as in HD mouse models [17,18]. Recently, the functional importance of NMDARs emerged from a study describing the role of NMDAR antagonist memantine to block the nuclear inclusion of Htt in yeast artificial chromosome (YAC) mice [19]. These data suggest that NMDARs play an important role in HD and may contribute to neuronal loss. NMDA replicate the neurophathological features of HD and have been used as models of the disease [20,21]. In the striatum of experimental mice, medium-sized aspiny interneurons expressing SST, NPY and NADPH-d/bNOS are selectively resistant to QUIN-induced excitotoxicity. Similarly, such interneurons are relatively well spared observations in the brains of HD patients [19,21,22,23,24,25,26]. Previous studies have also shown increased SST secretion and gene expression in HD brain and NMDA/ QUIN-induced excitotoxicity [21,23,27]. In support of the selective preservation of interneurons, it was argued that these neurons lack NMDARs [28]. In contrast, several recent studies have shown the presence of NMDARs in SST/NPY/NOS positive neurons in striatum of rat brain and cultured striatal neurons [4,29,30]. Most importantly, we have recently shown that immunoblockade of SST by using antisense oligoneucleotides and immunoneutralization of released SST by using SST specific antibodies potentiate neuronal loss in QUIN/NMDA-induced excitotoxicity in cultured striatal neurons, including NPY, NADPH-d and bNOS positive neurons [31]. Furthermore, selective sparing of SST positive neurons in bNOS knockout mice suggests that the presence of SST is essential for the survival of interneurons [32]. The presence of SST in the central and peripheral nervous system is associated with several physiological functions, which are attributed to different receptor subtypes, namely somatostatin receptor 1–5 (SSTR1-5), which are members of G-protein coupled receptor (GPCR) family. All five SSTR subtypes display overlapping distribution in different parts of brain and importantly couple to Gi protein and inhibit cAMP in a pertussis toxin sensitive manner. SSTRs are involved in the regulation of ion channels; inhibition of Ca2+ and activation of K+ channels involved in the release of several neurotransmitters and modulation of neuro- transmission [33]. These functional properties of SSTR subtypes can be further enhanced by interaction with members of their own family as well as other GPCRs, including dopamine and opioid receptors via heterodimerization [34,35,36,37]. Widespread dis- tribution of SSTRs in CNS is involved in various neurological diseases such as Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), epilepsy, HIV encephalitis, dementia and psychiatric disorders, including schizophrenia [22,38,39,40,41,42]. These studies cumulatively suggest the critical and pivotal role of SSTR subtypes in neurodegenerative diseases. We recently observed that the knock-down of SSTR 1 and SSTR 5 using antisense oligonucleotides accelerated neuronal death upon NMDA treatment in cultured striatal neurons (unpublished observations). Accordingly, in an attempt to elucidate the possible functions of SSTR1 and 5, the present study was undertaken to determine the expression of NMDARs, DARPP-32, calbindin, bNOS/SST and SSTRs in striatum of R6/2 and SSTR1/52/2mice. In addition, we also studied the downstream signaling cascades including calcineurin, calpain, PKC-a, ERK1/ 2, synapsin-IIa and enkephalin associated in the process of neurodegeneration in HD pathology as well as in experimental models of the disease. In the present study, for the first time, we describe that the SSTR1/5 complex is a critical regulator of NMDARs, DARPP-32 and downstream signaling cascades normally seen in R6/2 transgenic mice. Importantly, this study revealed that SSTR1/52/2 mice mimic neuro-and biochemical changes of presymptomatic HD transgenic mice. Somatostatin Receptor 1/5 Knockout and R6/2 Mice Results Decreased DARPP-32 expression and increased Calbindin D-28K expression in striatum of SSTR1/52/2 mice and R6/ 2 mice brain Neuronal population in striatum is largely composed of medium sized projection neurons, which are positive for DARPP-32 and constitute 85% of total neuronal population. Several previous studies have shown significant loss of DARPP-32 like immunore- activity in HD patients and/or in experimental models of disease [43,44]. Accordingly, here we examined the DARPP-32 like immunoreactivity using immunohistochemical and Western blot analysis in R6/2 and SSTR1/52/2 as well as respective wt mice brains. As illustrated in Figure 1a, consistent with previous studies, a marked decrease in signal intensity of DARPP-32 was observed in R6/2 when compared to wt mice. Like R6/2 mice, a similar decrease in DARPP-32 like immunoreactivity was observed in SSTR1/52/2 when compared to wt mice. DARPP-32 like immunoreactivity is mostly expressed as cytoplasmic protein. Consistent with our immunocytochemistry data, Western blot analysis showed a significant decrease in DARPP-32 expression in comparison to wt in the brains of both R6/2 and SSTR1/52/2 mice (Figure 1b). Similar to DARPP-32, calbindin D-28K immunoreactivity was detected in the striatum and has been used as an index of MSN. As illustrated in Figure 1c, increased expression of calbindin-like immunoreactivity was observed in R6/2 and SSTR1/52/2 when compared to wt mice. Western blot analysis showed consistent results as seen in immunohistochemistry with increased expression of calbindin in striatum of R6/2 and SSTR1/52/2 mice (Figure 1c). SST and bNOS positive neurons are selectively spared in striatum of R6/2 and SSTR1/52/2 mice Previous studies have shown that medium-sized aspiny interneurons positive for bNOS/ NADPH-d also co-express SST and NPY and are selectively preserved in HD patients as well as experimental models of disease [7,8]. Accordingly, we determined the expression of bNOS and SST-positive neurons in striatum of R6/2 and SSTR1/52/2 mice brain. As shown in Figure 2a, bNOS immunoreactivity was observed in sparsely distributed neurons all over the striatum with arborizing neuronal processes without discernable changes in R6/2 and SSTR1/52/2 mice when compared to wt mice. In addition, Western blot analysis also revealed no significant changes in bNOS expression levels in R6/2 and SSTR1/52/2 mice in comparison to their counterpart wt. As shown in Figure 2b, the neuronal population exhibiting SST like immunoreactivity was comparable in R6/2 and SSTR1/52/2 mice using immunohistochemistry and Western blot analysis. As shown in Figure 2c, further quantitative analysis revealed comparable changes in bNOS and SST positive neurons in R6/ 2 and SSTR1/52/2 mice as well as in comparison to wt. R6/2 transgenic mice exhibited 14 and 8% loss of bNOS and SST positive neurons, respectively. Conversely, SSTR1/52/2 mice showed 15 and 3% loss of bNOS and SST positive neurons respectively, not significantly different from wt mice. Expression of NMDA receptor subtypes in the striatum of SSTR1/52/2 and R6/2 mice The role of activated NMDARs in degeneration of medium spiny neurons in HD as well as in excitotoxicity is indisputable. Several previous studies have shown the activation of NMDAR subtypes in the brain of R6/2 transgenic mice [18]. We sought to determine PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24467