Chronic-Stress-Induced Behavioral Changes Associated with Subregion-Selective Serotonin Cell Death in the Dorsal Raphe

The current study examined the neurochemical mechanisms and neuroanatomical changes underlying coexisting behavioral effects associated with chronic-stress-induced alterations in serotonin (5HT) neurons. Chronic unpredictable stress (CUS) to adult male rats produced depression-like changes with cognitive dysfunction and selective cell death in the interfascicular nucleus of the dorsal raphe (DRif), resulting in decreased 5HTergic innervation of medial prefrontal cortex (mPFC). Twenty-one days of CUS decreased basal plasma levels of corticosterone and produced a shorter latency to immobility and longer durations of immobility in the force-swim test that persisted for 1 month after CUS. Deficits in acquisition, recall, perseveration, and reversal learning were evident 1 month after CUS. MK801 treatment during CUS blocked the changes in the forced-swim test and deficits in memory recall. These behavioral changes were associated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive soma and the eventual loss of 5HT neurons in the DRif and its projections to the mPFC as evidenced by fewer labeled cells in the DRif after retrograde tracer injections into the mPFC of stressed rats. Similar to the effects of MK801 on behavior, MK801 pretreatment during stress blocked the CUS-induced decreases in 5HT soma within the DRif and its projections to the mPFC. Finally, the depression-like behaviors were blocked by acute injection of the 5HT2A/C agonist (−)-2,5-dimethoxy-4-iodoamphetamine hydrochloride into the mPFC before forced-swim testing. These results identify a cause and mechanism of 5HTergic dysfunction of the mPFC and associated mood and cognitive behaviors., SIGNIFICANCE STATEMENT Chronic stress causes persistent mood and cognitive changes typically associated with dysregulated serotonin (5HT) transmission in the medial prefrontal cortex (mPFC), but the cause of this dysregulation is unknown. Prior studies have focused on 5HTergic terminals in this region, but this study shows that chronic stress causes NMDA-receptor-dependent and subregion-specific cell death of 5HT neurons in the dorsal raphe. The consequent decreased 5HT innervation of the mPFC was associated with mood and cognitive changes that persisted long after the termination of stress. These findings identify a mechanism of subregion-selective death of 5HT neurons in the dorsal raphe, a defined neuroanatomical pathway, and a behavioral phenotype that mirror stress-associated diseases such as major depressive disorder

MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A Receptor Induced PGE2 Signaling

MDMA is a widely abused psychostimulant which causes release of serotonin in various forebrain regions. Recently, we reported that MDMA increases extracellular glutamate concentrations in the dentate gyrus, via activation of 5HT2A receptors. We examined the role of prostaglandin signaling in mediating the effects of 5HT2A receptor activation on the increases in extracellular glutamate and the subsequent long-term loss of parvalbumin interneurons in the dentate gyrus caused by MDMA. Administration of MDMA into the dentate gyrus of rats increased PGE2 concentrations which was prevented by coadministration of MDL100907, a 5HT2A receptor antagonist. MDMA-induced increases in extracellular glutamate were inhibited by local administration of SC-51089, an inhibitor of the EP1 prostaglandin receptor. Systemic administration of SC-51089 during injections of MDMA prevented the decreases in parvalbumin interneurons observed 10 days later. The loss of parvalbumin immunoreactivity after MDMA exposure coincided with a decrease in paired-pulse inhibition and afterdischarge threshold in the dentate gyrus. These changes were prevented by inhibition of EP1 and 5HT2A receptors during MDMA. Additional experiments revealed an increased susceptibility to kainic acid-induced seizures in MDMA treated rats which could be prevented with SC51089 treatments during MDMA exposure. Overall, these findings suggest that 5HT2A receptors mediate MDMA-induced PGE2 signaling and subsequent increases in glutamate. This signaling mediates parvalbumin cell losses as well as physiologic changes in the dentate gyrus, suggesting that the lack of the inhibition provided by these neurons increases the excitability within the dentate gyrus of MDMA treated rats

Retrospective screening of high-resolution mass spectrometry archived digital samples can improve environmental risk assessment of emerging contaminants: A case study on antifungal azoles

Environmental risk assessment associated with aquatic and terrestrial contamination is mostly based on predicted or measured environmental concentrations of a limited list of chemicals in a restricted number of environmental compartments. High resolution mass spectrometry (HRMS) can provide a more comprehensive picture of exposure to harmful chemicals, particularly through the retrospective analysis of digitally stored HRMS data. Using this methodology, our study characterized the contamination of various environmental compartments including 154 surface water, 46 urban effluent, 67 sediment, 15 soil, 34 groundwater, 24 biofilm, 41 gammarid and 49 fish samples at 95 sites widely distributed over the Swiss Plateau. As a proof-of-concept, we focused our investigation on antifungal azoles, a class of chemicals of emerging concern due to their endocrine disrupting effects on aquatic organisms and humans. Our results demonstrated the occurrence of antifungal azoles and some of their (bio)transformation products in all the analyzed compartments (0.1–100 ng/L or ng/gd.w.). Comparison of actual and predicted concentrations showed the partial suitability of level 1 fugacitymodelling in predicting the exposure to azoles. Risk quotient calculations additionally revealed risk of exposure especially if some of the investigated rivers and streams are used for drinking water production. The case study clearly shows that the retrospective analysis of HRMS/MS data can improve the current knowledge on exposure and the related risks to chemicals of emerging concern and can be effectively employed in the future for such purposes

Use of fluorescent ANTS to examine the BBB-permeability of polysaccharide

Recently, some polysaccharides showed therapeutic potentials for the treatment of neurodegenerative diseases while the most important property, their permeability to the blood brain barrier (BBB) that sheathes the brain and spinal cord, is not yet determined. The determination has been delayed by the difficulty in tracking a target polysaccharide among endogenous polysaccharides in animal. We developed an easy way to examine the BBB-permeability and, possibly, tissue distribution of a target polysaccharide in animal. We tagged a polysaccharide with fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) for tracking. We also developed a simple method to separate ANTS-tagged polysaccharide from unconjugated free ANTS using 75% ethanol. After ANTS-polysaccharide was intra-nasally administered into animals, we could quantify the amounts of ANTS-polysaccharide in the brain and the serum by fluorocytometry. We could also separate free ANTS-polysaccharide from serum proteins using trichloroacetic acid (TCA) and 75% ethanol. Our method will help to track a polysaccharide in animal easily. • ANTS-labeling is less tedious than but as powerful as radiolabeling for tracking a target polysaccharide in animal. • Our simple method can separate structurally intact ANTS-polysaccharide from animal serum and tissues. • This method is good for the fluorometry-based measurement of ANTS-conjugated macromolecules in tissues

BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR

<div><p>An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer’s disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their <i>in vivo</i> efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aβ₄₂ peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD.</p></div

Midi-GAGR reverses neurite atrophy caused by 4HNE and H₂O₂.

<p>(A-E) Differentiated N2A cells were treated with different concentrations of 4HNE (0, 1, 5, 10 and 25 μM) for 48 h or H₂O₂ (0, 1, 10, 50, 100 and 200 μM) for 24 h and immunostained with α-tubulin antibody. The representative images of N2A cells treated with H₂O (vehicle) (A), 25 μM 4HNE (B), or 200 μM H₂O₂ (C). Scale bar = 75 μm. Bar graphs represent the average total neurite lengths of N2A cells in response to different concentrations of either 4HNE (D) or H₂O₂ (E). *, p < 0.05 and **, p < 0.001 compared to control. (F-I) Differentiated N2A cells were pre-treated with different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of midi-GAGR for 24 h, followed by incubation with either 25 μM 4HNE for 48 h or 200 μM H₂O₂ for 24 h and then immunostained with anti-α-tubulin antibody. The representative images of N2A cells pretreated with 1 μM midi-GAGR and then incubated with either 25 μM 4HNE (F) or 200 μM H₂O₂ (G). Scale bar = 75 μm. Bar graphs show the average total neurite lengths of N2A cells pre-treated with different concentrations of midi-GAGR followed by treatment with 25 μM 4HNE (H) or 200 μM H₂O₂ (I). Dotted lines correspond to the average total neurite lengths of N2A cells without any treatment. Data represent mean ± SEM of, at least, 40 cells/group from two independent experiments. *, p<0.05 and **, p<0.001 compared to 4HNE alone.</p