Probes for INS

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA+/+) that constitutively expresses only FN containing the EDA isoform.Eyes from C57BL6/J (wild-type), B6.EDA+/+ (constitutively active EDA), B6.EDA-/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm's canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts.Ultrastructure analyses show the TM of B6.EDA+/+ mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6 J mice (p < 0.05). SC basement membrane is thicker and more continuous in B6.EDA+/+ mice compared to C57BL/6 J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.05). IOP is significantly higher in B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.001), and significant ON damage (p < 0.001) and RGC loss (p < 0.05) detected at 1 year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA+/+ mice compared to C57BL/6 J control eyes (p < 0.01).B6.EDA+/+ mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA+/+ mice recapitulate many aspects of glaucomatous damage.

Behaviors associated with distress can affect the anxiety-like states in observers and this social transfer of affect shapes social interactions among stressed individuals. We hypothesized that social reactions to stressed individuals engage the serotonergic dorsal raphe nucleus (DRN) which promotes anxiety-like behavior via postsynaptic action of serotonin at serotonin 2C (5-HT2C) receptors in the forebrain. First, we inhibited the DRN by administering an agonist (8-OH-DPAT, 1 μg in 0.5 μL) for the inhibitory 5-HT1A autoreceptors which silences 5-HT neuronal activity. 8-OH-DPAT prevented the approach and avoidance, respectively, of stressed juvenile (PN30) or stressed adult (PN60) conspecifics in the social affective preference (SAP) test in rats. Similarly, systemic administration of a 5-HT2C receptor antagonist (SB242084, 1 mg/kg, i.p.) prevented approach and avoidance of stressed juvenile or adult conspecifics, respectively. Seeking a locus of 5-HT2C action, we considered the posterior insular cortex which is critical for social affective behaviors and rich with 5-HT2C receptors. SB242084 administered directly into the insular cortex (5 μM in 0.5 μL bilaterally) interfered with the typical approach and avoidance behaviors observed in the SAP test. Finally, using fluorescent in situ hybridization, we found that 5-HT2C receptor mRNA (htr2c) is primarily colocalized with mRNA associated with excitatory glutamatergic neurons (vglut1) in the posterior insula. Importantly, the results of these treatments were the same in male and female rats. These data suggest that interactions with stressed others require the serotonergic DRN and that serotonin modulates social affective decision-making via action at insular 5-HT2C receptors.

Social interaction allows for the transfer of affective states among individuals, and the behaviors and expressions associated with pain and fear can evoke anxiety-like states in observers which shape subsequent social interactions. We hypothesized that social reactions to stressed individuals engage the serotonergic dorsal raphe nucleus (DRN) which promotes anxiety-like behavior via postsynaptic action of serotonin at serotonin 2C (5-HT 2C ) receptors in the forebrain. First, we inhibited the DRN by administering an agonist (8-OH-DPAT, 1µg in 0.5µL) for the inhibitory 5-HT 1A autoreceptors which silences 5-HT neuronal activity via G-protein coupled inward rectifying potassium channels. 8-OH-DPAT prevented the approach and avoidance, respectively, of stressed juvenile (PN30) or stressed adult (PN50) conspecifics in the social affective preference (SAP) test in rats. Similarly, systemic administration of a 5-HT 2C receptor antagonist (SB242084, 1mg/kg, i.p.) prevented approach and avoidance of stressed juvenile or adult conspecifics, respectively. Seeking a locus of 5-HT 2C action, we considered the posterior insular cortex which is critical for social affective behaviors and rich with 5-HT 2C receptors. SB242084 administered directly into the insular cortex (5µM bilaterally in 0.5µL ) interfered with the typical approach and avoidance behaviors observed in the SAP test. Finally, using fluorescent in situ hybridization, we found that 5-HT 2C receptor mRNA ( htr2c) is primarily colocalized with mRNA associated with excitatory glutamatergic neurons ( vglut1 ) in the posterior insula. Importantly, the results of these treatments were the same in male and female rats. These data suggest that interactions with stressed others require the serotonergic DRN and that serotonin modulates social affective decision-making via action at insular 5-HT 2C receptors.

Prenatal infection increases risk for neurodevelopmental disorders such as autism in offspring. In rodents, prenatal administration of the viral mimic Polyinosinic: polycytidylic acid (Poly I: C) allows for investigation of developmental consequences of gestational sickness on offspring social behavior and neural circuit function. Because maternal immune activation (MIA) disrupts cortical development and sociability, we examined approach and avoidance in a rat social affective preference (SAP) task. Following maternal Poly I:C (0.5 mg/kg) injection on gestational day 12.5, male adult offspring (PN 60-64) exhibited atypical social interactions with stressed conspecifics whereas female SAP behavior was unaffected by maternal Poly I:C. Social responses to stressed conspecifics depend upon the insular cortex where corticotropin releasing factor (CRF) modulates synaptic transmission and SAP behavior. We characterized insular field excitatory postsynaptic potentials (fEPSP) in adult offspring of Poly I:C or control treated dams. Male MIA offspring showed decreased sensitivity to CRF (300 nM) while female MIA offspring showed greater sensitivity to CRF compared to sham offspring. These sex specific effects appear to be behaviorally relevant as CRF injected into the insula of male and female rats prior to social exploration testing had no effect in MIA male offspring but increased social interaction in female MIA offspring. We examined the cellular distribution of CRF receptor mRNA but found no effect of maternal Poly I:C in the insula. Together, these experiments reveal sex specific effects of prenatal infection on offspring responses to social affective stimuli and identify insular CRF signaling as a novel neurobiological substrate for autism risk.

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.This is a work of the U.S. Government and is not subject to