Distinct roles of arrestin 1 protein in photoreceptors during Drosophila development.

Arrestin regulates many facets of G-protein coupled receptor signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. Here we generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. We show that Arr1-eGFP is localized in the cytoplasm and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated rhodopsin 1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared with that of Arr2-eGFP. This slower kinetic activity may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, we show that in flies with reduced Arr1 prolonged depolarizing afterpotential can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation-deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors we failed to observe endocytosis of Arr1-eGFP. In contrast, we show that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, we propose that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae whereas it regulates the deactivation in adult.

Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila.

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We used enhanced green fluorescent protein-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpA(P24) photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA(P24) is the failure to activate CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca(2+) entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala(1) flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA(P24) photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.

Exploring Excitotoxicity and Regulation of a Constitutively Active TRP Ca2+ Channel in Drosophila.

Unregulated Ca2+ influx affects intracellular Ca2+ homoeostasis, which may lead to neuronal death. In Drosophila, following the activation of rhodopsin the TRP Ca2+ channel is open to mediate the light-dependent depolarization. A constitutively active TRP channel triggers the degeneration of TrpP365 /+ photoreceptors. To explore retinal degeneration, we employed a multidisciplinary approach including live imaging using GFP tagged actin and arrestin 2. Importantly, we demonstrate that the major rhodopsin (Rh1) was greatly reduced before the onset of rhabdomere degeneration; a great reduction of Rh1 affects the maintenance of rhabdomere leading to degeneration of photoreceptors. TrpP365 /+ also led to the up-regulation of CaMKII, which is beneficial as suppression of CaMKII accelerated retinal degeneration. We explored the regulation of TRP by investigating the genetic interaction between TrpP365 /+ and mutants affecting the turnover of diacylglycerol (DAG). We show a loss of phospholipase C in norpAP24 exhibited a great reduction of the DAG content delayed degeneration of TrpP365 /+ photoreceptors. In contrast, knockdown or mutations in DAG lipase (InaE) that is accompanied by slightly reduced levels of most DAG but an increased level of DAG 34:1, exacerbated retinal degeneration of TrpP365 /+. Together, our findings support the notion that DAG plays a role in regulating TRP. Interestingly, DAG lipase is likely required during photoreceptor development as TrpP365 /+; inaEN125 double mutants contained severely degenerated rhabdomeres.

Group II Metabotropic Glutamate Receptors Modulate Sound Evoked and Spontaneous Activity in the Mouse Inferior Colliculus.

Little is known about the functions of Group II metabotropic glutamate receptors (mGluRs2/3) in the inferior colliculus (IC), a midbrain structure that is a major integration region of the central auditory system. We investigated how these receptors modulate sound-evoked and spontaneous firing in the mouse IC in vivo We first performed immunostaining and tested hearing thresholds to validate vesicular GABA transporter (VGAT)-ChR2 transgenic mice on a mixed CBA/CaJ x C57BL/6J genetic background. Transgenic animals allowed for optogenetic cell-type identification. Extracellular single neuron recordings were obtained before and after pharmacological mGluR2/3 activation. We observed increased sound-evoked firing, as assessed by the rate-level functions (RLFs), in a subset of both GABAergic and non-GABAergic IC neurons following mGluR2/3 pharmacological activation. These neurons also displayed elevated spontaneous excitability and were distributed throughout the IC area tested, suggesting a widespread mGluR2/3 distribution in the mouse IC.

Generation of a ChATCre mouse line without the early onset hearing loss typical of the C57BL/6J strain.

The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChATCre mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23753A mutation (a.k.a., the ahl mutation). To develop ChATCre mice without accelerated hearing loss, we backcrossed ChATIRES-Cre mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChATIRES-Cre and the wild-type allele at the Cdh23 locus (ChATCre,Cdh23WT). In the new line, auditory brainstem response thresholds were ∼20 dB lower than those in 9 month old ChATIRES-Cre mice at all frequencies tested (4-31.5 kHz). These thresholds were stable throughout the period of testing (3-12 months of age). We then bred ChATCre,Cdh23WT animals with Ai14 reporter mice to confirm the expression pattern of ChATCre. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei. We conclude that the backcrossed ChATCre,Cdh23WT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChATCre,Cdh23WT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.

Addressing variability in the acoustic startle reflex for accurate gap detection assessment.

The acoustic startle reflex (ASR) is subject to substantial variability. This inherent variability consequently shapes the conclusions drawn from gap-induced prepulse inhibition of the acoustic startle reflex (GPIAS) assessments. Recent studies have cast doubt as to the efficacy of this methodology as it pertains to tinnitus assessment, partially, due to variability in and between data sets. The goal of this study was to examine the variance associated with several common data collection variables and data analyses with the aim to improve GPIAS reliability. To study this the GPIAS tests were conducted in adult male and female CBA/CaJ mice. Factors such as inter-trial interval, circadian rhythm, sex differences, and sensory adaptation were each evaluated. We then examined various data analysis factors which influence GPIAS assessment. Gap-induced facilitation, data processing options, and assessments of tinnitus were studied. We found that the startle reflex is highly variable in CBA/CaJ mice, but this can be minimized by certain data collection factors. We also found that careful consideration of temporal fluctuations of the ASR and controlling for facilitation can lead to more accurate GPIAS results. This study provides a guide for reducing variance in the GPIAS methodology - thereby improving the diagnostic power of the test.