Somatostatin Receptor 1/5 Knockout and R6/2 Mice Figure 4. Peroxidase immunohistochemistry illustrating the changes in distribution pattern of SSTR1-5 in striatum of R6/2 and SSTR1/52/2 mice. SSTR1(53 kDa) like immunoreactivity increased in striatum of R6/2 mice and abolished completely in SSTR1/52/2 mice (a). SSTR2 (57 kDa) like immunoreactivity was increased in R6/2 and SSTR1/52/2 mice (b). SSTR3 (60 kDa) expression was increased in R6/2 while it decreased in SSTR1/52/2 mice striatum (c). Comparable expression of SSTR4 (44 kDa) was observed in both R6/2 and SSTR1/52/2 mice brain (d). SSTR5 (58 kDa) expression was selectivley higher in R6/2 mice brain than wt and receptor like immunoreactivity was not detected in SSTR1/52/2 mice (e). Data presented as mean 6 SD (SSTR1/52/2, n = 5 and R6/2 mice, n = 3) in comparison to wt mice brain, *P,0.05, ** P,0.01. Scale bar = 20 mm panel a–e and 5 mm for inset. doi:10.1371/journal.pone.0024467.g004 Whether SSTR subtype regulates DARPP-32 phosphorylation is not known. Here we provide the evidence that in the absence of SSTR subtypes, DARPP-32 expression is decreased in striatum in a similar manner as seen in HD brain and/or HD transgenic mice. The loss of DARPP-32 like immunoreactivity in SSTR1/52/2 mice is not surprising; as previous studies have also shown that DARPP-322/2 mice resemble HD mice in dopamine signaling [43]. Consistent with the existing notion that bNOS positive neurons are preserved in HD, the expression pattern and quantification of bNOS positive neurons in R6/2 mice as well as SSTR1/52/2 mice is comparable. Similar is the pattern of SST expression in both strains. In line with previous observations, our results further strengthen the concept of selective preservation of medium-sized aspiny interneurons expressing SST/b-NOS [8]. Furthermore, our previous studies have shown that blockade of SST using antisense oligonucleotide leads to the loss of bNOS/NADPH-d positive neurons upon QUIN/NMDA treatment [31]. Taken together, these results support the notion that the presence of SST is likely responsible for the survival of aspiny interneurons in excitotoxicity. In HD, the activation of NMDAR is one of the leading causes of neuronal loss, in addition to the mutation in Htt. Functional and physiological significance of NMDARs has recently been de- scribed in pathophysiology of HD and reported that the NMDAR antagonist, memantine, blocks the nuclear inclusion of mutated Htt seen in HD [19]. Furthermore, recent studies have shown the distinct role of synaptic and extrasynaptic NMDARs in early and late onset of HD [56,57,58]. NMDAR positive neurons are most vulnerable in HD as well as in various mouse models of excitotoxicity [18,59,60,61]. Cumulatively, these studies indicate an increase NR1 and the loss of NR2B expression. However, studies for NR2A are controversial and such discrepancies may be due to the mouse strain used as a model. In the current study, an increased NR1 and NR2A expression, with the loss of NR2B immunoreactivity in both R6/2 and SSTR1/52/2 mice may be linked to the neurodegeneration of MSNs, which is attributed to an increased Ca2+ influx. Moreover, in SSTR1/52/2 mice, NR2A and NR2B immunoreactivity accumulates intracellularly while in R6/2 both receptors are well expressed at cell surface. In light of these results, we propose two different mechanisms for the role in NMDAR-mediated neurotoxicity. First, the membrane expression of NR2A and NR2B in HD transgenic mice allows receptor interaction at cell surface, which results in excitotoxicity. Consistent with the existing concept that NMDARs are function- ally active in heteromeric complex, the increased cell surface expression of NR1 and NR2A in R6/2 might be involved in excitotoxicity through heterodimerization. In the absence of SSTR subtypes, NMDAR trafficking might be impaired, leading to receptor accumulation intracellularly. Whether SSTR and NMDAR functionally interact with each other is not known and further studies are in progress to determine this. Studies are warranted to delineate the molecular mechanism for the intracellular accumulation of NMDAR in SSTR1/52/2 mice. Furthermore, the possibility of impaired mitochondrial function in these processes cannot be ruled out. The physiological response of cells upon the activation of SSTRs is receptor specific and can display multiple effects. SSTR2 is known to inhibit Ca2+ activated channels and increased neuronal Ca2+ is detrimental in excitotoxicity in vitro as well as in HD. Our results show increased expression of SSTR2 in the absence of SSTR1/5 and concomitantly in HD transgenic mice, suggesting a compensatory mechanism to inhibit Ca2+ due to enhanced excitatory input via the activation of NMDAR. In support, we have recently shown that SSTR2 and SSTR5 heterodimerize with significant changes in receptor pharmacolog- ical properties as well as enhanced signaling [62]. Furthermore, SSTR1/52/2 mice exhibit increased expression of D2R in comparison to wt (unpublished observations). Since SSTR5 and D2R functionally interact and exist in a heteromeric complex, the increased expression of D2R in SSTR1/52/2 mice supports the compensatory role in the absence of SSTR5 in vivo [36]. Increased expression of NR1 and NR2A might be linked with the decreased expression of calcineurin and increased expression of calpain in R6/2 and SSTR1/52/2 mice. Calcineurin is involved in the phosphorylation of DARPP-32, which further regulates the cell survival pathways. Calcineurin knockout mice show the inhibition of motor functions, loss of synaptic plasticity, learning and memory [63]. The loss of calcineurin expression in R6/2 and SSTR1/52/2 mice might correlate with the symptoms of HD. Furthermore, the loss of calcineurin and DARPP-32 expression can be correlated with the decreased expression of PKC-a. PKC-a plays a role in regulation of membrane associated signal transduction pathways mediated by Ca2+ homeostasis [64]. DARPP-32 phosphorylated at Thr34 (threonine) via PKC-a converts into protein phosphatase-1 (PPtase-1), which in turn inhibits the phosphorylated forms of calcium response element binding protein (CREB) and leads to cell survival [65]. Alternatively, the loss of PKC-a expression can also be correlated with increased apoptosis detected in R6/2 mice. However, decreased expression of PKC-a may also be associated with decreased expression of DARPP-32 in R6/2 and SSTR1/ 52/2 mice. Changes in pERK1/2 can be correlated with the variable expression of SSTRs. Increased pERK1/2 in SSTR1/ 52/2 mice may be due to the loss of SSTR5, which is known to inhibit pERK1/2 and increased expression of SSTR5 in R6/2 might exert inhibitory role on pERK1/2. Previous studies have shown increased calpain activity in HD human brain tissue and YAC transgenic mice [48,66,67]. Increased expressions of calpain in R6/2 and SSTR1/52/2 mice further suggest a role of NMDAR-mediated cell death [48]. Enkephalin primarily functions as an anti-nociceptive but is also disrupted in early-onset of HD [68]. In agreement with previous studies, we detected no changes in enkephalin expression in R6/2 mice [68]. However, these results contradict other findings, which report a decrease in enkephalin expression [69]. Such discrepan- cies may be due to the isoform of enkephalin targeted. In SSTR1/ 52/2 mice, increased expression of enkephalin, in part, may be indirectly due to changes in opioid receptors, since SSTRs share 40% structural homology and exhibit heterodimerization [37]. Post-mortem brains from HD patients and transgenic mice have shown augmented apoptosis in striatum [18]. Amongst all SSTRs, SSTR3 is the only known receptor subtype that can induce PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24467