Quantitative PCR analysis of 5-HT receptor expression in tissue from the inferior colliculus and cochlear nucleus of an animal model of tinnitus

Sarah Milton1, Ed Lobarinas2, Nikki West1, Paul Mawer1, Amy Butler1, Richard Salvi2, Nicholas Barnes1

1Cellular and Molecular Pharmacology Group, School of Experimental and Clinical Medicine, The Medical School, Vincent Drive, Birmingham, UK, 2Center for Hearing and Deafness, State University of New York at Buffalo, 137Cary Hall, Buffalo, NY, USA

Tinnitus affects 1% of the Western World’s population, often severely disrupting daily routine (Simpson and Davies, 2000), although there is poor understanding of the mechanisms involved. Possible links to an involvement of the central serotonin (5-hydroxytryptamine; 5-HT) with tinnitus and hyperacusis have been suggested (Marriage and Barnes, 1995; Simpson and Davies, 2000; Folmer and Shi, 2004). To investigate the potential involvement of the 5-HT system in an animal model of tinnitus, we have quantified transcripts for a variety of 5-HT receptors in the cochlear nucleus (CN) and inferior colliculus (IC) using quantitative PCR.

Male Sprague-Dawley rats underwent behavioural training to assess the onset of tinnitus after salicylate or saline control. Schedule-induced polydipsia avoidance conditioning (SIP-AC) was preformed as described in Lobarinas et al., (2004). Briefly, food-restricted rats were self-trained to lick for water between deliveries of food pellets. Animals that displayed SIP were trained to discriminate sound intervals from quiet intervals by delivering foot shock when licks occurred during sound intervals. After the rats learned to discriminate (>90% accuracy) sound from quiet periods, they received either salicylate (250 mg/kg, i.p.) or saline. Licks in quiet were significantly reduced only in animals treated with salicylate, indicating that these rats perceived the phantom sound of tinnitus. A separate group of rats were given salicylate (‘tinnitus’) or saline (‘control’) and the CN and IC tissue dissected and stored at -80°C. Total RNA was extracted using SV Total RNA Isolation System Kit (Promega). First strand cDNA synthesis was achieved using oligo dT primers and AMV reverse transcriptase (Promega) with 1 μg template RNA. Amplification of specific target was achieved using Quantace Syber® green (Bioline) and fluorescence levels detected by a Rotor-Gene 2000 (Corbett). Relative quantitation of 5-HT receptor expression was compared between saline and salicylate-treated animals and to expression levels of the reference gene, GAPDH.

Within the IC, no significant difference between ‘control’ or ‘tinnitus’ animals was found for the expression of the 5-HT1A, 5-HT2A, 5-HT2C, 5-HT4 or 5-HT7 receptors (although there was a trend of higher receptor expression in ‘tinnitus’ animals for both 5-HT2A and 5-HT2C receptor expression). Within the CN no significant difference in the expression of the 5-HT1B, 5-HT2A and 5-HT4 receptors was found, however a significant down regulation of 5-HT1A receptor transcript expression was detected in the CN of ‘tinnitus’ animals compared to control (Relative quantitation ratios, ‘control’ 1.1 ± 0.17, ‘tinnitus’ 0.43 ± 0.15; mean ± SEM, n = 4, P<0.05). The present results further implicate an involvement of the central 5-HT system in the manifestation of tinnitus.

(Supported in part by NIH grant (1R01DC009219-01).

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