using pulled glass capillaries and a microinjector (PV 820 Pneumatic PicoPump; World Precision Instruments, Sarasota, FL) with Fast Green (2 mg/ml; Sigma, St Louis, MO, USA) combined with the DNA constructs encoding GFP or mCherry and/or Smo-ΔN and/or Smo-CA and/or Cl-Sensor (molar ratio 1:2 between fluorescent protein DNA and other DNA constructs). Cl-sensor constructs was cloned into gw vector (Friedel et al., 2015). Smo constructs were provided by Jin Jiang and Philip Beachy Kim et al., 2009). Plasmids were further electroporated by delivering 40 V voltage pulses with a BTX ECM 830 electroporator (BTX Harvard Apparatus, Holliston, MA, USA). The voltage was discharged in five electrical pulses at 950-ms intervals via tweezer-type electrodes (Nepa Gene Co, Chiba, Japan) placed on the head of the embryo across the uterine wall. We performed in utero electroporation in embryonic rats at E15, corresponding to an active period of both radial and tangential migration of newborn neurons in the cortex (Kriegstein and Noctor, 2004). At birth, successfully electroporated pups were selected after transcranial visualization of the GFP reporter fluorescent protein only. Following experimentation, morphological analysis of electroporated tissues, selected by GFP or mCherry expression under a fluorescence stereomicroscope (Olympus SZX 16), was performed. The criteria commonly used for selection of animals were localization and cell density of transfected cortices and absence of abnormal cortex morphology (i.e. cortical disruption, thickening and abnormal cortical organization). Our analyses revealed no alterations in cortical layer position or morphology in GFP or mCherry, Smo-CA and Smo-ΔN conditions, although we cannot exclude the possibility that Smo constructs impact the neuronal cells at the cellular and subcellular levels. Nevertheless, ∼20% of electroporations failed due to the absence of transfected cells, as revealed by the lack of fluorescent cells (in GFP or mCherry and Smo conditions). These animals were excluded from the study.

Western blotting
Electroporated zones of the somatosensory cortex were homogenized in RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, pH 8, containing protease inhibitors (Complete Mini; Roche)]. Lysates were centrifuged (10,000 g for 10 min at 4°C) and the supernatant was heated at 90°C for 5 min with Laemmli loading buffer. Loading was 20 μg of protein, as determined using a modified Bradford reaction (Bio-Rad Laboratories). Proteins were separated by 7-15% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin (BSA) in TBS containing 0.1% Tween 20 (TBST) for 2 h at room temperature, then incubated with primary antibodies diluted in TBST containing 3% BSA overnight at 4°C or for 2 h at room temperature. Blots were probed with antibodies against KCC2 pSer 940 (1 mg/ml; rabbit, Novus Biologicals), KCC2 pThr 1007 (1 mg/ ml; sheep, Division of Signal Transduction Therapy Unit, University of Dundee) and KCC2 (1:2000;rabbit, US Biological). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies diluted in TBST containing 3% BSA for 60 min, washed with TBST and then developed using a G:BOX gel imaging system (Syngene). The appropriate exposure time of the digital camera for acquisition of chemiluminescent signals from immunoblots was adapted to avoid saturated pixels, and expression levels were estimated using ImageJ software (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/).

Immunocytochemistry and confocal microscopy
Under deep anesthesia with isoflurane prior to chloral hydrate (7% in 1 M PBS), P10 to P30 electroporated rats were intracardially perfused with cold PBS (1 M) followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed, post-fixed overnight at 4°C and rinsed in PBS. Coronal cortical sections (70 μm thick) were obtained using a vibratome (Microm HM 650 V). Sections were incubated first for 1 h in PBS with 1% BSA and 0.3% Triton X-100, then overnight at 4°C with rabbit anti-Smo (1:500; ab38686; Abcam), rabbit anti-caspase-3 cleaved (1:500; 9661S; Cell Signaling Technology), chicken anti-MAP2 (1:5000; ab5392; Abcam), mouse anti-NeuN (1:1000; MAB377; Chemicon), rabbit anti-FoxP2 (1:4000; ab16046; Abcam) or mouse anti-synaptophysin (1:1000; MAB5258; Chemicon). Sections were rinsed in PBS and incubated for 2 h with the corresponding Alexa488-(1:1000; FluoProbes), Cy5-or Cy3-conjugated (1:1000; Chemicon) secondary antibodies diluted in PBS). DAPI (Vector Laboratories H-1200) was applied to stain nuclei. Control tissues used to determine the level of nonspecific staining included tissues incubated without primary antibody. Sequential acquisition of immunoreactivity of GFP-positive pyramidal-like cells was performed using a laser scanning confocal microscope (Zeiss LSM 510 Meta) with a 40× or 63× oilimmersion objective. For each experimental condition, a semi-quantitative analysis of synaptophysin (Syn)-labeled area fraction per field was measured and reported relative to the area fraction of pixels positive for MAP2 staining. The surface areas of NeuN immuno-positive neurons expressing GFP or Smo mutants were measured manually using the Freehands selection tool in ImageJ. For neuronal migration, electroporated rat brains were removed at E20 and fixed in Antigenfix (Diapath) for 24 h before they were included in agar and sectioned (70 μm) using a vibratome (Leica VT 100 S). Analysis was performed as previously described (Elias et al., 2007), with an automated subdivision into regions of equal length, from the ventricle to the surface [i.e. ventricular/subventricular zones (VZ/SVZ), intermediate zone (IZ) and cortical plate (CP)]. In each set of images, laser light levels and detector gain and offset were adjusted to avoid any saturated levels. Optical sections were digitized (1024×1024 pixels) and processed using ImageJ software. All of the images were analyzed blind.

Relative quantitative expression of mRNA transcripts
Expressions of Gli1 and Ptch1 mRNA in the electroporated regions of the somatosensory cortex were measured using real-time RT-qPCR. Total RNA was isolated from cerebral cortices (P15 rats) using a Mini RNeasy kit (Qiagen) then converted to cDNA using 1 μg RNA and a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. Single-cell gene expression was performed on primary hippocampal neurons transfected with a mixture of constructs encoding mCherry (mock), Smo SA0-5, Smo-ΔN or Smo-CA with or without GANT61 (10 μM) for 48 h. Whole-cell configuration using patch pipettes was used to harvest the cytosol from 15 transfected neurons per condition, which were converted immediately to cDNA as described above. PCR was carried out with a LightCycler 480 SYBR Green I Master (Roche Applied Science) with 1 μL cDNA using the following oligonucleotides (QuantiTect Primer Assays, Qiagen): Gli1 (Gli1; QT01290324), Ptch1 (QT01579669), KCC2 (Slc12a5; QT00145327) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; QT001199633). Relative mRNA values were calculated using LC480 software with GAPDH as the housekeeping gene. PCR was performed in triplicate.

Sonic hedgehog protein immunoassay
Cortical tissues from electroporated rats at the indicated age were homogenized in RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, pH 8, containing protease inhibitors (Complete Mini; Roche)]. Lysates were centrifuged (5000 g for 5 min at 4°C). Loading was 200 μg of protein, as determined using a modified Bradford reaction (Bio-Rad Laboratories). Quantification of Shh was performed using a Rat Shh ELISA Kit (FineTest, Wuhan Fine Biotech Co. Ltd., China) in the concentrated solutions following the manufacturer's protocol. Experiments and analysis were performed blind.

Slice preparation and electrophysiological recordings
Electroporated cortical regions of P14 rats were identified using an atlas of the developing rat brain (Khazipov et al., 2015). Electroporated cortical regions of P20 and P30 rats were identified using the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2005). Based on these two atlases, the somatosensory regions were prepared for acute slice experiments from P14, P20 and P30 rats in ice-cold (2-4°C) oxygenated modified artificial cerebrospinal fluid (ACSF) (0.5 mM CaCl 2 and 7 mM MgSO 4 ; NaCl replaced by an equimolar concentration of choline). Slices (300-μm thick) were cut with a Vibratome (VT1000E; Leica, Nussloch, Germany) and kept at room temperature (25°C) for at least one hour before recording in oxygenated normal ACSF containing (in mM): 126 NaCl, 3.5 KCl, 2 CaCl 2 , 1.3 MgCl 2 , 1.2 NaH 2 PO 4 , 25 NaHCO 3 and 11 glucose, pH 7.4 equilibrated with 95% O 2 and 5% CO 2 . Slices were then transferred to a submerged recording chamber perfused with ACSF (3 ml/min) at 34°C.

Spiking activity and data analysis
Extracellular field potential recordings were performed from P14, P20 and P30 electroporated somatosensory cortical slices. Extracellular tungsten electrodes of 50 μm diameter (California Fine Wire, Grover Beach, CA, USA) were positioned in the V/VI cortical pyramidal cell layer of the transfected area to record the multiunit activity (MUA). Field potentials were recorded using a DAM80 Amplifier (World Precision Instruments, Sarasota, FL, USA) using a 1-3 Hz bandpass filter and were analyzed offline with the Axon package MiniAnalysis program (Jaejin Software, Leonia, NJ, USA). To determine the developmental changes in GABA A signaling, we used isoguvacine, a potent and selective GABA A receptor agonist (Krogsgaard-Larsen and Johnston, 1978). We determined the effect of isoguvacine on MUA as a ratio of the spiking frequency at the peak of the isoguvacine response to the spiking frequency in control.

Cl-Sensor fluorescence recordings from brain slices
To perform non-invasive monitoring of neuronal intracellular chloride concentration ([Cl − ] i ), we used a ratiometric genetically-encoded Cl −sensitive probe called Cl-Sensor (Markova et al., 2008;Waseem et al., 2010) that was co-expressed in the cells of interest together with other constructs as described above. The acquisition of fluorescence images was performed using a customized imaging setup with consecutive cell excitation at 430 and 500 nm and emission at 480 and 540 nm. The frequency of acquisition was 0.05 Hz. The duration of excitation was selected for each cell type and was selected to avoid use-dependent bleaching of the signal (Friedel et al., 2013). Results are expressed as fluorescence ratio measured at 430 and 500 nm excitation wavelengths (R 430/500 ). Experiments were performed on acute cortical slices from in utero electroporated rat pups on postnatal days P10 and P30. Individual slices were transferred to a specially designed recording chamber where they were fully submerged and superfused with oxygenated ACSF complemented with 1 μM tetrodotoxin, 0.3 μM strychnine and 10 μM NBQX to prevent spontaneous neuronal activity and noncontrolled [Cl − ] i changes at 30-32°C at a rate of 2-3 ml/min. The applications of the ACSF solution containing isoguvacine (30 μM) or KCl (25 mM)+ isoguvacine (30 μM) were performed with a perfusion system.

Seizure induction with pentylenetetrazol
To evaluate the susceptibility to seizures in control rats (GFP-transfected brains), and in Smo-CA-and Smo-ΔN-expressing animals at P30, pentylenetetrazol (PTZ; 25 mg/kg; Sigma) was administered via intraperitoneal injections every 10 min until generalized seizures occurred. Rats were placed in a plexiglass cage, and the time to the onset of the generalized seizure was measured by observation. There was no significant difference in weight and sex between groups of animals. Experiments were performed blind, and the electroporated region was verified after PTZ induction. Rats with a a very local electroporated cortical area of GFP fluorescence or fluorescence in other brain regions than somatosensory cortex were excluded.

Primary cultures and transfection of rat hippocampal neurons
Neurons from 18-day-old rat embryos were dissected and dissociated using trypsin and plated at a density of 70,000 cells cm −2 in minimal essential medium (MEM) supplemented with 10% NU serum (BD Biosciences, Le Pont de Claix, France), 0.45% glucose, 1 mM sodium pyruvate, 2 mM glutamine and 10 U ml −1 penicillin-streptomycin (Buerli et al., 2007). On days 7, 10 and 13 of culture incubation (DIV, days in vitro), half of the medium was changed to MEM with 2% B27 supplement (Invitrogen). For electrophysiology, neuronal cultures were plated on coverslips placed in 35mm culture dishes. Twelve hours before plating, dishes with coverslips were coated with polyethylenimine (5 mg/ml). Transfection of cultured neurons was performed with 300 μl Opti-MEM medium mixed with 7 μl Lipofectamine 2000 (Invitrogen), 1 μl Magnetofection CombiMag (OZ Biosciences) per μg of DNA and 1.5 μg premixed DNA encoding the constructs of interest. The mixture was incubated for 20 min at room temperature and thereafter distributed dropwise above the neuronal culture. Culture dishes (35-mm) were placed on a magnetic plate (OZ Biosciences) and incubated for 35 min at 37°C, 5% CO 2 . Transfection was terminated by the substitution of 80% of the incubation solution with fresh culture medium. Cells were used in the experiments 48-72 h after transfection. These experiments were based on co-transfection into the same cell of two different pcDNAs encoding a fluorescent marker of transfection (eGFP or mCherry, 0.3 μg), and Smo-related constructs (1.2 μg).

Gramicidin-perforated patch-clamp recordings
Gramicidin-perforated patch-clamp recordings were performed on primary hippocampal neurons, transfected with a mixture of Smo-related constructs (mCherry, in the presence or absence of GANT61 (10 μM, 48 h) and with or without KCC2-pH ext (Friedel et al., 2017). Measurements were performed 2 or 3 days after transfection (corresponding to 8 or 9 DIV). Coverslips with transfected neurons were placed onto the inverted microscope and perfused with an external HEPESbuffered solution (HBS: 140 mM NaCl, 2.5 mM KCl, 20 mM HEPES, 20 mM d-glucose, 2.0 mM CaCl 2 , 2.0 mM MgCl 2 , and 0.02 mM bumetanide, pH 7.4). For recording from neurons, external HBS contained 0.5 μM tetrodotoxin and 15 μM bumetanide. The recording micropipettes (5 MΩ) were filled with a solution containing 150 mM KCl, 10 mM HEPES and 20 μg/ml gramicidin A, pH 7.2. Isoguvacine (30 μM) was dissolved in an external solution and focally applied to recorded cells through a micropipette connected to a Picospritzer (General Valve Corporation, pressure 5 p.s.i.). Recordings were performed using an Axopatch-200A amplifier and pCLAMP acquisition software (Molecular Devices) in voltage-clamp mode. Data were low-pass filtered at 2 kHz and acquired at 10 kHz. Isoguvacine responses were recorded at voltages −110, −90, −70 and −50 mV, or at −70, −50, −30 and −10 mV depending on neuron GABA inversion potential. A linear regression was used to calculate the best-fit line of the voltage dependence of the isoguvacine responses.

Surface immunolabeling on living neurons and analysis of KCC2-pH ext proteins
For immunolabeling of KCC2-pH ext proteins on living neurons, rabbit anti-GFP antibody was dissolved in culture medium applied to neurons for 2 h at 37°C, 5% CO2 (Friedel et al., 2015). Neurons were then rinsed three times at room temperature with HBS, labeled with anti-rabbit Cy3-conjugated antibody for 20 min at 13°C and fixed in Antigenfix (Diapath). To reveal the intracellular pool of live-labeled proteins, cells were permeabilized with 0.3% Triton X-100, blocked by 5% goat serum and incubated for 1 h at room temperature with anti-rabbit Alexa647-conjugated antibody. For visualization of the total pool of overexpressed KCC2-pH ext , cells were labeled overnight (4°C) with mouse anti-GFP antibody and for 1 h at room temperature with anti-mouse Alexa488-conjugated antibody. For control of the cell membrane integrity during live-cell immunolabeling, one batch of cultures were routinely transfected with the KCC2-pH ext mutant ΔNTD-KCC2-pH ext , which does not incorporate into the plasma membrane (Friedel et al., 2017). A/A-KCC2-pH ext , a double phosphomimetic mutant of KCC2 for T906A/T1007A that maintains KCC2 at the cell surface (Friedel et al., 2015), was used as a positive control.
Images of labeled neurons were acquired with an Olympus FluoView 500 confocal microscope [oil-immersion objective 60× (NA 1.4); zoom 1-5]. We randomly selected and focused on a transfected cell by only visualizing Alexa488 fluorescence and then acquired z-stack images of Alexa488, Cy3 and Alexa647 fluorochromes. Each z-stack included ten planes of 1 μm optical thickness, taken at 0.5 μm distance between planes. The cluster properties and fluorescence intensities of each cell were analyzed with Metamorph software. First, we used the logical 'NOT' conversion of pairs of Alexa647 and Cy3 images to isolate in each focal plane the Alexa647 signal that was not overlapping with Cy3 fluorescence restricted to the plasma membrane. This gave rise to additional images reflecting the fluorescence of the internalized pool of labeled clusters, called thereafter 'NOT-conversion'. Second, the arithmetic summation for each z-stack and channel was performed to collect the whole fluorescence of the different signals (Alexa488 signal for total protein fluorescence; Cy3 signal for plasma membrane restricted fluorescence; NOT-conversion signal for internalized restricted fluorescence; and Alexa647 signal for all surface-labeled fluorescence). Third, a binary mask was created for each cell, using the Alexa488 image, to isolate the signal coming from the transfected neuron, and the fluorescence parameters (total fluorescence, single cluster fluorescence as well as density and brightness of clusters) were analyzed for each channel (Alexa488, Cy3, NOT-conversion and Alexa647) in regions overlapping with the binary mask. The analysis parameters were the same for each experiment, and all experiments were performed blind. After analysis, data were normalized to the mean value of cells transfected with KCC2-pHext+GFP.

Statistical analysis
No statistical methods were used to predetermine sample sizes. To ensure the consistency and reproducibility of our results, we conducted repeated trials in different cell cultures and acute brain slices prepared from at least three different animals for each experimental condition. If not stated otherwise, statistics are presented as the mean±s.d. for normally distributed data and as the median only for non-normally distributed data. Experiments with control and Smo-electroporated animals were processed at the same time to ensure homogeneity of experimental conditions.