Almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX (extracted from the ovary) acts as an attractive pheromone for some toxic puffers. To determine the putative pheromonal action of TTX, we examined whether grass puffers (Takifugu alboplumbeus) can detect TTX using electrophysiological, morphological, and behavioral experiments. Electroolfactogram results suggest that the olfactory epithelium of grass puffers responded in a dose-dependent manner to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons using a fluorescent dextran conjugate or immunohistochemistry against phosphorylated extracellular signal regulated kinase (pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was abundant cilia on the olfactory lamella. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that grass puffers can detect 5,6,11-trideoxyTTX using their nose and may positively use this functionally unknown TTX analog as an olfactory chemoattractant.

Retinal Pigment Epithelium Remodeling in Mouse Models of Retinitis Pigmentosa

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Phosphorescent Ir(III) complexes conjugated with oligoarginine peptides serve as optical probes for in vivo microvascular imaging

Imaging the vascular structures of organ and tumor tissues is extremely important for assessing various pathological conditions. Herein we present the new vascular imaging probe BTQ-Rn (n = 8, 12, 16), a phosphorescent Ir(III) complex containing an oligoarginine peptide as a ligand. This microvasculature staining probe can be chemically synthesized, unlike the commonly used tomato lectins labeled with a fluorophore such as fluorescein isothiocyanate (FITC). Intravenous administration of BTQ-R12 to mice and subsequent confocal luminescence microscope measurements enabled in vivo vascular imaging of tumors and various organs, including kidney, liver and pancreas. Dual color imaging of hepatic tissues of living mice fed a high-fat diet using BTQ-R12 and the lipid droplet-specific probe PC6S revealed small and large lipid droplets in the hepatocytes, causing distortion of the sinusoidal structure. BTQ-R12 selectively stains vascular endothelium and thus allows longer-term vascular network imaging compared to fluorescent dextran with a molecular weight of 70 kDa that circulate in the bloodstream. Furthermore, time-gated measurements using this phosphorescent vascular probe enabled imaging of blood vessel structures without interference from autofluorescence.

Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Sustained Alterations of Hypothalamic Tanycytes During Posttraumatic Hypopituitarism in Male Mice

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.