Repeated PTZ Treatment at 25-Day Intervals Leads to a Highly Efficient Accumulation of Doublecortin in the Dorsal Hippocampus of Rats

BACKGROUND: Neurogenesis persists throughout life in the adult mammalian brain. Because neurogenesis can only be assessed in postmortem tissue, its functional significance remains undetermined, and identifying an in vivo correlate of neurogenesis has become an important goal. By studying pentylenetetrazole-induced brain stimulation in a rat model of kindling we accidentally discovered that 25±1 days periodic stimulation of Sprague-Dawley rats led to a highly efficient increase in seizure susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: By EEG, RT-PCR, western blotting and immunohistochemistry, we show that repeated convulsive seizures with a periodicity of 25±1 days led to an enrichment of newly generated neurons, that were BrdU-positive in the dentate gyrus at day 25±1 post-seizure. At the same time, there was a massive increase in the number of neurons expressing the migratory marker, doublecortin, at the boundary between the granule cell layer and the polymorphic layer in the dorsal hippocampus. Some of these migrating neurons were also positive for NeuN, a marker for adult neurons. CONCLUSION/SIGNIFICANCE: Our results suggest that the increased susceptibility to seizure at day 25±1 post-treatment is coincident with a critical time required for newborn neurons to differentiate and integrate into the existing hippocampal network, and outlines the importance of the dorsal hippocampus for seizure-related neurogenesis. This model can be used as an in vivo correlate of neurogenesis to study basic questions related to neurogenesis and to the neurogenic mechanisms that contribute to the development of epilepsy

LAN Traffic Capture Applications Using the Libtins Library

Capturing traffic and processing its contents is a valuable skill that when put in the right hands makes diagnosing and troubleshooting network issues an approachable task. Apart from aiding in fixing common problems, packet capture can also be used for any application that requires getting a deeper understanding of how things work under the hood. Many tools have been developed in order to allow the user to study the flow of data inside of a network. This paper focuses on documenting the process of creating such tools and showcasing their use in different contexts. This is achieved by leveraging the power of the C++ programming language and of the libtins library in order to create custom extensible sniffing tools, which are then used in VoIP (Voice over IP) and IDS (Intrusion Detection System) applications

LAN Traffic Capture Applications Using the Libtins Library

Capturing traffic and processing its contents is a valuable skill that when put in the right hands makes diagnosing and troubleshooting network issues an approachable task. Apart from aiding in fixing common problems, packet capture can also be used for any application that requires getting a deeper understanding of how things work under the hood. Many tools have been developed in order to allow the user to study the flow of data inside of a network. This paper focuses on documenting the process of creating such tools and showcasing their use in different contexts. This is achieved by leveraging the power of the C++ programming language and of the libtins library in order to create custom extensible sniffing tools, which are then used in VoIP (Voice over IP) and IDS (Intrusion Detection System) applications

Diagrams.

<p>(<i>Upper panel</i>): Flow diagram of Exp. #1 regarding times and type of treatment with pentylenetetrazole. <i>(Lower panel):</i> Schematic diagram of the distribution of proliferative (BrdU-positive) cells in the brain at day 25 after a single administration of a convulsive dose of PTZ. Note the change from a widespread distribution (<b>A</b>) to a more restricted distribution at 25 days post-seizure (<b>B</b>). In control rats, there were only a few BrdU-positive cells, often in a duplex, mitosis-like state (<b>C, D, E</b>). Many proliferative cells in PTZ-treated animals appeared to enter the brain from the circulation via leptomeningeal blood vessels (<b>F</b>, arrow points to a mitosis-like state). (<b>G</b>, <b>H</b>): Quantitation of BrdU-positive cells. One episode of convulsive seizure causes, at day 3, dramatically increased BrdU-positive cell numbers in the hippocampus (15-fold over controls; p = 0.001) (<b>G</b>) and temporal neocortex (22.5-fold over controls; p = 0.001) (<b>H</b>). Although the numbers of BrdU-positive cells decreased dramatically by day 25, their number remained, nonetheless, at relatively high levels in the hippocampus (4.8-fold; p = 0.001)(<b>G</b>) and temporal neocortex (5.6-fold; p = 0.001) (<b>H</b>) over control levels. N = 15 rats for each timepoint. <i>Abbreviations</i>: <i>Te-II</i>, temporal neocortex layer II; <i>Ent</i>, entorhinal neocortex; <i>HC</i>, hippocampus; <i>lep</i>, leptomeninx. Bars: (<b>C, D, E</b>), 20 µm; (<b>F</b>), 30 µm.</p

Localization and quantification of DCX in the rat brain during kindling development.

<p>(<b>A, B</b>) Overview of DCX staining in the ventral (<b>A,</b> arrows) and dorsal (<b>B,</b> arrows) hippocampal hilus of the kindled animals. The dorsal hippocampus of kindled animals, was highly significant (p = 0.001) enriched in DCX⁺-cells (<b>C</b>). Note that the DCX antigens were localized both in cell bodies and extensions penetrating the densely packed granule cell neurons (<b>D</b>, arrows). (<b>E-F</b>): Phenotyping of DCX-cells. After 2× PTZ some DCX⁺ positive cells (green) in the dorsal hippocampus along the hilar border with the granule cell layer had a NeuN nucleus (red) (<b>E</b>, arrows). In kindled animals some of the DCX (green)/BrdU (red) double-labeled cells had a clonal appearance (<b>F</b>, inset, 3D-image) while other DCX⁺ cells (green) sometimes displayed a fragmented BrdU-positivity (<b>F</b>, arrow). By quantitative RT-PCR there was a 3-fold increase (p = 0.01) in the relative amount of DCX transcripts in kindled animals over that of controls (<b>G</b>). Note that the number of DCX⁺ cells also is maximal when PTZ is administered every 25^th day (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039302#pone-0039302-g004" target="_blank">Fig. 4</a>H, filled circles) as opposed to every 30^th day (<b>H</b>, open circles). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer; <i>hl</i>, hilus; <i>pml</i>, polymorphic layer. Bars: (<b>A,B</b>), 200 µm; (<b>D</b>), 100 µm.</p

Number and phenotyping of BrdU-positive cells after seizure activity.

<p>Each PTZ treatment led to an accumulation of BrdU⁺ cells in the dentate gyrus of the kindled rats (9-fold, p = 0.0001; <b>A</b>). (<b>B–E</b>): 3D projections of confocal BrdU(red)/NeuN(green) double-labeled images from PTZ-treated animals. A single episode of seizure activity led to the appearance of BrdU-positive cells in the polymorphic layer that were in a mitosis-like state (<b>B</b>). Occasionally some neurons in layers II and III of the temporal neocortex also displayed BrdU⁺ cells in close apposition to neurons (<b>B</b>, insets). After 2× PTZ some BrdU-positive cells have differentiated into neurons, particularly in the granule cell layer (<b>C</b>, arrows). In addition, some BrdU-positive nuclei were detected in the walls of large blood vessels (<b>C</b>, inset). The number of double-labeled BrdU(red)/NeuN(green) increased with the number of PTZ injections and reached a maximum in the granule cells layer of kindled animals (<b>D</b>, low power; <b>E</b>, higher power). <i>Abbreviations</i>: <i>Te</i>, temporal neocortex; <i>GCL</i>, granule cell layer; <i>BV</i>, Blood Vessel.</p

L-NAME treatment increased neurogenesis and seizure susceptibility.

<p>Daily treatment with L-NAME for 24 days (<b>A</b>) resulted in a significant increase in the number of fully seizing animals after the second PTZ administration on day 25 (<b>B</b>). In control animals, nestin immunoreactivity was detected in capillary walls (<b>C</b>). By quantitative RT-PCR there was a 7.8-fold (p = 0.001) in nestin mRNA levels at day 3 post-seizure (<b>D</b>). L-NAME-treated animals had significantly more (2.7-fold; p = 0.001) nestin mRNA than did animals treated with PTZ alone at day 25 (<b>D</b>) whereas kindled animals did not show an increased level of nestin mRNA (<b>D</b>). At the tissue level, nestin immunoreactivity at day3 in PTZ-treated animals was confined to radial glia-like cells in the inner molecular layer of the dentate gyrus, the polymorphic layer and, interestingly, to the CA2 region (arrow) (<b>E</b>, and inset). After 25 days, the nestin-like immunoreactivity was restricted to the polymorphic layer (<b>F</b> and inset). <i>Abbreviations</i>: <i>DG</i>, dentate gyrus; <i>pml</i>, polymorphic layer; <i>CA2</i>, hippocampal region.</p

Flow diagrams of the experimental design regarding times and type of treatment and EEG recording.

<p>After Exp. #2 (<b>A</b>) the proportion of rats that achieved full kindling status reached 80% (<b>C</b>; p = 0.001). By comparison, 31% of rats administered a subconvulsive dose of PTZ at all times (Exp. #3)(<b>B</b>) reached full kindling status after two injections and up to 81% of animals reached full kindling status after the fourth treatment (<b>C;</b> p = 0.001). (<b>D</b>–<b>F</b>): A subconvulsive PTZ treatment elicited intermittent non-ictal events that are typically dependent on the behavioral state of the animal (active or passive wakefulness)(<b>D</b>). After the second PTZ injection, the animals usually demonstrated mild multifocal body jerks (<b>E</b>). After the third PTZ injection, the animals showed typical seizure activity associated with motor arrest (<b>F</b>).</p

L-NAME treatment increased doublecortin levels in the rat hippocampus.

<p>L-NAME treatment increased DCX immunoreactivity on Western blots (<b>A</b>) by 2.2-fold (p = 0.02) at day 50 post-seizure (<b>B</b>). By immunohistochemistry, numerous DCX-positive cells were detected in the subgranular zone of the dorsal hippocampus by day 50 post-seizure (<b>C</b>). Quantitatively, L-NAME elicited significant increases (1.5-fold, p = 0.01) in the number of DCX-positive cells (<b>D</b>). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer.</p