For motor neuron studies, anesthetized animals were submitted to an intracardial perfusion of 40 ml of PBS solution at 1.2 ml/min followed by 40 ml of 4% PFA answer diluted in PBS answer at the same flow. in SMA results from alterations of systemic pathways that would ultimately target motor neurons. Although multiple neurotrophic factors are implicated in neuron survival, insulin-like growth factor-1 (IGF-1) is usually a critical factor for the maintenance and the survival of motor neurons (Neff et al., 1993; Ozdinler and Macklis, 2006). Interestingly, several recent investigations pointed toward an IGF-1 signaling pattern-alteration in SMA. First, the levels of circulating IGF-1 and hepatic mRNA expression were shown to display a marked reduction in a severe type of SMA-like model mice (Hua et al., 2011; Murdocca et al., 2012). Second, in skeletal muscles from type 1 SMA patients (Millino et al., 2009) or from SMA mice (Bosch-Marc et al., 2011), the IGF-1 receptor (IGF-1R) was found to be overexpressed. The level of this overexpression in human biopsies LY2979165 correlated with the severity of the disease (Millino et al., 2009). Interestingly, increasing IGF-1 availability for mouse SMA mouse muscles had no effect on IGF-1R expression (Bosch-Marc et al., 2011), suggesting that IGF-1R overexpression was constitutive and not the result of a compensatory mechanism for a putative decrease in serum IGF-1 concentration. Unfortunately, data around the IGF-1R expression in SMA mouse CNS are presently lacking. Third, among the three intracellular signaling pathways known to lie downstream of IGF-1R, the ERK1/2, JAK/STAT3, and AKT pathways (D’Mello et al., 1997; Prrizas et al., 1997; Yadav et al., 2005), the ERK cascade was LY2979165 found to be constitutively overactivated in the spinal cord and muscles (Millino et al., 2009; Biondi et al., 2010). In contrast, the AKT pathway was found to be underactivated in SMA mouse spinal cords (Biondi et al., 2010; Kye et al., 2014). Interestingly, IGF-1R overexpression was reported in the CNS and muscles from patients with amyotrophic lateral sclerosis (ALS) (Wilczak et al., 2003; Lunetta et al., 2012), another fatal motor neuron disease, contrasting with nearly identical serum IGF-1 concentration (Corbo et al., 2010). Moreover, recent data exhibited that the reduction of IGF-1 signaling through gene partial inactivation ((mice with B6.129-= 74) mice and (= 65) mice were LY2979165 referred to as control and = 62) and = 61) mice were designated as SMA and for 10 min at +4C. The serum was frozen, stored at ?80C, and used within 1 month. The quantity of IGF-1 was decided on 100 l of serum in duplicate using the Murine IGF-1 ELISA kit (900-K170; Peprotech) and following the manufacturer’s instructions. hybridization. To detect mRNAs, antisense oligonucleotide probes were synthesized as described previously (Oshima et al., 2002; Fukaya et al., 2005): forward 5-CACAGTCATCCGTGGCTGGA-3 and reverse 5-CGCTTCCCACACACACTTGG-3. Under deep pentobarbital (40 mg/kg) anesthesia, spinal cords were obtained from P12 SMA-like mice. Frozen sections (14 m thickness) were mounted on glass slides coated with 3-aminopropyltriethoxysilane. Each section was fixed immediately after cutting by immersion for 20 min in 2% paraformaldehyde in phosphate buffer (0.1 m, pH 7.4). Sections were then rinsed three times in PBS, dipped in water, and dehydrated with a graded ethanol series (70%, 95%, and 100%). The sections were then air-dried and stored at ?80C until further use. The hybridization mixture contained 50% formamide, 4 standard saline citrate (SSC), 1 Denhardt’s answer, 0.25 mg/ml yeast tRNA, 0.25 mg/ml sheared salmon sperm, 0.25 mg/ml polyA, 10% dextran sulfate, 100 mm DTT, and [35S]-dATP-labeled oligonucleotide probes (4.5 106 dpm/ml). One hundred microliters of Lep hybridization mixture was placed on each section. The sections were then covered with a Parafilm coverslip and incubated in a humidified chamber at +43C for 20 h. After hybridization, the sections were washed successively twice in 1 SSC made up of 0. 01 m DTT and twice in 0.5 SSC containing 0.01 m DTT for 15 min at +55C and finally in 0.5 SSC containing 0.1 m DTT for 15 min at room temperature. Sections were exposed to Nuclear Track emulsion (NTB-2; Kodak) for 8 weeks. Histological and immunohistochemical analysis. For motor neuron studies, anesthetized animals were submitted to an intracardial perfusion of 40 ml of PBS answer at 1.2 ml/min followed by 40 ml of 4% PFA answer diluted in PBS answer at the same flow. Then, spinal cords were dissected, incubated overnight in 4% PFA answer, and washed twice for 2 h with PBS. The lumbar spinal cords (L1 to L5) were cut using a vibratome VT-1000S (Leica) on the whole length of the sample at 50-m-thick cross sections. One of every five sections was processed for immunohistochemical analysis as described previously (Branchu.