Amyotrophic lateral sclerosis, gene deregulation in the anterior horn of the spinal cord and frontal cortex area 8: implications in frontotemporal lobar degeneration

Microarray analysis

Cofactors age and gender were not relevant for the analysis. 9,563 gene sequences were detected across all samples. Heat map indicates differences in transcripts expression levels between control and ALS cases in the anterior cord of the spinal cord and in frontal cortex area 8 (Figure 1). We identified 747 genes differentially expressed with p-value lower than or equal to 0.05 in the anterior horn of the spinal cord (up: 507 and down: 240) and 2,300 genes differentially expressed in the frontal cortex area 8 (up: 1,409 and down: 891) in sALS (Figure 1).

Supplementary Table 1 and Supplementary Table 2 identify all de-regulated genes. Post-analysis microarray data of differentially expressed genes assessed with enrichment analysis against Go Ontology database are shown in Tables 1 and 2.

Up-regulated genes in ALS anterior horn of the spinal cord cluster into inflammatory responses, metal ion regulation and hemostasis; whereas down-regulated genes cluster into neuronal axonal cytoskeleton and apoptosis.

In contrast, clusters of up-regulated genes were involved in neurotransmission, ion channels and ion transport, synapses, maintenance of axons and dendrites, intracellular signaling and synaptic vesicle mechanisms. The majority of down-regulated genes were encoded for proteins associated with myelin and glial cell regulation (Figure 2).

RT-qPCR validation

Sixty-six genes from different pathways were selected for validation by RT-qPCR.

Inflammatory gene expression in the anterior horn of the spinal cord

No modifications in the expression levels of glial fibrillary acidic protein gene (GFAP) or prostaglandin-endoperoxide synthase 2 gene (PTGS2) occurred in ALS when compared with controls (p=0.31 and p=0.55, respectively). However, expression levels of AIF1 and CD68 were significantly increased in the anterior horn of the spinal cord in ALS (p=0.044 and p=0.00023, respectively). Gene expression of toll-like receptors (TLRs) TLR2, TLR and TLR7 was significantly increased in the spinal cord in ALS cases (p=2.48E-05, p=0.00011 and p=0.00074, respectively), but TLR4 was not (p=0.669). IL1B was up-regulated (p=0.005), but IL6 and IL6ST were not (p=0.26 and p=0.76, respectively). In contrast, the expression of IL10 and its corresponding receptors IL10RA and IL10RB was increased in ALS (p=0.00046, p=0.022 and p=3.23E-05, respectively). TNFA expression was significantly increased whereas a trend was found for TNFRSF1B (p=0.04 and p=0.08, respectively). The expression of CTSC and CTSS was significantly increased in spinal cord in ALS (p=5.82204E-05 and p=0.00014, respectively). Levels of SLC11A1 were also significantly increased in spinal cord of ALS (p=0.014). HLA-DRB1, a protein coding gene for the Major Histocompatibility Complex Class II (MHC-II) DR β1 protein was markedly up-regulated in ALS (p=0.004365). PDCD1LG2, IFNγ and IL33 were significantly up-regulated in the anterior horn of the spinal cord in ALS (p=0.00153, p=0.03 and p=0.0032, respectively). Finally, IL8 (interleukin 8) and ITGB4 (integrin subunit beta 4) expression was similar in control and ALS cases (p=0.92 and p=0.40, respectively) (Figure 3).

Axonemal gene expression in anterior horn of the spinal cord

No modifications in the expression levels of NEFH, which codes for neurofilament heavy polypeptide protein, was seen in ALS when compared with controls (p=0.30). However, DNAAF1 levels were significantly reduced (p=0.019). Expression of DNAH2, DNAH5, DNAH7 and DNAH11 mRNA was significantly reduced in ALS (p=0.029, p=0.012, p=0.005 and p=0.023, respectively), whereas DNAH9 mRNA was not altered (p=0.14). DNAI1 mRNA expression was also significantly reduced in ALS (p=0.0086) (Figure 3).

SLC1A2 and SC17A7 expression in anterior horn of the spinal cord

SLC1A2 and SLC17A7 expression levels were significantly decreased in the anterior horn of the spinal cord in ALS anterior (p=0.000115 and p=0.000125, respectively). See Figure 3.

Neurotransmission-related gene expression in frontal cortex area 8

GRIA1, which codes for the ionotropic glutamate receptor AMPA 1, and GRIN2A and GRIN2B, coding for NMDA receptors, were significantly up-regulated (p=0.018, p=0.018 and p=0.029, respectively) in frontal cortex in ALS cases. GRM5, which codes for the glutamate metabotropic receptor 5, was also up-regulated (p=0.0079). However, no significant alteration was seen in the expression of NETO1 (p=0.165). Regarding the GABAergic system, GAD1 was up-regulated in ALS (p=0.034). Gene expression of GABA receptors GABRA1, GABRD, GABRB2 was increased (p=0.09, tendency, p=0.006 and p=0.0029, respectively). GABBR2 mRNA levels were also significantly elevated in the frontal cortex in ALS (p=0.01) (Figure 4).

Synaptic cleft gene expression in frontal cortex area 8

BSN, which codes for Bassoon, a pre-synaptic cytoskeletal matrix, was up-regulated in ALS (p=0.04). mRNA levels of PCLO, coding gene for Piccolo protein, and FRMPD4 were also increased in ALS (p=0.036 and p=0.029, respectively), Finally, NRN1, which codes for neuritin 1, but not DDN, which codes for dendrin protein, was up-regulated in the frontal cortex in ALS (p=0.04 and p=0.92, respectively) (Figure 4).

Myelin- and oligodendrocyte-related gene expression in frontal cortex area 8

Significant decrease in mRNA expression of myelin transcription factor (MYRF) (p= 0.028), OLIG2 (p=0.009), SOX10 (p=0.02), NKX2-2 (p=0.032), transferring (TF) (p=0.5), proteolipid protein 1 (PLP1) (p=0.040), myelin basic protein (MBP) (p=0.061), myelin-associated oligodendrocyte basic protein (MOBP) (p=0.019), oligodendrocyte glycoprotein (MOG) (p=0.05), Mal T-cell differentiation protein (MAL) (p=0.039), myelin associated glycoprotein (MAG) (p=0.035), and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP1) (p=0.017) was seen in frontal cortex in ALS cases compared with controls (Figure 4).

SLC1A2 and SLC17A7 expression in frontal cortex area 8

SLC1A2 expression was significantly increased (p=5.25e-5) whereas SLC17A7 mRNA showed a non-significant increase (p=0.42) in frontal cortex area 8 in ALS (Figure 4).

Immunohistochemistry in spinal cord

The anterior horn of the spinal cord in ALS cases showed decreased number of neurons and altered morphology of most remaining motor neurons including loss of endoplasmic reticulum (chromatolysis) and axonal ballooning (Figure 5a) and intracytoplasmic TDP-43-immunoreactive inclusions (Figure 5b). Immunohistochemistry was carried out in the lumbar spinal cord in control and sALS cases (Figure 5a and b). VDAC was reduced in a subpopulation of neurons in the anterior horn in ALS, but not in neurons of the Clarke’s column and posterior horn, when compared with controls (Figure 5c and d). Increased expression of GFAP was found in reactive astrocytes in the lateral columns and anterior horn of the spinal cord in ALS cases (Figure 5e and f). Marked differences were seen regarding microglial cell markers: IBA-1 and CD68 immunoreactivity was dramatically increased in the pyramidal tracts and anterior horn in ALS; moreover the morphology of microglia was modified in pathological cases with predominance of round, amoeboid microglia (Figure 5g-j). Similar immunoreactivity, distribution and morphology were found in reactive microglia using antibodies against HLA-DRB1 and HLA-DRB5 (Figure 5k-n). In contrast IL-10 and TNF-α immunoreactivity predominated in neurons; immunoreactivity was increased in neurons in ALS cases compared with controls (Figure 5o-r). Finally, GluT (SLC1A2), the transporter of glutamate from the extracellular space at synapses, was expressed in the membrane of neurons and in the neuropil; SLC1A2 immunoreactivity was decreased in neurons and neuropil of the anterior horn in ALS (Figure 5s, t).

Gel electrophoresis and western blotting in frontal cortex area 8

A few tested antibodies were eventually suitable for western blotting studies. No differences in the expression levels of glutamate receptor ionotropic, NMDA 2A (NMDAR2A) and glutamate decarboxylase 1 (GAD1) were observed between control and ALS cases. However, a significant increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 1 (AMPAR GluR-1) ** p < 0.01 and a tendency to increase in the expression of gamma-aminobutyric acid receptor subunit beta-2 (GABAAB2) (# p < 0.1) was found in the frontal cortex in ALS when compared to controls (Figure 6).

Inflammation in the anterior horn of the spinal cord

AIF1 gene codes for the Allograft Inflammatory Factor 1, a protein induced by cytokines and interferon which promotes macrophage and glial activation [32,33]. CD68 codes for the macrophage antigen CD68 glycoprotein which is expressed by microglial cells [35–37], the principal resident immune cell population in brain [38,39]. Microglia pro-inflammatory state activation can be initiated by engagement of germline-encoded pattern-recognition receptors such as Toll-like receptors (TLRs) which are expressed in glial cells [40]. TLR activation, in turn, activates phagocytosis [41–43] and pro-inflammatory responses [44]. Up-regulated interleukins in ALS are IL1B, the coding gene for interleukin 1B an important mediator of the inflammatory response [45], interleukin 10 (encoded by IL10) which has pleiotropic effects down-regulating the expression of Th1 cytokines, MHC class II antigens and co-stimulating the production of several molecules by macrophages through the activation of IL10 receptor subunit α and subunit β (encoded by IL10RA and IL10RB, respectively) [46]. However, IL6 mRNA, which encodes a specific pro-inflammatory cytokine with regenerative and anti-inflammatory activities in particular settings [47–50] is not modified. Tumor Necrosis Factor Receptor Superfamily Member 1A (encoded by TNFA) is involved in the regulation of a wide spectrum of biological processes including cell proliferation, cell differentiation, apoptosis, lipid metabolism and coagulation [50,51]. CTSC gene encodes Cathepsin C which is central coordinator of activation of many serine proteinases in immune cells [52]. CTSS codes for a protein of the same family, Cathepsin S, which acts as a key protease responsible for the removal of the invariant chain from MHC class II antigens [53]. SLC11A1 encodes natural resistance-associated macrophage protein 1, which acts as a host resistance to certain pathogens [54].

Major Histocompatibility Complex Class II (MHC-II) DR β1 protein, encoded by HLA-DRB-1, plays a central role in the immune system by presenting peptides derived from extracellular proteins [55,56] and participate in the activation of autophagosomes [57].

PDCD1LG2 codes for Programmed Cell Death 1 Ligand 2, a protein involved in co-stimulatory signals essential for T-cell proliferation and IFN-γ production [58]. IFNγ gene, which codes for the cytokine interferon-γ, is key player in antigen-specific immune responses [59]. Finally, interleukin 33, encoded by IL33, acts as a chemo-attractant for Th2 cells and functions as an ‘alarm’ that amplifies immune responses during tissue injury [60].

Increased inflammatory response in the anterior horn of the spinal cord has been further documented by immunohistochemistry showing increased expression of IBA-1, the protein encoded by AIF1, CD68, and HLA-DRB1 and HLA-DRB5 in reactive microglia. Reactive microglia has a round, amoeboid morphology and is also localized, as expected in the lateral and anterior pyramidal tracts. IL-10 and TNF-α are mainly localized in neurons of the spinal cord, and its expression is increased in remaining motor neurons of the spinal cord in ALS. These findings indicate a parallelism between gene expression and protein expression regarding inflammatory responses of assessed molecules. On the other hand the different localization of microglial markers, and IL-10 and TNF-α in neurons points to a cross-talk between microglia and neurons in the anterior horn of the spinal cord in ALS.

This is in contrast with other markers as glial fibrillary acidic protein and voltage dependent anion channel in which levels of mRNA differ from levels (or intensity) of protein expression. No modifications in the expression of GFAP mRNA are observed in the present study, but GFAP immunoreactivity is clearly increased in reactive astrocytes, as already reported in classical neuropathological studies. VDAC mRNA is not abnormally regulated in gene arrays; yet VDAC is decreased in motor neurons, but not in neurons of the Clarke’s column and neurons of the posterior horn, of the spinal cord in ALS. VDAC immunohistochemistry is in line with observations in human sALS showing deficiencies in mitochondria and energy metabolism [61,62].

Reduced expression of axolemal genes in anterior horn of the spinal cord

The expression levels of NEFH, which codes for neurofilament heavy polypeptide protein [63], are preserved in ALS. However, DNAAF1, which encodes dynein (axonemal) assembly factor 1, and mRNAs encoding several dynein axonemal heavy chains (DHC) are down-regulated thus suggesting impairment of motor ATPases involved in the transport of various cellular cargoes by ‘walking’ along cytoskeletal microtubules towards the minus-end of the microtubule [64–66].

Up-regulation of neurotransmission-related genes and synaptic cleft genes in frontal cortex

Genes involved in glutamatergic and GABAergic transmission are up-regulated in the frontal cortex in ALS. This applies to genes encoding the ionotropic glutamate receptor AMPA 1 (GRIA1), glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), the glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), and glutamate metabotropic receptor 5 (GRM5). Regarding the GABAergic system, GAD1, coding for glutamate decarboxylase 1, a rate-limiting enzyme that acts in the decarboxylation of glutamate essential for the conversion reaction of GABA from glutamate [67,68], is up-regulated, as are GABRA1, GABRD, GABRB2, which code for different subunits of ionotropic GABA-A receptors. GABBR2, which codes for the metabotropic receptor component Gamma-Aminobutyric Acid Type B Receptor Subunit 2 and forms heterodimers with GABBR1, thus resulting in the formation of the G-protein coupled receptor for GABA [69], is also up-regulated in ALS.

In line with increased expression of neurotransmitter-related genes, several genes encoding molecules linked with the synaptic cleft are also up-regulated in ALS. BSN codes for Bassoon, a pre-synaptic cytoskeletal matrix (PCM) protein acting as a scaffolding protein and essential for the regulation of neurotransmitter release in a subset of synapses [70,71]. PCLO codes for Piccolo protein, a component of the PCM assembled in the active zone of neurotransmitter release [72,73]. FRMPD4 codes for PSD-95-interacting regulator of spine morphogenesis protein which regulates dendritic spine morphogenesis and is required for the maintenance of excitatory synaptic transmission [74]. DDN and NRN1 code for dendrin protein and neuritin 1 protein, respectively which are involved in the remodeling of the postsynaptic cytoskeleton and neuritic outgrowth [75–77].

De-regulation of neurotransmitters and receptors is further supported by the demonstration of significant increase in the levels of GluR-1 and a tendency in those of GABAAB2 in the frontal cortex area 8 in ALS when compared with controls. It is worth stressing that only a few antibodies of the total assessed (eight) were suitable for western blotting.

Myelin and oligodendrocyte genes in frontal cortex area 8

Myelin transcription factor (encoded by MYRF) regulates oligodendrocyte differentiation and is required for central nervous system myelination [78–81]. The basic loop- helix protein OLIG2 mediates motor neuron and oligodendrocyte differentiation [22,82]. High mobility group protein SOX10 modulates myelin protein transcription [83,84]. NKX2.2 homeodomain transcription factor is a key regulator of oligodendrocyte differentiation [85]. Transferrin encoded by TF participates in the early stages of myelination [86,87]. Proteolipid protein 1 (encoded by PLP1) plays a role in the compaction, stabilization, and maintenance of myelin sheaths, as well as in oligodendrocyte development and axonal survival [88,89]. Myelin basic protein (encoded by MBP) is the second most abundant myelin-associated protein, constituting about 30% of total myelin protein [90]. Myelin-associated oligodendrocyte basic protein (encoded by MOBP) constitutes the third most abundant protein in CNS myelin and it acts by compacting and stabilizing myelin sheaths [91]. Myelin oligodendrocyte glycoprotein (encoded by MOG) is a cell surface marker of oligodendrocyte maturation [92]. Myelin associated glycoprotein (encoded by MAG) is a type I membrane protein and member of the immunoglobulin super-family involved in the process of myelination and certain myelin-neuron cell-cell interactions [93]. Mal T-cell differentiation protein (encoded by MAL) is involved in myelin biogenesis [94]. Finally, 2',3'-cyclic nucleotide 3' phosphodiesterase (encoded by CNP1) participates in early oligodendrocyte differentiation and myelination [95–97].

Concluding comments

Results of the present study validate gene expression of individual studies performed in a limited number of samples identifying a limited number of de-regulated genes in the anterior horn of the spinal cord [17,20,21,25]. Present results are more close to those carried out by using laser micro-dissection of anterior horn spinal motor neurons [27] thus reinforcing the consistence of observations in both studies. Whether some changes are related to the variable progression of the disease need further study with a larger number of cases of rapid or slow clinical course. In this line, altered mitochondria, protein degradation and axonal transport predominate in the 129Sv-SOD1(G93A) transgenic mouse with rapidly progressive motor neuron disease, whereas increased immune response is found in the C57-SOD1(G93A) transgenic mouse with more benign course [98].

The most important aspect of the present study is the description of altered gene expression and identification of altered clusters of genes in the frontal cortex area 8 in sALS cases without apparent cognitive impairment. It is worth stressing that altered clusters differ in the spinal cord and frontal cortex in sALS at terminal stages thus providing valuable information of molecular abnormalities which can also be present within the spectrum of FTLD-TDP. Noteworthy, altered regulation of transcription related to synapses and neurotransmission covering neurotransmitter receptors, synaptic proteins and ion channels in the frontal cortex in the absence of overt clinical symptoms of cognitive impairment are particularly important to identify early molecular alterations in frontal cortex with the spectrum of ALS/FTLD-TDP.

Tissue collection

Post-mortem fresh-frozen lumbar spinal cord (SC) and frontal cortex (FC) (Brodmann area 8) tissue samples were from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank following the guidelines of Spanish legislation on this matter and the approval of the local ethics committee. The post-mortem interval between death and tissue processing was between 2 and 17 hours. One hemisphere was immediately cut in coronal sections, 1-cm thick, and selected areas of the encephalon were rapidly dissected, frozen on metal plates over dry ice, placed in individual air-tight plastic bags, numbered with water-resistant ink and stored at -80°C until use for biochemical studies. The other hemisphere was fixed by immersion in 4% buffered formalin for 3 weeks for morphologic studies. Transversal sections of the spinal cord were alternatively frozen at -80°C or fixed by immersion in 4% buffered formalin. The whole series included 18 sALS cases and 23 controls. The anterior horn of the spinal cord was examined in 14 sALS (mean age 57 years; 6 men and 8 women) and the frontal cortex area 8 in 15 sALS (mean age 54 years; 11 men and 4 women). Spinal cord and frontal cortex were available in 11 cases. Lumbar anterior spinal cord was dissected on a dry-ice frozen plate under a binocular microscope at a magnification x4. TDP-43-immunoreactive small dystrophic neurites and/or TDP-43-positive granules and/or small cytoplasmic globules in cortical neurons in the contralateral frontal cortex area 8 were observed in 11 of 18 cases, but only abundant in three cases (cases 29, 30 and 31 in Table 3). Spongiosis in the upper cortical layers was found only in one case (case 28 in Table 3). Cases with frontotemporal dementia were not included in the present series. Patients with associated pathology including Alzheimer’s disease (excepting neurofibrillary tangle pathology stages I-II of Braak and Braak), Parkinson's disease, tauopathies, vascular diseases, neoplastic diseases affecting the nervous system, metabolic syndrome, hypoxia and prolonged axonal states such as those occurring in intensive care units were excluded. Cases with infectious, inflammatory and autoimmune diseases, either systemic or limited to the nervous system were not included. Age-matched control cases had not suffered from neurologic or psychiatric diseases, and did not have abnormalities in the neuropathologic examination, excepting sporadic neurofibrillary tangle pathology stages I-II of Braak and Braak. No C9ORF72, SOD1, TARDBP and FUS mutations occurred in any case. Table 3 shows a summary of cases.

Whole-transcriptome array

RNA from frozen anterior horn of the lumbar spinal cord and frontal cortex area 8 was extracted following the instructions of the supplier (RNeasy Mini Kit, Qiagen® GmbH, Hilden, Germany). RNA integrity and 28S/18S ratios were determined with the Agilent Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) to assess RNA quality, and the RNA concentration was evaluated using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). Selected samples were analyzed by microarray hybridization with GeneChip® Human Gene 2.0 ST Array and WT Labeling Kit and microarray 7000G platform from Affymetrix® (Santa Clara, CA, USA). Microarray service was carried out at the High Technology Unit (UAT) at Vall d’Hebron Research Institute (VHIR), Barcelona, Spain.

Microarray data and statistical analysis

Microarray data quality control, normalization and filtering were performed using bioconductor packages in an R programming environment for genes [99] which enabled data preprocessing for differential gene expression analysis and enrichment analysis. Gene selection was based upon their values using a test for differential expression between two classes (Student’s t-test). Genes differentially expressed showed an absolute fold change > 2.0 in combination with a p-value ≤ 0.05.

RT-qPCR validation

Complementary DNA (cDNA) preparation used High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided by the supplier. Parallel reactions for each RNA sample run in the absence of MultiScribe Reverse Transcriptase to assess the lack of contamination of genomic DNA. TaqMan RT-qPCR assays were performed in duplicate for each gene on cDNA samples in 384-well optical plates using an ABI Prism 7900 Sequence Detection system (Applied Biosystems, Life Technologies, Waltham, MA, USA). For each 10μL TaqMan reaction, 4.5μL cDNA was mixed with 0.5μL 20x TaqMan Gene Expression Assays and 5μL of 2x TaqMan Universal PCR Master Mix (Applied Biosystems). Table 4 shows identification numbers and names of TaqMan probes. The mean value of one house-keeping gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT1), was used as internal control for normalization of spinal cord samples, whereas the mean values of the three house-keeping genes, alanyl-transfer RNA synthase (AARS), glucuronidase Beta (GUS-β) and X-prolyl aminopeptidase (aminopeptidase P) 1 (XPNPEP1) were used as internal controls for normalization of frontal cortex samples [100,101]. The parameters of the reactions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Finally, capture of all TaqMan PCR data used the Sequence Detection Software (SDS version 2.2.2, Applied Biosystems). The double-delta cycle threshold (ΔΔCT) method was used to analyze the data; results with T-student test. The significance level was set at * p < 0.05, ** p < 0.01 and *** p < 0.001, and tendencies at # < 0.1. Pearson’s correlation method assessed a possible linear association between TDP-43 pathology in frontal cortex area 8 and gene deregulation in the same region; significant correlations were not found.

Immunohistochemistry

De-waxed sections, 4μm thick, of the lumbar spinal cord from control and ALS cases were processed in parallel for immunohistochemistry. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂O₂ for 15 min followed by 3% normal horse serum. Then the sections were incubated at 4ºC overnight with one of the primary antibodies: rabbit polyclonal antibodies to IBA-1 (019-19749, Wako Chemicals Gmbh, Neuss, GE) were used at a dilution of 1:1,000; VDAC (voltage dependent anion channel, ab15895, Abcam, Cambridge, UK) at 1:100; HLA-DRB1 (GTX104919, GeneTex, Barcelona, Spain) at 1:100; HLA-DRB5 (NBP2, Novusbio, Littleton, Colorado, USA) at 1:100; IL-10 (AP52181PU, ACRIS, ProAlt, Madrid, Spain) at 1:100; and GFAP (glial fibrillary acidic protein, RP014-S, Diagnostic Biosystem, Palex Medica, Sant Cugat, Spain) at 1:400. Mouse monoclonal antibodies to CD68 (ab955, Abcam, Cambridge, UK) and TNF-α (ab1793, Abcam, Cambridge, UK), were used at dilutions of 1:200 and 1:150, respectively. Antibodies to GluT: SLC1A2 (ab1783, Millipore, Billerica, MA, USA) were used at a dilution of 1:100. Following incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase (Dako, Agilent, Santa Clara, CA, USA) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂O₂. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Sections were slightly stained with haematoxylin.

Gel electrophoresis and western blotting

Frozen samples of the somatosensory cortex were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). The homogenates were centrifuged for 20 min at 12,000 rpm. Protein concentration was determined with the BCA method (Thermo Scientific). Equal amounts of protein (20μg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Amersham, Freiburg, GE). Non-specific bindings were blocked by incubation in 3% albumin in PBS containing 0.2% Tween for 1 h at room temperature. After washing, membranes were incubated overnight at 4°C with antibodies against glutamate receptor ionotropic, NMDA 2A (NMDAR2A, 130 kDa, rabbit, 1:200, Abcam, Cambridge, UK), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 1 (AMPAR GluR-1, 100 kDa, rabbit, 1:200, Cell Signaling Technology, Danvers, MA, USA), glutamate decarboxylase 1 (GAD1, 67 kDa, rabbit, 1:200, Cell Signaling Technology, Danvers, MA, USA) and gamma-aminobutyric acid receptor subunit beta-2 (GABAAB2, 59 kDa, mouse, 1:1000, Abcam, Cambridge, UK). Protein loading was monitored using an antibody against β-actin (42 kDa, 1:30,000, Sigma). Membranes were incubated for 1 h with appropriate HRP-conjugated secondary antibodies (1:2,000, Dako); the immunoreaction was revealed with a chemiluminescence reagent (ECL, Amersham). Densitometric quantification was carried out with the ImageLab v4.5.2 software (BioRad), using β-actin for normalization. Seven samples of FC area 8 per group were analyzed. These antibodies were selected on the basis of a larger screening which included antibodies against proteins whose RNA levels were de-regulated as revealed by RT-qPCR. Only antibodies working for western blotting were eventually assessed. The significance level was set at ** p < 0.01 and tendencies at # < 0.1