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Expression and Localization of Somatostatin Receptor Types 3, 4 and 5 in the Wild-Type, SSTR1 and SSTR1/SSTR2 Knockout Mouse Cochlea

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Expression and Localization of Somatostatin Receptor Types 3, 4 and 5 in the Wild-Type, SSTR1 and SSTR1/SSTR2 Knockout Mouse Cochlea Cell Tissue Res (2014) 358:717–727 DOI 10.1007/s00441-014-1977-7 REGULAR ARTICLE Expression and localization of somatostatin receptor types 3, 4 and 5 in the wild-type, SSTR1 and SSTR1/SSTR2 knockout mouse cochlea Vesna Radojevic & Daniel Bodmer Received: 5 November 2013 /Accepted: 24 July 2014 /Published online: 23 August 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Somatostatin (SST) is a peptide hormone that ex- actions of SST. Five distinct SST receptor types (SSTR1-5) erts inhibitory effects mediated through binding to specific have been identified (Patel 1999). cell surface G protein-coupled receptors, of which five distinct The distribution of mRNA expression for all five SST recep- subtypes (SSTR1-SSTR5) have been characterized. Our study tor types has been investigated extensively using different tech- performed on mouse cochlear hair cells shows the expression niques in rats and humans (Harrington et al. 1995;Kongetal. and localization of the three receptors (SSTR3-SSTR5) in 1994; Schindler et al. 1995). Published studies are in agreement wild-type (WT), single-knockout (SSTR1 KO) and double- and suggest that all SSTR types are variably expressed wide- knockout SSTR1/SSTR2 (DKO) mice. Similar SSTRs ex- spread throughout the brain. In rats, SSTR1 mRNA is the most pression were observed in the inner hair cells (IHC), outer abundant, followed by SSTR2, SSTR5, SSTR3 and SSTR4 hair cells (OHC) and supporting cells of cultivated P7 mouse (Patel 1999;Brederetal.1992; Kong et al. 1994;Thossetal. organ of Corti (OC) explants as well as in cultivated cochlear 1995;Beaudetetal.1995). SSTR3 immunoreactivity is widely neuroepithelial supporting cells (NEsc). We found differences distributed throughout the human brain and is particularly in the expression of SSTR3-5 in WT, SSTR1 KO and DKO enriched in the cortex, hippocampus, amygdala, hypothalamus mouse cochlea, which might be explained as a compensatory and cerebellum (Hervieu and Emson 1999; Schindler et al. effect in the cochlea after the loss of SSTR1 and/or SSTR2. 1995). Immunohistochemistry of adult rat brain sections showed that SSTR4 is distributed in both the cell body and neuronal Keywords Cochlea . Hair cells . Knockout mice . Organ of processes in the cerebral cortex, hippocampus and several Corti . Somatostatin . Somatostatin receptor nuclei of the brainstem (Selmer et al. 2000). Schreff et al. (2000) showed that SSTR4 is strictly somatodendritic and most likely functions in a postsynaptic manner. SSTR4 may have a previously unappreciated function during neuronal degenera- Introduction tion/regeneration. SSTR5 is prominently expressed in the basal forebrain, suggesting that it is involved in the mediation of the SST, a regulatory peptide with two bioactive forms, SST-14 effects of SST on the sleep-wake cycle through its association and SST-28, is produced in neuroendocrine cells in the brain with cortically projecting subcortical systems (Stroh et al. 1999). and periphery and acts on a number of tissue targets to Most SSTR subtypes have been shown to modulate voltage- modulate neurotransmission, cell secretion and cell prolifera- gated Ca2+ channels and glutamate receptor channels in several tion (Reichlin 1983; Epelbaum et al. 1994; Patel 1999). A organ systems (Cervia and Bagnoli 2007). Excessive Ca2+ family of transmembrane receptors mediates the physiological influx into cells is thought to be a major contributor to cell death in ischemic and excitotoxic models of neuronal injury (Smaili V. Radojevic (*) : D. Bodmer Department of Biomedicine, University Hospital Basel, Petersgraben et al. 2009). SSTRs are also involved in the regulation of ion 2+ + 4, 4031 Basel, Switzerland channels, the inhibition of Ca channels and activation of K e-mail: [email protected] channels, involved in the release of several neurotransmitters and the modulation of neurotransmission (Patel 1999; Petrucci V. Radojevic : D. Bodmer Clinic for Otolaryngology, Head and Neck Surgery, University et al. 2001; Johnson et al. 2001). All five subtypes of SSTRs are Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland expressed in the mammalian retina and are distributed to specific 718 Cell Tissue Res (2014) 358:717–727 cell populations, including subtypes of photoreceptor, bipolar, RNA extraction amacrine and ganglion cells (Thermos 2003;KeandZhong 2007; Vasilaki and Thermos 2009). Studies have shown that The SG, brain (Br) and cell lysate (Jayanthi et al. 2005)from SST analogues may be effective therapeutic agents in retinal P7 mice, the OC from WT mouse pups (P0–P21) and OC of diseases (Vasilaki and Thermos 2009). DKO mouse pups by age P14 and P21 were placed separately However, in contrast to the situation in the retina, little in RNAlater (Qiagen, Hombrechtikon, Switzerland). RNA information is available on the expression or function of isolation of the brains and inner ear components was per- somatostatin and its receptors in the inner ear. Tachibana formed using the RNAeasy Minikit (Qiagen) and an Ultra- et al. (1979) reported on somatostatin-like immunoreactivity Turrax T8 tissue homogenizer (IKA-Werke, Staufen, in the medial geniculate body, cochlear nucleus, inferior Germany) according to the manufacturer’s instructions, in- colliculus, auditory cortex and in the cochlea. Also, cluding DNase treatment. The quantity and quality of isolated somatostatin-like immunoreactivity could be observed in the RNA was determined with a NanoDrop ND 1000 (NanoDrop cochlear nuclei of postnatal rats and it was suggested that Technologies, DE, USA). The 260-/280-nm ratio of all sam- somatostatin might be important for the development of the ples was between 1.8 and 2.1. auditory system (Takatsuki et al. 1982). Our previous study demonstrated that SSTR1 and SSTR2 Reverse transcription polymerase chain reaction mRNA and protein are expressed in the mouse cochlea and localized in IHC and OHC and in supporting cells of the OC Total RNA (500 ng) was reverse transcribed into cDNA using (Radojevic et al. 2011). The present study is the first attempt to the first strand cDNA synthesis kit for reverse transcription determine SSTR3, -4 and -5 expression in the cochlea from polymerase chain reaction (RT-PCR) (Roche Applied Biosci- WT and DKO mice. ence, Rotkreuz, Switzerland) according to the supplier’sin- structions. PCR was performed using the PCR Master Mix (Roche Applied Bioscience) with primers specific for SSTR3, SSTR4 and SSTR5. β-actin primers were used as a positive Materials and methods control for cDNA synthesis. The primers were designed to cross at least one exon junction for the specific amplification Animals of cDNA and to recognize contamination with genomic DNA. The primer sequences were: Experiments were performed on mouse cochleae from WT C57BL/6 mice of both sexes. The patterns of SSTR3-5 ex- SSTR1 forward (fwd): 5’-CAGGGTAGCCATTCTC pression were also observed at postnatal ages (n=10 per ATC-3’, postnatal day for mRNA isolation). Immunohistochemistry SSTR1 reverse (rev): 5’-AGCGTGGAAGTGACCAGA was performed on four mice per postnatal day. The procedures − − AAG-3’, used to generate SSTR1 / (homozygous SSTR1 KO)/ − − SSTR2 (fwd): 5’-CGCATGGTGTCCATCGTAGT-3’, C57BL6J mice (Kreienkamp et al. 1999) and SSTR2 / (ho- SSTR2 (rev): 5’-GGATTGTGAATTGTCTGCCTTGA-3’, mozygous SSTR2 KO)/C57BL6J mice have been described SSTR3 (fwd): 5’-CTGGCTGTCAGTGGCATCTT-3’, previously (Zheng et al. 1997). SSTR1 KO mice were crossed SSTR3 (rev): 5’-CACATGAGATCCGAAGGGC-3’, with SSTR2 KO mice to produce DKO mice. Age-matched SSTR4 fwd: 5’-CAGCGGCATGGTCACTATC-3’, WT mice were produced from C57BL6J mice used to stabilize SSTR4 rev: 5’-CCGTCCACGCTAAGCACTG-3’, the genetic backgrounds of SSTR1 KO and SSTR2 KO mice SSTR5 fwd: 5’-TTCCTGGCAACGCAGAATG-3’, (Bodmer et al. 2012). SSTR5 rev: 5’-CAAGAGCGGCAGAGACATGAG-3’, Animal care and use were conducted in conformity with β-actin fwd, 5’-ACGGTCAGGTCATCACTATCGG the European Communities Council Directive of 24 Novem- CA-3’, ber 1986 (86/609/EEC) and reviewed and permitted by the β-actin rev, 5’-ATCCTGTCAGCAATGCCTGGGT-3’ Kantonales Veterinäramt, Basel, Switzerland. (Microsynth, St. Gallen, Switzerland). Tissue extraction and culture PCR reactions were performed in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). For SSTR3, The mice were decapitated and cochlear microdissections SSTR4 and SSTR5, an initial denaturing step at 94 °C for 30 s were performed under a light microscope to isolate the OC was followed by 30 PCR cycles consisting in denaturation at and the spiral ganglion (SG). Brain tissue was removed from 94 °C for 15 s, annealing at 60 °C for 30 s and extension at the same animals. During microdissection, the different tis- 72 °C for 15 s. The PCR was completed with a final extension sues were maintained in ice-cold PBS. step at 72 °C for 3 min. β-actin was subjected to 30 PCR Cell Tissue Res (2014) 358:717–727 719 cycles consisting in denaturation at 94 °C for 30 s, annealing SSTR5-fwd: 5′-GGCTTCCACACCTAGCTGG-3′, at 58 °C for 45 s and extension at 72 °C for 90 s. The PCR was SSTR5-rev: 5′-AGCACAGGCACTAATACCGC- completed with a final extension step at 72° for 5 min. PCR 3′(Microsynth). products were stained with SYBR Green I (Molecular Probes, OR, USA), separated by electrophoresis on a 2 % agarose gel Each reaction contained 300 nM of primer. The reaction and visualized under ultraviolet light. Omission of RT-PCR occurred at 95 °C for 10 min, followed by 40 cycles of 95 °C was used as the no template control. for 15 s and 60 °C for 60 s. We calculated the relative quantities of specifically amplified cDNA using the compar- Real-time PCR ative threshold cycle method. GAPDH was used as an endog- enous reference (Microsynth) (Bodmer et al. 2012;Halletal. Total RNA (500 ng) was reverse transcribed into cDNA using 2007; Chen and Ruan 2009). Template-free and reverse the First Strand cDNA synthesis kit (Roche Applied Biosci- transcription-free controls excluded non-specific amplifica- ences, Foster City, USA) according to the manufacturer’s tion and DNA contamination. instructions.
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