High Affinity Decynium-22 Binding to Brain Membrane Homogenates and Reduced Dorsal Camouflaging after Acute Exposure to it in Zebrafish.

Organic cation transporters (OCTs) are expressed in the mammalian brain, kidney, liver, placenta, and intestines, where they facilitate the transport of cations and other substrates between extracellular fluids and cells. Despite increasing reliance on ectothermic vertebrates as alternative toxicology models, properties of their OCT homologs transporting many drugs and toxins remain poorly characterized. Recently, in zebrafish (Danio rerio), two proteins with functional similarities to human OCTs were shown to be highly expressed in the liver, kidney, eye, and brain. This study is the first to characterize in vivo uptake to the brain and the high-affinity brain membrane binding of the mammalian OCT blocker 1-1'-diethyl-2,2'cyanine iodide (decynium-22 or D-22) in zebrafish. Membrane saturation binding of [3H] D-22 in pooled zebrafish whole brain versus mouse hippocampal homogenates revealed a high-affinity binding site with a KD of 5 ± 2.5 nM and Bmax of 1974 ± 410 fmol/mg protein in the zebrafish brain, and a KD of 3.3 ± 2.3 and Bmax of 704 ± 182 fmol/mg protein in mouse hippocampus. The binding of [3H] D-22 to brain membrane homogenates was partially blocked by the neurotoxic cation 1-methyl-4-phenylpyridinium (MPP+), a known OCT substrate. To determine if D-22 bath exposures reach the brain, zebrafish were exposed to 25 nM [3H] D-22 for 10 min, and 736 ± 68 ng/g wet weight [3H] D-22 was bound. Acute behavioral effects of D-22 in zebrafish were characterized in two anxiety-relevant tests. In the first cohort of zebrafish, 12.5, 25, or 50 mg/L D-22 had no effect on their height in the dive tank or entries and time spent in white arms of a light/dark plus maze. By contrast, 25 mg/L buspirone increased zebrafish dive tank top-dwelling (p < 0.05), an anticipated anxiolytic effect. However, a second cohort of zebrafish treated with 50 mg/L D-22 made more white arm entries, and females spent more time in white than controls. Based on these findings, it appears that D-22 bath treatments reach the zebrafish brain and have partial anxiolytic properties, reducing anti-predator dorsal camouflaging, without increasing vertical exploration. High-affinity binding of [3H] D-22 in zebrafish brain and mouse brain was similar, with nanomolar affinity, possibly at conserved OCT site(s).

Neuroendocrine Mechanisms Governing Sex Differences in Hyperalgesic Priming Involve Prolactin Receptor Sensory Neuron Signaling.

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

Prolactin Regulates Pain Responses via a Female-Selective Nociceptor-Specific Mechanism.

Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

Acute dietary tryptophan manipulation differentially alters social behavior, brain serotonin and plasma corticosterone in three inbred mouse strains.

Clinical evidence indicates brain serotonin (5-HT) stores and neurotransmission may be inadequate in subpopulations of individuals with autism, and this may contribute to characteristically impaired social behaviors. Findings that depletion of the 5-HT precursor tryptophan (TRP) worsens autism symptoms support this hypothesis. Yet dietetic studies show and parents report that many children with autism consume less TRP than peers. To measure the impact of dietary TRP content on social behavior, we administered either diets devoid of TRP, with standard TRP (0.2 g%), or with 1% added TRP (1.2 g%) overnight to three mouse strains. Of these, BTBRT(+)Itpr3(tf)/J and 129S1/SvImJ consistently exhibit low preference for social interaction relative to C57BL/6. We found that TRP depletion reduced C57BL/6 and 129S social interaction preference, while TRP enhancement improved BTBR sociability (p < 0.05; N = 8-10). Subsequent marble burying did not differ among diets or strains. After behavior tests, brain TRP levels and plasma corticosterone were higher in TRP enhanced C57BL/6 and BTBR, while 5-HT levels were reduced in all strains by TRP depletion (p < 0.05; N = 4-10). Relative hyperactivity of BTBR and hypoactivity of 129S, evident in self-grooming and chamber entries during sociability tests, were uninfluenced by dietary TRP. Our findings demonstrate mouse sociability and brain 5-HT turnover are reduced by acute TRP depletion, and can be enhanced by TRP supplementation. This outcome warrants further basic and clinical studies employing biomarker combinations such as TRP metabolism and 5-HT regulated hormones to characterize conditions wherein TRP supplementation may best ameliorate sociability deficits.