Mechanisms of serotonin and serotonin receptor biology in normal and glaucomatous retinas

PROJECT SUMMARY Glaucoma is a progressive disease of retinal ganglion cells (RGCs) and their axons. Some evidence suggests that oral SSRIs (selective serotonin reuptake inhibitors), which increase serotonin (5-HT) levels, may have a protective effect against progression of open angle glaucoma. To understand the mechanism of this potential protection, we assessed RGC expression of serotonin receptors (HTRs) in mouse and human scRNA-seq datasets. In mice, Htr1b is expressed in RGCs at a high level and preferentially expressed in alpha RGCs (αRGCs). In humans, HTR1B is expressed in RGCs, suggesting a conserved function. In mouse retinal tissue, we detected Htr1b transcript in the ganglion cell layer (GCL) and HTR1B protein in the GCL and inner plexiform layer near excitatory post-synaptic sites. Using electroretinogram (ERG), optokinetic response (OKR), and multielectrode array (MEA) testing in conjunction with histology in Htr1b-/- and control mice, we found that Htr1b-/- retinas are anatomically normal, but Htr1b is required for several aspects of normal inner retinal function. We also found that HTR1B agonists and antagonists impact ON and OFF RGCs differently. Finally, SSRI administration in drinking water mitigated some IOP-induced phenotypes without reducing IOP. These data suggest that 5-HT and HTRs play a previously unrecognized role in RGC function and IOP/glaucoma susceptibility. We will test the hypothesis that Htr1b is required for normal RGC and αRGC function and acts through downstream effects on membrane excitability. We will also ask if increased 5-HT signaling through Htr1b promotes RGC resistance to injury whereas decreased 5-HT signaling through Htr1b increases RGC susceptibility in the setting of elevated IOP. There are three aims: (1) Determine the cell-type specific function of Htr1b in the inner retina; (2) Elucidate the intracellular mechanisms of action of Htr1b in four αRGC subtypes; and (3) Assess the impact of altered 5-HT signaling on glaucoma susceptibility. In Aim 1, we will combine a novel conditional Htr1b knockout with Cre-expressing lines specific for RGCs, αRGCs, or amacrine cells to dissect Htr1b function at the retinal circuitry level (ERG/MEA), behavioral level (OKR), and brain circuitry level (mapping of RGC axon transport to the superior colliculus). We will also use a novel system of adult mouse RGC isolation and culture to test cell-autonomous anatomic responses to 5-HT. In Aim 2, we will use pharmacologic techniques alongside whole-cell patch clamp recording to compare 5-HT and Htr1b biology among the four αRGC subtypes and to test the hypothesis that HTR1B acts via regulation of potassium channels. We will also use fibronectin intrabodies generated with mRNA display to map HTR1B to excitatory and inhibitory post-synaptic sites. In Aim 3, we will provide SSRI in the drinking water of mice with elevated IOP and use both physiologic and anatomic studies to determine if increased 5-HT causes RGC neuroprotection. We will then determine if concurrent Htr1b loss of function or overexpression prevents or enhances neuroprotection, respectively.