Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals

Reduced GABAergic inhibition in the basolateral amygdala and the development of anxiety-like behaviors after mild traumatic brain injury.

Traumatic brain injury (TBI) is a major public health concern affecting a large number of athletes and military personnel. Individuals suffering from a TBI risk developing anxiety disorders, yet the pathophysiological alterations that result in the development of anxiety disorders have not yet been identified. One region often damaged by a TBI is the basolateral amygdala (BLA); hyperactivity within the BLA is associated with increased expression of anxiety and fear, yet the functional alterations that lead to BLA hyperexcitability after TBI have not been identified. We assessed the functional alterations in inhibitory synaptic transmission in the BLA and one mechanism that modulates excitatory synaptic transmission, the α7 containing nicotinic acetylcholine receptor (α7-nAChR), after mTBI, to shed light on the mechanisms that contribute to increased anxiety-like behaviors. Seven and 30 days after a mild controlled cortical impact (CCI) injury, animals displayed significantly greater anxiety-like behavior. This was associated with a significant loss of GABAergic interneurons and significant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitory postsynaptic currents (IPSCs). Decreases in the mIPSC amplitude were associated with reduced surface expression of α1, β2, and γ2 GABAA receptor subunits. However, significant increases in the surface expression and current mediated by α7-nAChR, were observed, signifying increases in the excitability of principal neurons within the BLA. These results suggest that mTBI causes not only a significant reduction in inhibition in the BLA, but also an increase in neuronal excitability, which may contribute to hyperexcitability and the development of anxiety disorders

Delayed loss of GABAergic interneurons in the BLA within the first week after mild CCI.

<p>(A) Representative photomicrographs of GAD-67 immunohistochemically stained GABAergic interneurons in the BLA of sham (left), 1-day CCI (middle), and 7-day CCI (right) animals. Total magnification is 630x; scale bar, 50 µm. (B) group data showing the mean and standard error of the stereologically estimated total number of GAD-67-positive cells in the BLA 1- and 7-days after CCI compared with sham. Only 7-days after CCI was there a significant bilateral reduction in GAD-67-positive cells indicating a delayed loss of GABAergic interneurons. ***p<0.001; n = 10 for each group.</p

Total Number of Interneurons.

<p>**p<0.01, ***p<0.001.</p

Mild TBI increases anxiety-like behaviors in the open field test.

<p>(A) No differences in percent time spent in the center of the open field were found between CCI (n = 19) and sham (n = 19) animals 24 hours after injury. However, CCI rats spent significantly less time in the center of the open field 7- and 30-days after injury compared to sham animals. No significant differences were found between the sham and CCI animals in distance traveled (B), vertical activity (C), or movement time (D) at any of the time points. Bars show the mean ± SE of the percentage of time spent in the center (A), distance traveled (B), vertical activity (C), and movement time (D). *p<0.05.</p

Surface expression of α1, β2, and γ2 GABA_A receptor subunits is reduced in the BLA of CCI animals 7 days after mild CCI.

<p>Western blot for subunits of (A) α1, (B) β2, and (C) γ2 subunits, respectively, was performed using biotinylated proteins isolated from the ipsilateral and contralateral sides of Sham and CCI 7-day animals. Group data showing the mean ± SE of the ratio between each subunit and GLUT1 optical densities. Top panel: representative Western blot for α1 (A), β2 (B), and γ2 (C) subunits of GABA_A receptors, respectively. Bottom panel: representative Western blot for GLUT1, used as a loading control. Note that surface expression of GABA_A α1, β2 and γ2 subunits are reduced in CCI animals when compared to Sham animals. *<i>p</i><0.01; n = 4 for each group.</p

Mild CCI does not cause a significant loss of neurons in the BLA 24 hours or 7 days after injury.

<p>(A) Panoramic photomicrograph of Nissl-stained brain slice. Indicated are the sites of impact and the ipsilateral BLA. (B) Representative photomicrographs from Nissl-stained sections showing BLA cells from the ipsilateral (Top) and contralateral (Bottom) sides of sham, 1-day CCI, and 7-day CCI animals, respectively. Total magnification is 630X; scale bar, 50 µm. (C) Group data (mean ± SE; n = 8 for each group) of stereological estimation of the total number of Nissl-stained neurons in the BLA.</p