THE EFFECTS OF COCAINE SELF-ADMINISTRATION ON STRUCTURAL PLASTICITY OF ASTROCYTES

One of the hallmark features of cocaine use disorder is susceptibility toward relapse following cessation of use. It is thus imperative to elucidate neurobiological factors of relapse, to inform more targeted treatment approaches. Previous studies have reported decreases in expression of astrocyte-associated proteins in the nucleus accumbens core following rat cocaine self-administration and extinction training, pointing to possible glial control of relapse vulnerability. Recent results from our laboratory have further revealed remarkable decreases in morphometric features of accumbens astrocytes, as well as decreased colocalization with neuronal synaptic elements. Here I explore the regional specificity of astrocytic responsiveness to cocaine self-administration, as well as the necessity of the withdrawal for its manifestation. Results indicate that the reduced astrocytic phenotype is specific for astrocytes in nucleus accumbens and are not observed in other brain reward centers following cocaine self-administration and extinction. Further, the extinction period was necessary for the development of the atrophic phenotype, with rats undergoing self-administration only not showing morphometric impairments. To understand the subcellular nature of the atrophic phenotype, the second part of the study explored astrocyte branching complexity as well the number and density of accumbens astrocyte processes associated with two commonly utilized cocaine self-administration paradigms. To this end, novel 3-dimensional branching and Sholl analyses were employed as well as end-point and segment analyses, in order to delineate between qualitative and quantitative changes to astrocyte structure following cocaine self-administration. Results showed similar directional changes to subcellular features of astrocytes, with a more severe atrophic phenotype across the analyzed features associated with longer cocaine access and prolonged abstinence. Lastly, segment analysis revealed changes in segment number but not average length, indicating atrophic phenotype is not due to astrocyte shrinkage but rather loss of branching complexity and segments. In summary, this dissertation expands knowledge regarding structural plasticity of astrocytes following cocaine use and withdrawal, revealing specific vulnerability of nucleus accumbens astrocytes to cocaine self-administration, and a common observation of decreased interactions between astrocytes and synapses across self-administration paradigms, which is exacerbated by prolonged cocaine access and abstinence.Doctor of Philosoph

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia

Graphical Abstract Highlights d Derivation of human neocortical and spinal cord neuroepithelial stem (NES) cells d Zika virus (ZIKV) infects NES cells and radial glia, impairing mitosis and survival d ZIKV induces mitochondrial sequestration of centrosomal phospho-TBK1 d Nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death In Brief Onorati et al. establish neuroepithelial stem (NES) cells as a model for studying human neurodevelopment and ZIKV-induced microcephaly. Together with analyses in human brain slices and microcephalic human fetal tissue, they find that ZIKV predominantly infects NES and radial glial cells, reveal a pivotal role for pTBK1, and find that nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death

Cell viability versus concentration of TiO₂-NTs in absence or presence of UV radiation.

<p>Absorbance of MTS was measured as described in Materials and methods section. (A) Each non-irradiated data set (red symbols) is compared to a sample irradiated with UV light (blue symbols) at given concentration of TiO₂-NTs. All Measurements are presented in a boxplot representing the distribution of the measured cell viability and described in Materials and methods. Differences in the cell viability between irradiated and non-irradiated cells are shown with the black arrows at each concentration of TiO₂-NTs. The differences in the median values between the two groups at 0 and 1000 μg/mL and the two groups at 1 and 50 μg/mL of nanotubes are considered to be significant at P = <0.001, and P = <0.05, respectively. (B) Differences of median values of MTS absorbance between irradiated and non-irradiated cells, shown with the black arrows within the frame A, are plotted with the open bars. Solid black line represents the prediction of the difference in cell viability, based on the effect of the UV irradiation and phototoxic effect due to irradiated nanotubes not covered by serum proteins. Both contributions are shown separately in the frame C. (C) Solid dark blue line shows the transmittance of UV light versus the concentration of the nanotubes obtained experimentally (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s002" target="_blank">S2 Information</a>. Optical properties of TiO₂-NTs dispersion.), while dashed red line represents the concentration of the free nanotubes calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s003" target="_blank">S3 Information</a>. Model of albumin binding to TiO2-NTs and best fit parameter values. (D) The relative amount of TiO₂-NTs covered by the serum proteins (red line) and the relative amount of serum proteins bound to TiO₂-NTs (yellow line) are shown as predicted by the model described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.s003" target="_blank">S3 Information</a>. Model of albumin binding to TiO2-NTs and best fit parameter values.</p

Protein Corona Prevents TiO₂ Phototoxicity

<div><p>Background & Aim</p><p>TiO₂ nanoparticles have generally low toxicity in the <i>in vitro</i> systems although some toxicity is expected to originate in the TiO₂-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO₂ nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.</p><p>Methods & Results</p><p>Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface.</p><p>Conclusion</p><p>These results imply that TiO₂ nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired – as for efficient photodynamic cancer therapy.</p></div

Labelling of TiO₂-NTs with fluorophore Alexa via the two steps-reaction of the TiO₂-NTs surface modification.

<p>Firstly, 3-(2-aminoethylamino)propyltrimethoxysilane (AEAPMS) is attached to free–OH groups of the nanotubes’ surface (fTiO₂-NT). Secondly, the Alexa 488 SDP ester is covalently linked to the free amino groups of silane molecules (A-TiO₂-NT).</p

Measurements of hydroxyl radical formations by UV irradiation of the TiO₂-NTs.

<p>Spin trap DMPO was used to detect production of the hydroxyl radicals generated by UV irradiated TiO₂-NTs. TiO₂-NTs were mixed with DMPO spin traps and cell medium with 10% FBS or without FBS. The sample was irradiated for 5 min with UV light (wavelength of 356 nm) or left in dark (control), followed by EPR measurements immediately after the addition of the cell medium. In parallel experiments the dispersion of the TiO₂-NTs in the cell medium without or with the serum proteins (FBS) was put in the dark for one hour, than the spin trap DMPO was added and samples were irradiated with the UV light. (A) Representative EPR spectrum of a trapped hydroxyl radical in the presence of the FBS or (B) absence of the FBS. (C) The experimental EPR spectrum was simulated with hyperfine splitting constants: A_N = 1.49 G and A_H = 1.49 mT typical for OH radical. Simulation was done with EPRSim Wizard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129577#pone.0129577.ref029" target="_blank">29</a>]. EPR spectrum intensity peak normalized to the experiment with highest intensity peak, of nanomaterial in the cell medium without the FBS and with the FBS.</p

Fluorescence quenching due to binding of the bovine serum albumin to fluorescently labelled A-TiO₂-NTs.

<p>Intensity of fluorescence emission of Alexa labeled nanotubes excited at 250 nm was measured on spectrofluorimeter. Fluorescently labeled nanotubes (A-TiO₂-NTs) were dispersed in water (300 μg/mL) and sonicated on ultrasonic bath, then 100 μL of dispersion was titrated with FBS in 2 μL steps to final volume 150 μL. The analog experiment with 0.6 μM Alexa has been done for reference to prove that the free Alexa has a distinguishable titration curve from the bound Alexa.</p