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.
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 employed eGFP-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpAP24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpAP24 is due to a failure to activate CaMKII and RDGC because of a loss of light-dependent Ca2+ 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 ala1 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 norpAP24 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. Taken together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.
Background:We investigated both trafficking and function of Arrestin 1 (Arr1) in Drosophila. Results: We show that Arr1 translocates to rhabdomeres to modulate deactivation of phosphorylated rhodopsin in adult photoreceptors but becomes internalized in pupal photoreceptors. Conclusion: Arr1 plays distinct roles in pupal and adult photoreceptors. Significance: Trafficking and function of arrestin may be regulated depending on the physiological context.
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 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-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.
SignificanceGlutamate is the primary excitatory neurotransmitter in the brain. It binds to both ionotropic and metabotropic glutamate receptors. Fast ionotropic receptors generate rapid synaptic transmission, whereas slower metabotropic glutamate receptors (mGluRs) modulate this synaptic transmission. Here, we discovered that activation of group II mGluRs enhances sound-evoked and spontaneous neuronal firing in the inferior colliculus-the hub of the central auditory system. We used transgenic mice which allowed for identification of excitatory and inhibitory neurons and found that both these cell types are modulated by group II mGluRs. Our results provide better understanding of mGluR modulatory roles, which is crucial in opening avenues for using mGluR-targeting drugs to treat hearing Glutamate is the primary excitatory neurotransmitter in the brain. It binds to both ionotropic and metabotropic glutamate receptors (mGluRs). Ionotropic receptors, which are ligand-gated cation channels, are important for rapid signal transmission, whereas mGluRs act via G-protein coupled second messenger cascades and are relatively slower. In general, mGluRs have been thoroughly investigated due to their potential as therapeutic targets in several psychiatric and neurological disorders
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 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-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.
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