Sensory hair cells in our inner ear are necessary to detect sound, and hair cell death is the primary cause of hearing loss. A certain class of antibiotics, including neomycin, induces hair cell death. Understanding the mechanisms involved in preventing hair cell death may yield pharmacological advancements to protect sensory hair cells when these antibiotics are administered. Zebrafish are often used to study hair cells because fish have readily accessible hair cells on their exterior that are analogous to sensory hair cells within our inner ear. This project investigated the effects of heat shock on neomycin-induced hair cell death. Heat shock can trigger several signaling pathways known to protect cells against cellular insults. We hypothesized that applying heat shock prior to exposing the hair cells to neomycin would inhibit damage by increasing cellular defense mechanisms as seen in other organisms. The data were obtained by placing 5-day-old zebrafish in a 38 degree Celsius water bath for one hour (heat shock), followed by an hour break for recovery, and then a 30-minute incubation period in neomycin. We quantified the degree of damage to the hair cells 5 hours after neomycin treatment by scoring (ranking) hair cell clusters based on cluster size and counting the number of hair cells located in specific areas. Our results suggest that heat exposure partially inhibits neomycin-induced hair cell damage. Further research may yield insight toward pharmacological advances that prevent antibiotic-induced hair cell damage.

Despite much investigation, there has been relatively little progress in the development of novel analgesic compounds to treat debilitating chronic pain conditions that affect millions of people. Inherent problems in behavioral screens to identify analgesic compounds include the use of expensive and time consuming low throughput rodent based assay as well as the use of behaviors that only measure the presence or absence of acute behavioral responses to noxious stimuli, whereas assays that measure an animal’s ability to choose between an innocuous and noxious environment overcome these setbacks. In this study, we aim to develop a high throughput two-choice thermal discrimination assay utilizing zebrafish (Danio rerio) larvae that can be used to screen for novel analgesic compounds. In order to do this, we will profile the ability of zebrafish larvae to discriminate between two temperatures. Individually arrayed 5 days post fertilization larvae will be placed in wells containing two distinct thermal zones and assayed to determine if they display a thermal preference between a testing temperature and a normal rearing temperature (28.5^oC). The testing temperature will range in one degree increments from noxious cold (10^oC) to noxious heat (38^oC). We will also test the limits of thermal preference by testing the ability of larvae to discriminate between two noxious temperatures, for example 36 vs 38^oC. To conclude if thermal preference is malleable based on the thermal environment to which larvae are acclimated, we will determine if prior incubation at a selected temperature other than 28.5^oC will alter the thermal preference of zebrafish larvae. Finally to determine if our assay may be appropriate for the use in a screen for novel analgesic compounds, we will investigate whether or not known noxious or analgesic compounds alter the thermal preference of zebrafish larvae.

The hair cells of the inner ear are the functional sensory units of hearing and balance in the auditory and vestibular system, respectively, and are therefore a major cause of deafness and balance deficiencies when lost after damage. Unlike in reptiles, birds and fish, existing studies suggest that the mammalian sensory epithelia of the inner ear lack the ability to regenerate hair cells after such damage. Here we focus on the cristae, which are a component of the vestibular system, and are responsible for sensing angular acceleration and rotation of the head.  Previous research has suggested that Notch activity, a crucial signaling pathway necessary for patterning the sensory epithelium, continues in adult mouse cristae, suggesting the potential for regeneration after damage.  We characterized a lesion protocol for this system through the administration of the ototoxin iminodipropionitrile (IDPN) to test whether spontaneous regeneration of hair cells is present in the mammalian cristae. We used confocal microscopy to image the layers of the cristae's sensory epithelium  after immunohistochemistry staining with markers specific to hair cells.  Each cristae's hair cells were counted, and an average was obtained for cristae for each of several time points after IDPN injection. Preliminary findings show a severe reduction in total hair cell number in the mouse cristae one week after IDPN injection, and continued loss of hair cells in subsequent weeks.  While we saw no evidence of spontaneous regeneration in the mouse cristae, this lesion protocol may be used in further hair cell regeneration studies using various manipulations or drugs, such as inhibition of Notch signaling.