compensatory mechanism of SSTR5. Our observations raise a potential question about the role of SSTR1 or SSTR5 independently. We did not eliminate this question from our discussion and propose that knocking out SSTR1 or SSTR5 might have same effect as seen in double ko mice, albeit to the lesser degree. We have recently characterized the SSTR subtypes for homo-and heterodimerization in detail with distinct pharmaco- logical and enhanced functional properties [34,35,72]. Taken together, our results indicate that SST and SSTRs might play an important neuroprotective role in neurodegenerative diseases. Targeting this pathway can provide novel insights in understanding the pathophysiology of neurodegenerative diseases. This is the first study showing similar disease characteristics related to neurochemical changes upon ablation of the GPCR gene (i.e., SSTRs). We here reinforced the concept that mice in the absence of SSTR subtype might have greater susceptibility to neurode- generative diseases as well as excitotoxicity. We propose that in HD transgenic mice as well as in HD patient’s pharmacological restoration and maintenance of somatostatinergic system might serve as a beneficial therapeutic approach in treatment of HD. Materials and Methods Materials Rabbit polyclonal antibodies for SSTR subtypes have been previously characterized [42,45,73]. Mouse monoclonal antibodies for DARPP-32 and calcineurin were purchased from BD Biosciences (Mississauga, ON, Canada). Antibodies against NMDAR-1, NMDAR-2A, and NMDAR-2B were obtained from Millipore (CA, USA) and anti-calbindin D-28K from Sigma- Aldrich (MO, USA). PKC-a, ERK1/2, synapsin-IIa and Met/Liu enkephalin were obtained from Cell Signaling Technology (MA, USA). The peroxidase vectastain ABC kit was purchased from Vector Laboratories (CA, USA). Nitrocellulose Hy-bond mem- brane and enhanced chemiluminescence (ECL) detection kit was obtained from Amarsham Ltd. (Oakdale, ON, Canada). All other chemicals of analytical grade were obtained from various commercial sources. Animals Perfused and frozen brains from 8-week old HD (C57BL/6 X CBA)F1(B6CBA)-TgN (HDexon1)62 (R6/2) carrying 120 +/2 5 CAG repeat expansions and [wild-type(WT)]B6CBAF1/J mice were obtained from Jackson Laboratory (Sacramento, CA, USA). Similarly, frozen and perfused brains from 8 weeks old SSTR1/ 52/2 mice and wt were kindly provided by Dr. F. C. Brunicardi (Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA). Further details for the creation and characterization of SSTR1/52/2 mice have been described in detail previously [74]. The protocols regarding animal care were followed in compliance with the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. The wt and SSTR1/52/2 mice were housed at Baylor college of Medicine, Houston, Texas, USA with 12-hour light/ dark cycle. Immunohistochemistry studies In the present study, three male adult mice from each group, wt and R6/2 and five male adult mice from wt and SSTR1/52/2 mice were used. 30 mm thick coronal brain sections were cut on Leica vibratome. Immunostaining was performed on free-floating sections using the avidin–biotin peroxidase Vectastain ABC kit as described previously [45,73,75]. Briefly, brain sections were incubated with 0.3% hydrogen peroxide followed by incubation Somatostatin Receptor 1/5 Knockout and R6/2 Mice with 0.02% Triton X-100. Following three washes, sections were incubated with 5% NGS for 1 h at room temperature (RT) and followed by overnight incubation with specific primary antibodies against rabbit-anti-SSTR1-5 (1:500), rabbit-anti-SST (1:600), mouse anti-DARPP-32 (1:250), mouse anti-NMDAR-1, NMDAR-2A, NMDAR-2B and calbindin D-28K (1:500) and rabbit anti-bNOS (1:400) in 1% NGS at 4uC in humid atmosphere. The sections were incubated with the biotinylated secondary antibodies; goat-anti-rabbit to detect SSTRs, SST, and bNOS and goat anti-mouse to detect DARPP-32, calbindin and NMDARs and followed by incubation in ABC complex for 30 min. The final color was developed by adding a mixture containing 0.001% hydrogen peroxide and 0.2 mg/ml of 3,3- diaminobenzidine (DAB) for 3–4 min in 50 mM Tris buffer. Sections were washed in PBS, mounted onto slides and viewed and photographed under Leica microscope attached with the Retiga 2000R camera. Western blot analysis Striatal tissues isolated from R6/2 or SSTR1/52/2 mice and respective wt brains were homogenized in homogenizing buffer containing (62.5 mM Tris-HCl, 50 mM dithiothretiol [DTT], 10% glycerol, 2% SDS). 15 mg of total protein was fractioned by electrophoresis on 10% SDS polyacrylamide gel, transferred onto 0.2 mm-nitrocellulose membrane. The membrane was blocked with 5% non-fat dried milk at RT for 1 h and incubated overnight at 4uC with specific primary antibodies against– SSTRs1-5, NMDARs, bNOS (1:600), SST, enkephalin, synapsinIIa, calpain, calcineurin, calbindin, phospho and total ERK1/2, PI3K (1:1000), DARPP-32 (1:250) and PKC-a (1:500). Following three subse- quent washes, the membrane was incubated with peroxidase conjugated goat anti-rabbit or a goat anti-mouse secondary antibody at RT for 1 h. The bands were detected using chemiluminescence reagent and images were taken using an Alpha Innotech FluorChem 8800 (Alpha Innotech Co., San Leandro, CA) gel box imager. b-actin was used as the housekeeping protein for loading control. The bands intensity was quantified using densitometric analysis and the changes in protein expression were calculated as the ratio of band of interest and the density of b-actin. Quantitative analysis Quantitative analysis on brain sections was performed using NIH Image J software as described earlier [45]. Neurons were considered immunoreactive if the labeling of their cell bodies was distinctly higher than the background staining obtained in the presence of pre-absorbed or in the absence of primary antibodies. For quantitative analysis five mice were taken from wt, SSTR1/ 52/2 mice and three for wt and R6/2 mice. 12–15 randomly selected areas from 6–7 sections from individual mouse brain were used for neuronal counting. Since our immunohistochemical studies provide semiquantitative analysis of immunoreactivity in R6/2 or SSTR1/52/2 and respective wt mice we took all the possible precaution to keep our experimental conditions consistent in all aspects, i.e., incubation timings with antibodies (18 h) and 3, 3-diaminobenzidine for the final color development (4 mins). Statistical analysis Data are presented as percentage changes in neuronal population for positive immunoreactivity from 6–7 brain sections per mice. Total of (6000–6500) neurons were counted in each condition. Bars represent the mean 6 SEM (n = 3 for R6/2 and n = 5 for SSTR1/52/2). Statistical comparison between wt and R62 (n = 3) and wt and SSTR1/52/2 (n = 5) mice were analyzed PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24467