[1] 16w Wnt signaling pathway improves central inhibitory synaptic transmission in a mouse model of Duchenne muscular dystrophy

[1] 141w The dystrophin-associated glycoprotein complex (DGC) that connects the cytoskeleton, plasma membrane and the extracellular matrix has been related to the maintenance and stabilization of channels and synaptic receptors, which are both essential for synaptogenesis and synaptic transmission. The dystrophin-deficient (mdx) mouse model of Duchenne muscular dystrophy (DMD) exhibits a significant reduction in hippocampal GABA efficacy, which may underlie the altered synaptic function and abnormal hippocampal long-term plasticity exhibited by mdx mice. Emerging studies have implicated Wnt signaling in the modulation of synaptic efficacy, neuronal plasticity and cognitive function. We report here that the activation of the non-canonical Wnt-5a pathway and Andrographolide, improves hippocampal mdx GABAergic efficacy by increasing the number of inhibitory synapses and GABA A receptors or GABA release. These results indicate that Wnt signaling modulates GABA synaptic efficacy and could be a promising novel target for DMD cognitive therapy.

[1] 82w Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive neuromuscular disease that is characterized by the deficiency of dystrophin, a gene on Xp21, which is responsible for the early onset of the genetic disease (Rodino-Klapac et al., 2013). Lack of dystrophin in the brain has been associated with impairments in behavioral and cognitive function (Anderson et al., 2002;Bresolin et al., 1994), mainly with impaired memory retention, procedural learning and spatial memory (Muntoni et al., 1991;Vaillend et al., 1995Vaillend et al., , 2004).

[2] 99w In the central nervous system (CNS), dystrophin is expressed in postsynaptic densities of hippocampal, amygdale and cortical neurons (Bies et al., 1992;Comim et al., 2011). It is localized on the inner-side of the plasma membrane as part of a protein complex called the dystrophin-associated glycoprotein complex (DGC) that connects the cytoskeleton, plasma membrane and the extracellular matrix (Blake and Kroger, 2000;Perronnet and Vaillend, 2010). DGC has been associated with the maintenance and stabilization of channels and receptors, which are both essential to synaptogenesis and synaptic transmission (Albrecht and Froehner, 2002;Haenggi and Fritschy, 2006;Perronnet and Vaillend, 2010;Waite et al., 2009).

[3] 315w The dystrophin-deficient (mdx) mouse model of DMD exhibits a significant reduction in the number and size of GABA A receptor (GABA A -R) clusters in pyramidal neurons and Purkinje cells (Grady et al., 2006;Knuesel et al., 1999;Kueh et al., 2011) without a change in the total number of GABA A -Rs (Kueh et al., 2008). In CA1 hippocampal synapses, long-term plasticity is abnormally enhanced. These deficits in GABA efficacy may underlie altered synaptic function and abnormal longterm potentiation (LTP) (Dallerac et al., 2011;Vaillend and Billard, 2002;Vaillend et al., 1999). The Wnt signaling pathway plays a key role in the neuronal development and maintenance of the nervous system by modulating synaptic structure and function (Inestrosa and Arenas, 2010;Oliva et al., 2013a;Rosso and Inestrosa, 2013;Salinas and Zou, 2008). Recently, it has been reported that Wnt-5a ligands, through the activation of a non-canonical pathway, modulate the excitatory and inhibitory transmission at the postsynaptic region (Cerpa et al., 2010;Cuitiño et al., 2010;Varela-Nallar et al., 2010;Vargas et al., 2014;Box 1). In inhibitory synapses, Wnt-5a enhances the amplitude of the inhibitory postsynaptic current (IPSC) through the insertion and clustering of GABA A -Rs, increasing receptor recycling without affecting the endocytic process (Cuitiño et al., 2010). Together, these findings indicate that Wnt-5a increases the assembly of GABA A -Rs receptors and modulates the synaptic plasticity of inhibitory circuits. Because of the essential role of the Wnt signaling pathway in GABA A -R cell surface stability and activity, we hypothesize that the activation of the non-canonical Wnt pathway might improve the GABAergic deficit of the mdx mouse model of DMD. It has been suggested that andrographolide (ANDRO), the major constituent of Andrographis paniculata, has some protective properties in the brain. Recent results from our group suggest that ANDRO can protect against brain impairment during Alzheimer's disease progression (Serrano et al., 2014) and reduce the muscle impairment in mdx mice (Cabrera et al., 2014).

[4] 53w In the present study, we show that the activation of the noncanonical Wnt-5a pathway or ANDRO improve mdx hippocampal GABAergic efficacy by increasing the number of inhibitory synapses and GABA A -Rs. These findings indicate that Wnt signaling modulates GABA synaptic efficacy and could be a promising novel target for mdx cognitive therapy.

[1] 507w Experimental evidence suggests that an important factor for regulating the efficacy of GABAergic inhibition is the number and stability of postsynaptic GABA A -Rs (Luscher and Keller, 2004). Thus, a direct relationship has been demonstrated between the number of synaptic GABA A -Rs and the strength of the inhibitory synapses (Nusser et al., 1998). Previous studies indicate that the dystrophin-deficient (mdx) mouse model of DMD includes alterations in the clustering of GABA A -R subtypes in the postsynaptic neurons of the hippocampus (Knuesel et al., 1999;Levi et al., 2002). In addition, we previously reported that the activation of a non-canonical Wnt pathway (Wnt/Ca 2+ pathway) by Wnt-5a or Foxy-5 induces a rapid and sustained increase in the cluster number of surface GABA A -Rs at 15 min of treatment (Cuitiño et al., 2010). Foxy-5 is a formulated hexapeptide derived from the sequence of the Wnt-5a ligand that mimics the full molecule in hippocampal neurons (Cuitiño et al., 2010;Vargas et al., 2014) and in other systems (Safholm et al., 2006). Using the immunofluorescence assay, we analyzed the effect of the activation of the non-canonical Wnt pathway on the surface amount of GABA A -Rs in hippocampal pyramidal neurons obtained from the embryos of mdx mice. We analyzed the GABA A -Rs composed of γ2 subunits using a specific antibody directed toward the external epitope of this subunit. Importantly, the γ2 subunit is critical for the expression, trafficking, clustering and synaptic localization of the major hetero-pentameric receptor expressed in the brain (Essrich et al., 1998). We observed that hippocampal pyramidal neurons obtained from mdx mice have a lower cluster number on the surface of GABA A -Rs than hippocampal neurons obtained from wild-type (WT) mice (cluster/20 μm was 4.71 ± 0.37, whereas in mdx, it was 2.06 ± 0.07; Fig. 1A, B), but the total amount of this subunit did not change (Fig. 1C). Additionally, the cluster size remained the same when we compared WT and mdx mice (Fig. 1A, B). According to previous reports (Grady et al., 2006;Knuesel et al., 1999), our results indicated that the alterations in mdx mice were specific to GABA A -R cell surface expressions. Interestingly, when hippocampal neurons from mdx were treated with the Wnt-5a ligand, we observed that 15 min of the treatment induced an increase in the cluster number (3.61 ± 0.20 cluster/20 μM) of γ2 subunitcontaining GABA A -Rs on the surface in comparison with untreated mdx neurons without affecting the total amount of this GABA A -R subunit (Fig. 1A-C). Interestingly, the localization and size of the clusters and the total levels of the scaffold protein of GABAergic postsynaptic sites, gephyrin, were similar between WT and mdx neurons, and did not change in the presence of Wnt-5a (Fig. 1 A-C). Our data suggest that the activation of the non-canonical Wnt-5a pathway induces an improvement in the GABA transmission of mdx mice via an increase in the number of functional GABA clusters, most likely by stabilizing the α-1 subunit GABA A -Rs in perisomatic postsynaptic densities (Panzanelli et al., 2011).

[2] 266w Next, we wondered whether the activation of the non-canonical Wnt-5a pathway, that increased the surface amount of GABA A -Rs, may also lead to an increase of functional receptors. Using electrophysiological tools, we studied the effects of Foxy-5 on GABA A -R-mediated IPSC in WT and mdx mice. We recorded the isolated IPSC after blocking the ionotropic glutamatergic receptors that included NMDARs and AMPARs with 50 μM D-AP5 and 20 μM CNQX, respectively. Bath application of Foxy-5 (50 μM) induced a fast and long-lasting increase in IPSC peak amplitude in both untreated WT (136.22 ± 9.3%, n = 6; p b 0.05) and mdx (130.45 ± 4.86%, n = 8; p b 0.05) mice (Fig. 1A and B). To establish whether the locus of expression of IPSC increases occurs in the pre-or postsynaptic regions, we analyzed the modifications of the PPR quantified by ((R2/R1) × 100). This effect is illustrated by a representative recording in the top panel (Fig. 2A). The superimposed recordings showed changes in the amplitude of eIPSC before and during the Foxy-5 administration. However, no difference in the paired pulse index was recorded for mdx (PPR mdx : 71.3 ± 6.4%; PPR mdx-foxy : 76.5 ± 12.5; n = 6; p = 0.28) nor WT mice (PPR WT : 73.5 ± 6.4%; PPR WT-foxy : 76.4 ± 9.1%; n = 6; p = 0.08; Fig. 2C). In terms of the postsynaptic locus of synaptic potentiation, Foxy-5 was unable to induce changes in the GABA probability release, suggesting that the locus of GABA straightening is caused by an increase of postsynaptic GABA A -Rs.

[3] 433w To further establish a postsynaptic locus of expression of this form of GABA potentiation and to discard changes in the presynaptic excitability, we recorded control miniature inhibitory postsynaptic currents (mIPSC) in the presence of TTX (500 nM). First, we observed that the mIPSC frequency in mdx mice is lower than in WT mice (mdx: 0.61 ± 0.09 Hz and WT: 1.16 ± 0.1 Hz; n = 6; p b 0.05), while the amplitude of mIPSC in mdx and WT were similar (mdx: 29.57 ± 4.09 pA and WT: 32.3 ± 1.64 pA; n = 6; p = 0.27). These differences in mdx and WT mIPSC frequency were observed when comparing the histogram and cumulative probability plots (Fig. 3B-F). Then, we analyzed the effect of the activation of non-canonical Wnt signaling in the mdx and WT mIPSC. After 15 min of baseline of mdx or WT mIPSC recordings, we applied Foxy-5. Under this condition, the frequency of mdx mIPSC increased from 0.56 ± 0.09 to 1.12 ± 0.25 Hz (n = 6; p b 0.05), and the amplitude of mdx mIPSC changed from 29.57 ± 4.09 to 37.4 ± 2.6 pA (n = 6; p = 0.11). However, in WT the amplitude and frequency of mIPSC was not affected by Foxy-5. Thus, the amplitude was 33.20 ± 0.50 pA in control and 36.01 ± 2.7 pA under Foxy 5 (n = 8; p N 0.05), while the frequency was 1.51 ± 0.29 Hz in WT and 1.44 ± 0.21 Hz; under Foxy 5 (n = 8; p N 0.05). The changes in both the amplitude and frequency of mdx mIPSC was also observed after comparing both cumulative probability and fraction plots of the mIPSC frequency and amplitude before and after the application of Foxy-5 (Fig. 3E and F). In addition, the rise time and decay time constant of mIPSC in mdx and WT were unaffected by Foxy-5 (data not shown). Changes in mIPSC frequency have been attributed to presynaptic mechanisms (Manabe and Nicoll, 1994), suggesting that enhanced GABA efficacy occurs presynaptically as a consequence of an increase in GABA release probability. However, we observed an increase in the amplitude of mIPSC, suggesting that in addition to the increase in the probability of GABA release, the amount of GABA receptors (see Figs. 1 and 2) were increased by the activation of the non-canonical Wnt-5a pathways. Moreover, we observed that mIPSC were GABA A -mediated because they were totally blocked by picrotoxin. Together, these data suggest that the activation of the non-canonical Wnt-5a pathway induced an increase in the efficacy of GABAergic transmission of mdx mice.

[4] 12w Foxy increases mIPSC amplitude without affecting PPR, however, increases the mIPSC frequency.

[5] 587w An important factor that modulates the strength of synaptic transmission is the number of release sites connecting two neurons (Hsia et al., 1998). It has been demonstrated that in hippocampal GABAergic synapses, the developmental expansion of GABA connectivity is not caused by an increase in the number of release sites but by the formation of new stereotypically connected cell pairs (Groc et al., 2003). Because the clusters of GABA A -Rs are significantly reduced in mdx mice, we examined the multiplicity index, an approach that enables the estimation of synaptic network connectivity (Groc et al., 2003;Hsia et al., 1998) between GABAergic interneurons and CA1 pyramidal neurons in the hippocampus. We compared the effect of noncanonical Wnt-5a signaling activation on the mdx interneuron-CA1 connectivity by comparing the spontaneous IPSC (sIPSC) and mIPSC. For each cell, we recorded the sIPSC, and mIPSC during 500 nM TTX perfusion (n = 6). First, we compared the multiplicity index between mdx and WT. As shown in the representative recording in Fig. 4A, the TTX application produces a clear reduction in both the frequency and amplitude of the IPSC. The IPSC frequency in WT was reduced from 2.91 ± 0.20 to 1.2 ± 0.13 Hz (Fig. 4A, C and D; n = 6; p = 0.012), whereas in mdx mice, the frequency of the IPSC was reduced from 1.30 ± 0.10 to 0.54 ± 0.17 Hz (Fig. 4A, C and D, n = 6; p b 0.05). In addition, the amplitude of the mIPSC in both mdx and WT was on average ~56% and ~51% of the amplitude of sIPSC (mdx sIPSC 60.00 ± 5.29 pA; mdx mIPSC 34.00 ± 3.33 pA; n = 6, and WT sIPSC 59.33 ± 3.10 pA; WT mIPSC 30.33 ± 0.76 pA; n = 6). These data indicate that in both mdx and WT mice, the action potential-dependent GABA IPSC actually originated from more than one release site (Groc et al., 2003;Hsia et al., 1998). The multiplicity index was then calculated for each neuron (see methods). We determined that the multiplicity index was, on average 2.5 ± 0.4 in mdx mice and 2.4 ± 0.6 in WT mice (n = 6, Fig. 4B). Moreover, we found that the multiplicity index was not affected by the Wnt-5a analog perfusion, reaching values similar to controls (3.0 ± 0.9; Fig. 4B). These data indicate that the number of release sites at GABAergic synapses on CA1 pyramidal neurons of mdx is essentially the same as in WT mice, and that number of GABA release sites was not affected by the activation of the non-canonical Wnt-5a pathway. However, we observed that the frequency of sIPSC and mIPSC in mdx mice was significantly reduced compared with the WT mice, whereas the amplitude of sIPSC and mIPSC of mdx and WT were not significantly different. Interestingly, the perfusion of Foxy-5 increased the frequency of sIPSC (to 2.43 ± 0.30 Hz; n = 6; p = 0.03) and mIPSC in mdx slices (to 1.0 ± 0.35 Hz; n = 6; p = 0.03) without affecting the amplitude of either the sIPSC (to 62.68 ± 2.93; n = 6; p = 0.16) or the mIPSC (to 38.92 ± 3.04 pA; n = 6; p = 0.27). Taken together, these data suggest that mdx shows a lower number of interneuron-CA1 contacts but with the same number of release sites. The activation of the non-canonical Wnt-5a signaling enhances the number of GABAergic synapses on CA1 pyramidal neurons, whereas the number of GABA release sites seems to remain constant.

[6] 76w Non-canonical Wnt-5a signaling involves at least two pathways: the planar cell polarity (PCP) or the Wnt/JNK pathway and the Ca 2+ pathway (Box 1). The PCP pathway activates small GTPases, including Rho and Rac and the protein kinase JNK (Farias et al., 2009). The activation of the Wnt/Ca 2+ pathway triggers an increase in intracellular Ca 2+ levels and activates the protein kinases CaMKII and protein kinase C (PKC) (Cuitiño et al., 2010;Inestrosa et al., 2012).

[7] 90w Andrographolide (ANDRO), a labdane diterpene, is an active compound of Andrographis paniculata. Traditionally, ANDRO has been used for the treatment of some human illnesses, including acute hepatitis, meningitis, and many other acute inflammatory conditions, through the inhibition of NF-κB signaling (Xia et al., 2004). Some studies suggest that ANDRO might exert some protective effects in the brain, i.e., against oxidative stress induced by nicotine (Das et al., 2009), in cerebral ischemia (Chan et al., 2010) and more recently, in a mouse model of Alzheimer's disease (AD) (Serrano et al., 2014).

[8] 200w Previous studies showed that a Wnt-5a ligand is able to activate both non-canonical pathways with different temporality (Farias et al., 2009), and this effect is also observed in the presence of Foxy-5. To analyze the temporality of the activation of the non-canonical Wnt-5a pathway, hippocampal slices from two-month-old wild-type mice were exposed to 25 μM ANDRO, conditioned medium of Wnt-5a ligand or Foxy-5 (50 μM) for different time periods of up to four hours. Fig. 5A and B show the activation kinetics of non-canonical Wnt-5a signaling induced by the Wnt-5a ligand, with a peak of activation at 60 min for both CaMKII and JNK kinases. Fig. 5C and D show the effect of Foxy-5 on the activation of the Wnt/Ca 2+ and Wnt/JNK pathways, respectively, which displayed similar kinetics of activation as the Wnt-5a ligand. In addition, Fig. 5E and F show the effect of ANDRO on non-canonical Wnt signaling, activates the CaMKII kinase and the JNK kinase with a plateau at 60 min. These results indicate that ANDRO activates the non-canonical Wnt-5a pathway with a rather similar kinetic than the Wnt-5a ligand and Foxy-5, suggesting that ANDRO could activate this signaling pathway with at similar mechanism than ligand-receptor binding.

[9] 347w Because the activation of non-canonical Wnt-5a signaling increases the number of inhibitory synapses (Cuitiño et al., 2010 and present results), we hypothesized that the non-canonical Wnt activator ANDRO would enhance GABAergic efficacy synaptic responses if the increases in synapse density corresponded to the formation of functional synapses. First, we analyze the effects of ANDRO (4 μM), on GABA A -R-mediated evoked IPSC in WT and mdx mice. Under 50 μM D-AP5 and 20 μM CNQX, we observed that bath application of ANDRO induced a long-lasting increase in IPSC peak amplitude in both WT (161.96 ± 22.34%, n = 6; p b 0.05) and mdx (153.50 ± 17.00%, n = 6; p b 0.05) mice (Fig. 6). Previously, we demonstrated that Wnt-5a and Foxy-5 act as noncanonical ligands in mature hippocampal neurons, activating both Wnt signaling pathway, Wnt/JNK and Wnt/Ca 2+ (Farias et al., 2009;Cuitiño et al., 2010). To dissect whether ANDRO increase the inhibitory efficacy by activation of the Wnt/JNK or the Wnt/Ca 2+ pathways, we used inhibitors for the three well known effectors of these pathways: TAT-TI-JIP to inhibit JNK activity of the Wnt/JNK pathway and KN-93 and Go¨6976 to inhibit CaMKII and PKC activities of the Wnt/Ca2 + pathway. KN-93 or Go¨6976 did not affect significantly the amplitude of IPSC induced by ANDRO However, when we analyzed the contribution of the JNK, we found that only in the presence of the TAT-TI-JIP inhibitor, the effect of ANDRO on the IPSC amplitude was completely blocked (Fig. 6 B). To establish whether the locus of expression of IPSC increases occurs in the preor postsynaptic regions, we analyzed the modifications in the PPR index. We found no difference in the PPR in mdx or WT mice in both control condition and under ANDRO perfusion (PPR mdx : 70.73 ± 7.48%; PPR mdx-ANDRO 74.97 ± 2.26; n = 6; p = 0.47) nor WT mice (PPR WT : 69.42 ± 6.53%; PPR WT-ANDRO : 79.28 ± 1.10%; n = 6; p = 0.18; Fig. 6C). Also TAT-TI-JIP, KN-93 or Go¨6976 has not effect on PPR index (data not shown).

[10] 253w In addition to discard changes in the presynaptic excitability, we analyze the effect of ANDRO on mIPSC. First, we observed that the mIPSC frequency in mdx is lower than in WT mice (mdx: 0.90 ± 0.15 Hz and WT: 1.16 ± 0.18 Hz; n = 6; p = 0.025), while the amplitude of mIPSC in mdx and WT were similar (mdx: 33.00 ± 1.15 pA and WT: 35.66 ± 2.66 pA; n = 6; p = 0.27). These differences in mdx and WT mIPSC frequency were observed when comparing the histogram and cumulative probability plots (Fig. 7). Then, we analyzed the effect of ANDRO (4 μM) in the frequency and amplitude of both mdx and WT mIPSC. After 15 min of baseline of mdx or WT mIPSC recordings, we applied ANDRO. Under this condition, the frequency of mdx mIPSC increased to 1.76 ± 0.27 Hz (n = 6; p b 0.05), whereas the amplitude was not modified the mIPSC amplitude (mdx mIPSC reach 34.6 ± 0.88 pA (n = 6; p = 0.11). In addition, ANDRO induced only an increase in the amplitude of WT mIPSC; the frequency was not affected (1.51 ± 0.29 Hz and 1.44 ± 0.21 Hz; n = 6; p = 0.86). The changes in both amplitude and frequency of mdx mIPSC was also observed after comparing both cumulative probability and fraction plots of the mIPSC frequency and amplitude before and after the application of ANDRO (Fig. 7E and F). As with Foxy-5, ANDRO increase the hippocampal GABAergic release.

[11] 269w To evaluate whether this Wnt-dependent inhibitory synaptic enhancement is due to changes in baseline synaptic efficacy, we analyzed isolated (under 50 μM D-APV and 20 μM CNQX), the fIPSP input/output relations under ANDRO treatment in WT and mdx mice. Input/output curves are displayed to represent the relationship between the fIPSP amplitude and stimulus intensity in both mice groups. After obtaining a stable baseline for at least 15 min, the stimulus intensity was decreased until the amplitude of the fIPSP was zero. Afterward, the current delivered to the slice was increased by a step of 20 μA at a time (Fig. 8 A). For each intensity applied, the fIPSP amplitude was plotted against the stimulus in the WT (n = 6) animals, indicating a decrease in the efficacy of the inhibitory synaptic transmission of the mdx mice. For example, at a stimulus intensity of 60 μA (that activates ~50% of the maximal fIPSP in WT), the amplitude of fIPSP was 140.5 ± 0.6 μV in WT (n = 6), whereas in mdx, it reached 60.4 ± 0.1 μV (n = 6). Interestingly, treatment with ANDRO enhanced the basal inhibitory synaptic transmission in mdx mice. In fact, the fIPSP amplitude elicited by a supraumbral stimulus (up to 30 μA) is increased in ANDRO-treated mdx mice. In this condition, one stimulus of 60 μA evoked a fIPSP that reached values of 130.0 ± 0.4 μV (n = 6; Fig. 8 A and B). Altogether, these data strongly suggest that in vivo activation of non-canonical Wnt signaling induces an enhancement in the number of basal inhibitory synapses, increasing the mdx GABAergic synaptic efficacy.

[1] 39w In the present study, we showed that the activation of the noncanonical Wnt-5a pathway improves the hippocampal GABAergic deficit by increasing the number of inhibitory synapses and GABA A R in the mdx mouse model of Duchenne muscular dystrophy.

[2] 111w The proper functioning of neuronal circuits depends on an adequate balance between inhibitory and excitatory transmission (Choquet and Triller, 2013). Our results show that morphological and electrophysiological alterations in GABA transmission in mdx mice had a postsynaptic origin. We showed that the paired pulse depression and multiplicity index were unaffected, indicating that the probability of GABA release and GABAergic synaptic connectivity in mdx mice were normal. However, our data also indicated that mdx animals presented an important decrease in the frequency and amplitude of mIPSC compared with WT mice. The decrease in synaptic efficacy was associated with a reduction in the number of GABA A -R clusters in the pyramidal neurons.

[3] 87w The alterations in the inhibitory synaptic efficacy that were observed in the hippocampal neurons of mdx mice could explain some cognitive impairment reported in DMD. Knuesel and colleagues reported a significant decrease in the number and size of GABA A -R clusters immunoreactive for α1 and α2 subunits in the cerebellum and hippocampus of mdx mouse (Knuesel et al., 1999). The α1 and α2 subunit-containing receptors have also been shown to play an important role in working memory and cognition (Hashimoto et al., 2009;Zheng et al., 2007).

[4] 132w It has been proposed that the dystrophin-associated glycoprotein complex (DGC) acts in stabilizing the receptors, limiting their lateral diffusion out of the synaptic region by a signaling pathway that is dependent on neuroligin and neurexin (Craig and Kang, 2007;Fritschy and Brunig, 2003;Sudhof, 2008). We observed that the GABA A -R cluster alteration was specifically in the inhibitory receptors and independent of the scaffold protein gephyrin, suggesting that alterations in the inhibitory transmission of the mdx mice would be a consequence of the loss in a GABA A -R cluster subtype in the CA1 pyramidal neurons. Furthermore, an ultra-structural study revealed an increase in the number of CA1 inhibitory synapses in mdx mice, suggesting a reorganization of the inhibitory circuits as a consequence of impaired GABA A -R clustering (Miranda et al., 2009).

[5] 281w The activation of the non-canonical Wnt signaling pathway by the Wnt-5a ligand has been implicated in various aspects of neural development and the modulation of synaptic function (Cuitiño et al., 2010;Farias et al., 2009;Varela-Nallar et al., 2010;Vargas et al., 2014; Box 1). Our results show that treatment with Wnt-5a significantly increased the amount of functional GABAA-Rs on the neuronal cell surface, increasing cluster numbers and the inhibitory GABAergic efficacy in both mdx and WT mice. We observed that Foxy-5 also increased the GABA efficacy in both mdx and WT mice. In agreement with previous results (Cuitiño et al., 2010), we found that Wnt-5a acted in the postsynaptic region, favoring the insertion of GABA A -Rs via the activation of the non-canonical Wnt pathway (Fig. 1). In terms of the postsynaptic locus of the synaptic enhancement of GABA efficacy, we observed that Foxy-5 increased the amplitude of both WT and mdx evoked IPSC without affecting the probability of GABA release, suggesting that the locus of GABA strengthening was caused by an increase in functional GABA A -Rs (Figs. 2 and 9). However, Foxy-5 generated an increase in the frequency of mIPSC, which might indicate pre-synaptic changes. Present result showed a puzzling effect of Foxy-5 on mdx mice GABA transmission. In mdx mice Foxy-5 increases also the mIPSC amplitude, without effect on PPR suggesting a postsynaptic locus of GABA modifications. Taking into account that the developmental expansion of GABA connectivity is caused by the formation of new stereotypically connected cell pairs (Groc et al., 2003) it is possible to resolve this point with the use of the multiplicity index, an approach that estimates synaptic network connectivity (Groc et al., 2003;Hsia et al., 1998).

[6] 96w We observed that the multiplicity indices were similar in mdx and WT mice and were not affected by Foxy-5. Thereby, these results indicated that an average number of release sites at GABAergic synapses on CA1 pyramidal neurons of mdx were essentially the same as in the WT mice, and that number of GABA release sites was not affected by the activation of the non-canonical Wnt pathway. Taken together, our data suggest that the activation of non-canonical Wnt-5a signaling triggers an increase in the number of GABAergic synapses, whereas the number of GABA release sites remain constant.

[7] 276w The use of natural compounds is an emergent conceptual strategy in the search of drugs with therapeutic potentials for the treatment of several neuropathologies. Recent results from our lab suggest that ANDRO through the reduction in the active state of GSK-3β, can plays a role in the protection of synaptic proteins related to synaptic processes, as well as improve the synaptic potentiation and spatial memory function in AβPPswe/PS-1 Alzheimer's mouse model (Serrano et al., 2014). Here, we show that ANDRO acted as a non-canonical ligand via the activation of both the Wnt/Ca 2+ (CaMKII) and Wnt/PCP (JNK) signaling pathways, thereby increasing basal inhibitory synaptic transmission, strengthening of the hippocampal GABAergic efficacy. We observed that ANDRO increase the amplitude of evoked IPSC as well as an increase of frequency of mIPSC in mdx mice. Also we found that ANDRO improved the deficit in basal GABA transmission in the mdx hippocampus. GABA A -Rs are involved in the control of the establishment, maturation and stabilization of functional synapses, as well as, in excitability, cognition and learning and memory (Ben-Ari et al., 2007). Therefore, an alteration in the trafficking, surface expression of GABA A -Rs and synaptic efficacy can contribute to the manifestation of neurological and psychiatric diseases (Gonzalez-Burgos et al., 2011;Jacob et al., 2008). In the present study, we have described how the activation of non-canonical Wnt-5a pathways can increase the surface expression of GABA A -Rs and improve synaptic efficacy in mature hippocampal circuits in mdx mice, conferring to the Wnt signaling pathway a key role in the homeostasis of GABAergic synapses, which might improve the synaptic plasticity and cognitive impairment expressed by dystrophic mice (Fig. 9).

[1] 77w The Wnt-5a analog, Foxy-5 (Safholm et al., 2006) was obtained from Genemed Synthesis (San Antonio, Texas), and ANDRO was obtained from Sigma-Aldrich (St. Louis, MO). The primary antibodies used were rabbit Anti-GABA A receptor (γ2 subunit, #G0545 Sigma), rabbit anti-SAPK/JNK (#9252 Cell Signaling, Danvers, MA), rabbit antiphospho-SAPK/JNK (#9251S Cell Signaling), mouse anti-calcium calmodulin-dependent protein kinase II (CaMKII) (sc-32,288 Santa Cruz Biotechnology Inc., Dallas, TX) mouse anti-phospho-CaMKII (sc-32,289) and mouse anti-β-actin clone AC-15 (A1978 Sigma-Aldrich, St. Louis, MO).

[2] 66w Parental strains of wild-type (C57BL/10) and mdx C57BL/10ScSn-Dmdmdx (mdx) mice were obtained from Jackson Laboratories (BarHarbor, MEHight). The animals were kept at room temperature with a 24-h night-day cycle and fed pellets and water ad libitum. All protocols were conducted under strict accordance with and with the formal approval of the Animal Ethics Committee of the Pontificia Universidad Católica de Chile and Universidad de Valparaíso, Chile.

[3] 212w Procedures for animal care, surgery, and slice preparation were in accordance with the "Guidelines for the Care and Use of Laboratory Animals" adopted by the Society for Neuroscience. The procedures will be described briefly because they have been extensively detailed previously (Fuenzalida et al., 2007;Vargas et al., 2014). Wild-type and mdx mice (2-3 months of age) were decapitated, and the brain was immediately removed and submerged in cold (~4 °C) artificial CSF (ACSF; in mM: 124.00 NaCl, 2.69 KCl, 1.25 KH 2 PO 4 , 2 Mg 2 SO 4 , 26 NaHCO 3 , 2.50 CaCl 2 and 10.00 glucose). The pH was stabilized at 7.4 by bubbling the ACSF with carbogen (95% O 2 , 5% CO 2 ). Transverse hippocampal slices (300-350 μm thick) were cut with a Vibroslice microtome (VSL, WPI, Sarasota, FL) and incubated in ACSF (1 h, at room temperature, 20-22 °C). The slices were transferred to a 2 ml chamber fixed to a binocular stereo microscope (MSZ-10, Nikon). The slices were super-fused with carbogen-bubbled ACSF (2 ml/min) and maintained at room temperature. All recordings were made under 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM, CNQX) and 2amino-5-phosphonovaleric acid (50 μM, APV) (Sigma) in ACSF perfusion media to suppress excitatory α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPA) and N-methyl D-aspartate receptor (NMDAR) transmission.

[4] 422w Single cell recordings were made in the whole-cell configuration with fire-polished pipettes (3-5 MΩ) filled with intracellular solution (see below), connected to an EPC-7 patch-clamp amplifier (Heka Instruments), filtered at 3.0 kHz, sampled at 4.0 kHz using an A/D converter (ITC-16, InstruTech), and stored with Pulse FIT software (Heka Instruments). Single-electrode voltage-clamp recordings were obtained from pyramidal neurons of CA1. In the voltage-clamp configuration, the series resistance was compensated to ~70%, and recordings were accepted only when the seal resistance was ~1 GΩ and the series resistance (7-14 MΩ) did not change ~10% during the experiment. The intracellular solution contained (in mM): 97.5 K-gluconate, 32.5 KCl, 10.0 HEPES, 1 MgCl 2 , 5 EGTA, and 4-sodium salt (Na-ATP), pH 7.2. The experiments started after a 5-10 min stabilization period following entry into the intracellular compartment with the patch electrodes. The voltage-clamp recordings were rejected when the access resistance (7-15 MΩ) increased 20% during the experiment. Recordings of mini inhibitory postsynaptic currents (mIPSC) were made under tetrodotoxin (TTX) (1 μM), and the data were analyzed offline using analysis software (Minianalysis, Synaptosoft). Considering the intra-and extracellular chloride concentration, the reversal potential of the IPSC was ~60 mV. Then, to dissect IPSC from excitatory postsynaptic currents (EPSC), all the cells were voltage clamped at 0 mV (holding potential). The evoked IPSC was elicited using concentric electrodes (platinum/ iridium, FHC Inc.), which were placed at the stratum radiatum close to the pyramidal layer (~10-20 μm). GABAergic neurons were activated by bipolar cathodic stimulation through an isolation unit (Isoflex, A.M.P.I.). Voltage-clamp data were high-pass filtered at 3.0 kHz and sampled at rates between 6.0 and 10.0 kHz through a Digidata 1322A (Molecular Devices). The paired pulse ratio (PPR) was calculated as (R2/R1) × 100, where R1 and R2 are the peak amplitudes of the first and second IPSCs, respectively. Sixty traces (5-min recording) were digitally averaged for calculations. The field inhibitory postsynaptic potentials (fIPSP) (isolated under ionotropic glutamate receptors antagonist (50 μM D-AP5 and 20 μM CNQX)), was elicited using concentric electrodes (platinum/iridium, FHC Inc.), which were placed at the stratum radiatum close to the pyramidal layer (~10-20 μm). The extracellular glass recordings pipettes (2-4 MΩ, filled with ACSF) was placed in the stratum pyramidale and connected to an AC amplifier (P-5 Series, Grass), with a gain of 10,000×, LP filters of 3.0 kHz and an HP filter of 0.30 Hz. This fIPSP was blocked by GABA A R antagonist Picrotoxin (10 μM), confirming that the event depend on GABA A R activation and was synaptically generated.

[5] 54w Multiplicity was calculated as the mean amplitude of action potential-driven events (a) divided by the mean quantal size (q: mean amplitude of mEPSC recorded in TTX). The a value was determined for each cell, which subtracted the contribution of mEPSC to the pool of events collected in absence of TTX using the following expression:

[6] 68w where f b and f q denote the mean frequency values from events recorded before and after the addition of TTX to the perfusion media, respectively, and b is the mean amplitude of both action potential-driven sIPSC and mIPSC. To determine the effect of Wnt signaling activation on inhibitory synaptic efficacy, Foxy-5 (50 μM) was added to the ACSF perfusion media after 10 min of a stable recording.

[7] 115w Hippocampal neurons were obtained from 18-d old wild-type and mdx mice embryos. Hippocampi were aseptically dissected and trypsinized for 20 min. After centrifugation for 1 min, the neurons were seeded in phenol-red-free DMEM (plus 10% horse serum) into 1% poly-L-lysine-coated plates. After 120 min, the medium was removed, and Neurobasal medium was added, containing 1% B27 supplement from Invitrogen. On day 3 of the culture, hippocampal neurons were treated with 2 μM 1-β-D-arabinofuranosylcytosine (AraC) for 24 h. Fifteen-to eighteen-day old neuronal cultures were used for various experiments; the average number of neurons in each experiment was ~95% of the cells present in the cultures (Alvarez et al., 2004;Farias et al., 2007Farias et al., , 2009)).

[8] 170w The samples were dissected on ice and immediately frozen in liquid nitrogen or processed as previously detailed (Carvajal et al., 2013). Briefly, tissues were homogenized in RIPA buffer (10 mM Tris-Cl, pH 7.4, EDTA 5 mM, 1% NP-40, 1% sodium deoxycholate, and 1% SDS) supplemented with a protease inhibitor mixture (1 mM PMSF, 2 μg/mL aprotinin, 1 μg/mL pepstatin and 10 μg/mL benzamidine) and phosphatase inhibitors (25 mM NaF, 100 mM Na 3 VO 4 , 1 mM EDTA and 30 μM Na 4 P 2 0 7 ) using a Potter homogenizer and then passed sequentially through different caliber syringes. Protein samples were centrifuged at 14,000 rpm at 4 °C twice for 15 min. Protein concentrations was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL). The samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane. The reactions were followed by incubation with anti-mouse, anti-goat named above or anti-rabbit IgG peroxidase conjugated antibodies (Pierce) and developed using an ECL kit (Western Lightning Plus ECL, PerkinElmer).

[9] 38w Animals were administered 4 mg/kg ANDRO suspended in saline solution (or saline solution as a vehicle) intraperitoneally three times per week for 2 months. The animals were housed in groups of three with food and water ad libitum.

[10] 69w The statistical analysis was performed using the statistical software Prism 5 (GraphPad Software, Inc.). All reported values are expressed as the mean ± SEM. The statistical significance of differences was assessed with the non-paired Student's t-test or analyses of variance (ANOVA) after normal distribution of the data were verified using the Shapiro-Wilk normality test (GraphPad Software, Inc.). Non-normally distributed data were analyzed using either the Mann-Whitney or Kruskal-Wallis test.

[1] 105w To generate secreted Wnt ligand, HEK-293 cells were stably transfected by Lipofectamine 2000 (Invitrogen, Grand Island, NY), according to manufacturer's instructions, with constant and equal amounts of empty vector pcDNA or pcDNA containing sequences encoding Wntligand coupled to the sequence encoding a hemagglutinin (HA) tag (Cuitiño et al., 2010). For Wnt-conditioned or control media, transfected HEK-293 cells were grown to 85% confluence and maintained in Neurobasal medium without supplements for 60 h. Wnt secretion was verified by Western blot using a HA-specific antibody (Millipore, Billerica, MA). For the electrophysiological studies, the media containing Wnt ligands was dialyzed against ACSF for 16 h at 4 °C.

[2] 251w Hippocampal neurons were cultivated at a density 30,000 cells/coverslip. For cell surface GABA A -R staining, neurons were fixed with 4% paraformaldehyde/4% sucrose for 20 min at room temperature. The fixed cells were then washed and incubated with an antibody to γ2 GABA A -R subunit (Sigma-Aldrich, St. Louis, MO). For the staining of gephyrin, neurons were fixed as described above and permeabilized by incubation in PBS-0.2% Triton X-100. Then, the nerve cells were incubated with Alexa 543 and/or Alexa 488 (Pierce) for 30 min at 37 °C. To analyze receptor clustering, we quantified the number of clusters per neurite length with the ImageJ program [National Institutes of Health (NIH), Bethesda, MD]. Neurons on coverslips were imaged using a confocal microscope LSM 5 Pascal with a 63×/1.4 numerical aperture oilimmersion objective. The images used for quantification were taken with identical microscope settings and analyzed using the ImageJ software (NIH). GABA A -R images from 10 microscope fields for each condition (three independent experiments) were registered. Each field containing processes for 1 neuron was studied, in which 3 neurites were selected per neuron using the phalloidin staining to label neuronal processes. To quantify GABA A -R clusters, images of individual neurites were isolated, and the background for neurite-free fields were subtracted and adjusted to the threshold. GABA A -R cluster number and size were obtained with the Particle Analysis tool using a size particle limit of 0.05-1 μm 2 . The cluster number was normalized against neurite length to obtain cluster density.