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Voter fraud is a huge issue in almost every major election. Of course the presidential election is going to be no different. Which is why this story should come as no surprise. A woman who lives in Texas who is an illegal citizen was arrested and taken into custody this past Friday for illegally voting in Dallas County on five occasions.
The woman is 35-year-old Rosa Maria Ortega and she resides now in Tarrant County Jail where she is on a $10,000 bond. This was all reported by the Dallas Fort Worth NBC affiliate Channel 5 News. They reported the following about the incident,
"Ortega is married to an American and is living legally in this country, but is not a citizen and therefore, not qualified to vote, said Harry White, who supervises public integrity and white collar crime investigations for the Tarrant County District Attorney.
Ortega applied to vote in Tarrant County and acknowledged on the application form that she was not a citizen, White said. The county rejected her application and notified her she was not qualified to vote."
While she initially applied to register to vote she re-applied five months later after being denied yet she claimed she was American citizen but this was a lie. Everyone should know better, you do not mess with the federal government. Records indicate that Ortega never voted in Tarrant County, however she did on five separate occasions in Dallas County.
Her first election was in the 2004 Republican primary and the last recorded one was in the 2014 Republican primary. Illegal voting is a second degree felony which is punishable by a large sentence that ranges from 2 to 20 years.
The Democrats and Republicans are currently battling over the issue of voter ID fraud and the laws that dictate such events. Thirty different states have various forms of requirements when it comes to voter identification.
It has been mostly conservatives blaming Democrats for encouraging voter fraud because of the belief that illegal immigrants will vote. Which would skew in favor of Democratic candidates who are more lenient in amnesty and other issues towards illegal immigrants. But it seems the tide has turned in that department.


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Amani ya Juu
Boy's Button-Down Shirt
$ 26.00
$ 28.00
A bright collared dress shirt handmade in Africa. The Boy's Button-Down Shirt is handmade at Amani Liberia out of Lapa, a vibrant, traditional West African fabric used for clothing. Each has an animal stitched to the back. Available in Yellow/Red with a sparrow on the back and Blue/Green with a fish on the back.

subset: commoncrawl


text:
Not valid on everyday great price items, Insane Deals, Deal Flash items, fragrance, cosmetics, footwear, fine jewelry, baby gear and furniture, Lands' End® merchandise, Levi's®, Argan Woman, Prima Derm, JanSport, UNITED FACE, Ramonti, Harbor Bay, Oak Hill, True Nation, Gold Series, Synrgy, Island Passport, Society of One, Rochester, 555 Turnpike, Outlet Store purchases, and Gift Cards. Redeemable at Sears stores and sears.com only. One coupon per purchase. Void if copied, transferred, or obtained via unapproved means. Any other use constitutes fraud. Cash value 1/20¢. On return, coupon savings deducted from refund. Online code limited to one time use only and applies to items marked sold by Sears. To redeem online enter CLOTHING into "promo code" field at checkout. Savings shown in cart. Sears Holdings reserves the right to terminate or modify this offer at any time for failure to comply with its terms and/or due to any operational malfunction of the software, hardware or equipment required to process this offer or if due to any other reason. Sears card offer subject to credit approval. Excludes Sears Commercial One® and Sears Home Improvement Account ℠ accounts. Sears cards issued by Citibank, N.A. Sales Associate: If unable to scan, manually enter the coupon number. Valid 8/16/17-8/19/17 in the U.S.A. only; not to be used with any other coupon, associate discount, or during Family and Friends or member events.
Whether your young man is getting ready for a day at school or drifting off to sleep at night, boys' clothing will keep him comfortable all day. Sears carries a full line of everyday boys' apparel and formal wear, so he'll look sharp no matter where he's headed. Treat any boy to a new shirt and boys' shoes for the first day of school, or consider a stylish suit for a family wedding. He'll have a blast showing off clothing that expresses his unique personality.
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text:
There's a New Kid In Town@Denver Message Board › 58 RSVP'd YES for Meetup's Holiday Party@Chloe Dec 21 2012 7pm Free Admissi
|Rima & R.
user 5332686
Group Organizer
Denver, CO
Our Motto:"This is the place where everyone is treated like you treat your best friend"
Hey gang we will be at the Chloe, for our Party inside at the Lounge by the Fireplace which is located at 1445 Market in Lodo with Free Admission on Friday Dec 21, 2012, at 7:00PM, most people are there by 7:30pm. We will greet you in the Lounge. This is a really nice place to meet some new friends. Then the party will flow into the ballroom where you can dance the night away. Normally they will give you a free hand stamp if you com to our meetup. Come one, come all, bring all your friends and a smile............
Let's make this our "Bring a friend" to our meetup. Tell all your friends on Twitter and Facebook about our meetup on Friday Dec 21, 2012 at 7PM@ Chole
What is Friendship:
It is something what is very valuable but you can't buy it. It must be protected. You have to take care of friendship because it is very easy to lose a friend but after that it is much more difficult to recover it again. It is like a treasure. You must keep it in safety. If you don't, it will not last anymore. Friendship is soft. It's someone who you get on well with. Who you like to be with and who you would miss if you lost him. It's somebody who stands by you if you have some problems, who gives you advice, who has always time for you.
Free Admission
New members are Welcomed!!!!
Free Admission Now you can't beat that on a Friday Night
Here are the event details:
*Meet N Greet begins at 7:00pm, most arrive by 7:30PM and the party ends whenever
*All night Social networking event.
*Come alone or bring all your friends
*Greeted at the door!
Chloe's Discotheque opens a select three nights a week, Thursday, Friday, and Saturday. The Discotheque is bottle service oriented with 20 coveted tables available to those who enjoy a luxurious night out. The discotheque also boasts a 22 foot-LED wall, LED lights on ceiling, and custom made sound system. Because of the limited capacity and high-demand, reservations are suggested.
This is what they are saying about Chloe:
"I definitely missed the memo -- the one that stipulated that stilettos; perfectly coiffed hair; jaw-dropping, butt-hugging and breast-spilling black dresses; and fresh-from-the-salon manicures were prerequisites for last night's VIP launch party of Chloe Mezze Lounge, a new nightclub/discotheque, lounge-restaurant and social garden (complete with its own bar and a fire pit) that now resides in the former Ruth's Chris square footage at 1445 Market Street."
"The cosmopolitan concept, the fourth from Francois Safieddine, whose Lotus Concept enterprises also include Suite 200, 24k and Oak Tavern, opens tonight -- and judging from last night's red-carpet crowd, which was nothing short of a people-watching paradise, there will be more than enough flesh to give your eyes candy for a month."
We really hope you can make it
Rima and Rodney
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text: Ora Pro Nobis by RJ Chevalier published on 2010-12-15T21:21:41Z Thought I'd bring this out of Soundcloud retirement for a bit as I wait for other tracks to near completion. Genre cinematic triphop Comment by RJ Chevalier @songshining: Wow! That is some comment. Thanks so much :))) 2015/12/04 19:20:48 +0000 Comment by Songshining I really have to say this this music gives me passion for life. best music for me than any music 2015/12/01 13:23:41 +0000 Comment by atr bwa nice... 2014/11/01 22:48:37 +0000 Comment by RJ Chevalier @weezergeezer-1: Thanks man. Appreciate it. 2014/06/15 12:03:33 +0000 Comment by Gate Eleven powerful piece.... 2014/06/14 21:31:14 +0000 Comment by RJ Chevalier @syntheticfrog: Bang indeed. Thanks for listening. 2014/03/18 22:47:25 +0000 Comment by syntheticfrog bang mmmmmm 2014/03/18 22:02:43 +0000 Comment by ZackeryLarsen1 this is a great piece of art 2014/03/11 03:21:25 +0000 Comment by RJ Chevalier Lol. Thanks man. 2014/02/01 08:39:00 +0000 Comment by Jack Frost 72 STAHP MAKIN ME CRY STAAAHHHP 2014/02/01 04:55:52 +0000 Comment by RJ Chevalier @ohdamngirl: Is this spam? 2013/07/04 14:51:12 +0000 Comment by OONNIID amazing intro!!! 2013/05/19 21:36:26 +0000 Comment by RJ Chevalier @celesital-soundwave: Thanks so much for taking a listen. I actually still like this one too. 2013/05/06 20:33:49 +0000 Comment by Celestial Soundwave so good 2013/05/06 08:16:14 +0000 Comment by xsinisax hm 2012/12/20 23:07:42 +0000 Comment by RJ Chevalier @elisabetta-vassallo: Very kind. Thank you, from London :) 2012/12/18 16:59:21 +0000 Comment by Elisabetta Vassallo amazing! from Italy 2012/12/18 11:56:24 +0000 Comment by RJ Chevalier @igor-demeter: Thanks as always Igor. This one is one of my favourites that I've created. 2012/12/08 13:28:11 +0000 Comment by mrsgcnaylor This is dubstep! I love the way you gracefully join these types of music! 2012/09/13 03:31:49 +0000 Comment by RJ Chevalier @milton-j: Cool comment, man. Thanks. 2012/08/24 17:01:32 +0000 Comment by Milton.j great sound is like walking without think in nothing thanks for keep out of reality with your music. I love it 2012/08/24 15:49:08 +0000 Comment by RJ Chevalier Thought I'd bring this out of Soundcloud retirement for a bit as I wait for other tracks to near completion. 2012/08/23 19:00:55 +0000 Comment by RJ Chevalier @alemwp: Cheers mate, I appreciate your comments. 2011/12/29 18:40:47 +0000 Comment by RJ Chevalier @vicky-lala: Thanks Vicky. 2011/12/29 18:40:30 +0000 Comment by Vicky LALA @emmazen: amazing!!! 2011/12/29 06:26:18 +0000 Comment by alemwp Wow! This is spacious and so richly powerful!! Dream music! Love the analog touches!!! So well produced!!! 2011/12/25 17:31:58 +0000 Comment by Umbrella Harbor what software do you use? I've used a few, but none have sit quite right with me. 2011/12/21 13:09:37 +0000 Comment by RJ Chevalier @justinwaylonmorgan-1: Just keep tinkering and altering things until your ears are pleased. I just guess and play around a bit with effects. 2011/12/21 13:06:22 +0000 Comment by Umbrella Harbor This is what I want my music to be, but I cant get a firm grasp on music software. 2011/12/21 13:04:17 +0000 Comment by RJ Chevalier @gonap: That's a great idea, but no doubt everyone is trying to do it. Thanks for your kind words. 2011/11/27 12:26:36 +0000


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Recent experimental and modeling results demonstrated that surviving mossy cells in the dentate gyrus play key roles in the generation of network hyperexcitability. Here we examined if mossy cells exhibit long-term plasticity in the posttraumatic, hyperexcitable dentate gyrus. Mossy cells 1 wk after fluid percussion head injury did not show alterations in their current-firing frequency (I-F) and current-membrane voltage (I-V) relationships. In spite of the unchanged I-F and I-V curves, mossy cells showed extensive modifications in Na+, K+ and h-currents, indicating the coordinated nature of these opposing modifications. Computational experiments in a realistic large-scale model of the dentate gyrus demonstrated that individually, these perturbations could significantly affect network activity. Synaptic inputs also displayed systematic, opposing modifications. Miniature excitatory postsynaptic current (EPSC) amplitudes were decreased, whereas miniature inhibitory postsynaptic current (IPSC) amplitudes were increased as expected from a homeostatic response to network hyperexcitability. In addition, opposing alterations in miniature and spontaneous synaptic event frequencies and amplitudes were observed for both EPSCs and IPSCs. Despite extensive changes in synaptic inputs, cannabinoid-mediated depolarization-induced suppression of inhibition was not altered in posttraumatic mossy cells. These data demonstrate that many intrinsic and synaptic properties of mossy cells undergo highly specific, long-term alterations after traumatic brain injury. The systematic nature of such extensive and opposing alterations suggests that single-cell properties are significantly influenced by homeostatic mechanisms in hyperexcitable circuits.
Head trauma is a major cause of epilepsy in the adult population and represents the etiology of ≤20% of epilepsy cases in the general population (Hauser et al. 1991). Although the link between head trauma and temporal lobe epilepsy is well established, the mechanisms of epileptogenesis following head injury have not been fully elucidated (Garga and Lowenstein 2006). Fluid percussion injury (FPI), a rodent model of head trauma, has been instrumental in revealing various synaptic and cellular processes that mechanistically link the traumatic event to the development of persistent limbic hyperexcitability. Importantly, the dentate gyrus in FPI exhibits many pathological hallmarks of human head trauma and temporal lobe epilepsy, including mossy fiber sprouting, hilar cell loss, and increased in vitro and in vivo hyperexcitability (Lowenstein et al. 1992; Santhakumar et al. 2000, 2001; Toth et al. 1997).
Mossy cells, excitatory neurons located in the dentate hilus, are known to be some of the most vulnerable neurons in the entire mammalian brain, and significant mossy cell loss is observed in human epileptic tissue as well as after experimental head trauma (Blumcke et al. 2000; Lowenstein et al. 1992; Toth et al. 1997). However, several lines of evidence suggest that some mossy cells survive trauma (Blumcke et al. 2000; Ratzliff et al. 2002; Toth et al. 1997) and that these surviving mossy cells may spread or amplify dentate network hyperexcitability. First, mossy cells that survive traumatic head injury have been shown to respond to perforant path stimulation with an increased number of action potentials (Santhakumar et al. 2000). Second, mossy cells and granule cells form a recurrent excitatory loop as the vast majority of mossy cell terminals synapse onto granule cell dendrites, and granule cells project to mossy cells (Wenzel et al. 1997). Not only can mossy cells spread excitability to intralamellar granule cells, but they are in a unique position to spread excitability throughout the hippocampus due to their long-distance associational and commissural projections (Blasco-Ibanez and Freund 1997; Buckmaster et al. 1996; Frotscher et al. 1991; Ratzliff et al. 2002; Ribak et al. 1985; Soltesz et al. 1993). Recent results revealed that the net physiological effect of mossy cells is to excite intralamellar and extralamellar granule cells (but see Sloviter et al. 2003) because the acute deletion of mossy cells from horizontal and longitudinal hippocampal slices leads to decreased granule cell activity (Ratzliff et al. 2004). Finally, consistent with the experimental data indicating that mossy cells contribute to granule cell hyperexcitability in an epileptic brain, a recent anatomically and physiologically realistic computational modeling study showed that the spread of granule cell hyperexcitability was decreased if mossy cells were "killed" in the model (Santhakumar et al. 2005). These data support the idea that mossy cells can spread the hyperexcitability of granule cells through the dentate network.
In this study, we found that mossy cells did not show significant changes in their I-F and I-V curves after trauma despite the presence of a hyperexcitable dentate network. Yet on closer inspection, extensive, opposing alterations were found in various membrane currents that together resulted in the unchanged I-F and I-V relationships observed in posttraumatic mossy cells. Miniature and spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) also displayed wide-ranging, but nonrandom, systematic and opposing changes. These results point to homeostatic regulatory mechanisms influencing the properties of single cells in the hyperexcitable dentate gyrus.
Mossy cell labeling and fluid percussion injury
All procedures were performed under protocols approved by the University of California Irvine Institutional Animal Care and Use Committee. Mossy cell labeling (Ratzliff et al. 2004) and preparation for lateral fluid percussion injury (FPI) head trauma (Dixon et al. 1987; Lowenstein et al. 1992; Santhakumar et al. 2001; Toth et al. 1997) were performed on juvenile (postnatal days 13–15) Wistar rats (Charles River, Wilmington, MA) in one combined surgery. Briefly, the rats were placed in a stereotaxic frame under ketamine-xylazine anesthesia. For the mossy cell labeling (Ratzliff et al. 2004), a 2 × 2-mm hole was trephined into the right parietal bone, and Hamilton syringes were used to inject 1.5 μl of 7.5% (wt/vol) fast 3,3′-dilinoleyloxacarbocyanine perchlorate in DMSO (DiO; Invitrogen, Carlsbad, CA) at two sites in the right dentate gyrus. The hole was sealed with bone wax and a plastic cap to prevent movement of the brain outside the skull during the injury. The animals were then prepared for FPI: on the left side, a second 2-mm hole was trephined to the skull at 3 mm caudal to bregma, 3.5 mm lateral from the sagittal suture. Two steel screws were placed 1 mm rostral to bregma and 1 mm caudal to lambda. A Luer-Loc syringe hub with a 2.6 mm ID was placed over the exposed dura and bonded to the skull with cyanoacrylate adhesive. Dental acrylic was poured around the injury tube and skull screws and allowed to harden. Neopredef was applied to the wound, and the animal was returned to its home cage. One day later, animals were anesthetized with halothane and attached to a fluid percussion device (Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA) where a pendulum was dropped, to deliver a brief (20 ms), 2.0–2.2 atm impact on the intact dura. This resulted in a moderate level of injury that has been shown to cause a highly reproducible pattern of ∼50% hilar cell loss (Santhakumar et al. 2000; Toth et al. 1997). For sham injury, the animals were anesthetized and attached to the fluid percussion device, but the pendulum was not dropped. The above-described technique of prelabeling ventral mossy cells via an in vivo contralateral injection of the fluorescence axonal tracer DiO into the dorsal hippocampus has previously been established (Ratzliff et al. 2004). As a control, to ensure that this method is also valid after FPI, cell counts of DiO-labeled cells in tissue stained with an antibody to GluR2/3 were performed, as strong GluR2/3 immunoreactivity has been shown to be a specific label for mossy cells in the hilus (Leranth et al. 1996; Ratzliff et al. 2004; Toth et al. 1997). Data on cell counts indicate that almost all DiO-labeled cells were GluR2/3 positive after FPI just as in control animals (supplementary material1 ). Note that the similar firing patterns between FPI and control hilar cells DiO-labeled from the contralateral side (see Fig. 1) also indicate that this technique labels a similar population of cells before and after trauma.
Slice preparation
One week (5–8 days) after the injury or sham injury (Toth et al. 1997), the rats were anesthetized with halothane and decapitated. Horizontal brain slices (350 μm) were cut using a vibratome tissue sectioner (VT1000S; Leica, Nussloch, Germany) as described previously (e.g., Ross and Soltesz 2001). The slices were sagittally bisected, and the slices from the left hemisphere (ipsilateral to the side of injury and contralateral to injection site) were submerged in 32°C oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) composed of (in mM) 126 NaCl, 2.5 KCl, 2 MgCl2, 26 NaHCO3, 2 CaCl2, 1.25 NaH2PO4, and 10 glucose, for 1–6 h. Only slices from the ventral hippocampus were used (V, −5.6 to −6.6 mm relative to bregma).
In vitro electrophysiology
The slices were transferred to the submerged recording chamber and perfused with oxygenated ACSF at 33 ± 1°C (mean) or at room temperature (for sodium currents). Drugs used in each experiment are listed in the appropriate figure legends and were purchased from Tocris (Ellisville, MO). The following pipette solutions (pH 7.2–7.25, 265–275 mOsm) were used (concentration in mM). For most experiments, unless otherwise specified: 140 K-gluconate, 2 MgCl2, and 10 HEPES; for Na currents: 140 CsCl, 10 EGTA, 10 HEPES, 2 MgCl2, and 2 ATP; for K-currents: 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl2, and 20 EGTA; for evoked EPSCs in granule cells: 140 Cs-gluconate, 2 MgCl2, and 10 HEPES; for mIPSCs and sIPSCs: 140 CsCl, 2 MgCl2, and 10 HEPES; for depolarization-induced suppression of inhibition (DSI): 140 CsCl, 2 MgCl2, 10 HEPES, 2 EGTA, 3 QX-314, 0.2 EGTA, 1 ATP, and 0.2 GTP. Whole cell recordings were performed using IR-DIC visualization techniques (Stuart et al. 1993) with a Zeiss Axioskop FS microscope, using a ×40 or ×60 water-immersion lens. For all experiments that examined firing rate, input resistance, and action potential waveforms, the cells were maintained at −60 mV with small current injections. The test pulse consisted of 500-ms current injections, except for Fig. 2B, where single action potentials were elicited with 20-ms current injections. Nucleated patches were pulled from whole cell patches, as described previously (Martina and Jonas 1997). Recordings were made using an AxoPatch 200B amplifier, filtered at 4 kHz using a Bessel filter, and digitized at either 10 or 100 kHz with a Digidata 1320A analog–digital interface (Molecular Devices, Sunnyvale, CA). To induce DSI, cells were voltage clamped at −60 mV and depolarized to 0 mV for 100 or 500 ms (Chen et al. 2003; Pitler and Alger 1992). For evoked EPSC experiments in granule cells, synaptic events were evoked with an ACSF-filled patch pipette, using an A360 stimulus isolator (WPI, Sarasota, FL). The stimulation frequency was 0.1Hz delivered with a monopolar stimulation electrode placed in the inner molecular layer (30–50 μm from the granule cell layer at a lateral distance of ≥100 μm from the recording site). Stimulus intensities were adjusted to 30–50% of the maximal response, typically between 100 μΑ and 1 mA. Series resistance was monitored periodically (initial series resistances ranged from 8 to 12 MΩ), and recordings were rejected if it changed by >15%.
Measurement of action potential threshold
Three methods were used to detect the AP threshold. The first method, "change in dV/dt" is to take the dV/dt of the voltage trace, and take the SD of the dV/dt for 50 ms before the AP. The threshold is reached once the dV/dt reaches: mean(dV/dt) +2*SD(dV/dt) (Atherton and Bevan 2005). The second method, "dV/dtt threshold" is to set a cutoff point of the dV/dt, such as 30 mV/ms, as the AP threshold (Fig. 2A, dotted line) (Cooper et al. 2003; Metz et al. 2005). The final method, "maximum of second derivative" is to take the maximum of the second derivative of the voltage trace with respect to time (Fig. 2A, gray line) (Mainen et al. 1995).
Data analysis
Synaptic currents were analyzed for frequency, amplitude, rise time, and τ decay using MiniAnalysis 6.0 (Synaptosoft, Decatur, GA). DSI was analyzed with a custom-written software program that measures the synaptic charge transfer after the depolarization as a percentage of baseline charge transfer for 3 s before the depolarization. Analysis of action potential waveforms and threshold was performed with scripts written in Matlab 7.0 (The MathWorks, Natick, MA). Statistics were performed using either Student's t-test or the Kolmogorov-Smirnov test, with significance set at P < 0.05. N refers to number of cells (for physiology) or number of animals (cell counts). Sodium channel permeability was calculated from the Goldman-Hodgkin-Katz current equation PNa = INa*(RT)/(z2EF2)*[1 − exp(−zFV/RT)]/{[Na]i − [Na]o[exp(−zFV/RT)]}, (Martina and Jonas 1997). The sodium channel activation curve was fitted with a Boltzmann function raised to the third power: f = 1/{1 + exp[−(V − V)/k]}3, and the steady-state inactivation curve was fitted with a simple Boltzmann function f = 1/{1 + exp[−(V − V)/k]} (Martina and Jonas 1997).
Computational modeling
The model network was implemented using the NEURON 5.6 simulation environment (Hines and Carnevale 1997). The large-scale, anatomically and biophysically realistic model network used in this study was identical to the one described previously (Dyhrfjeld-Johnsen et al. 2006). Briefly, the multi-compartmental single-cell models were taken from the 500-cell network described in Santhakumar et al. (2005) and were incorporated into a network including 50,000 granule, 1,500 mossy, 500 basket, and 600 HIPP cells, i.e., in biologically realistic proportions as described in Dyhrfjeld-Johnsen et al. (2006). The distribution and synaptic conductances were based on those in Santhakumar et al. (2005) and were scaled to reflect the larger number of cells (Dyhrfjeld-Johnsen et al. 2006). The parameters used in the model were based on available physiological and anatomical data from our lab and from the literature (for details, see Dyhrfjeld-Johnsen et al. 2006; Santhakumar et al. 2005). Perforant path stimulation was simulated by a single synaptic input to 5,000 granule cells, 10 mossy cells, and 50 basket cells (situated in the middle lamella of the model network) at t = 5 ms after the start of the simulation. For the "FPI only" network, strictly identical to the "50% sclerosis" functional model network in Dyhrfjeld-Johnsen et al. (2006), mossy fiber sprouting and hilar cell loss were added to the model. Mossy fiber sprouting was modeled by adding synaptic connections from granule cells to the proximal dendrites of granule cells. Because FPI produces moderate mossy fiber sprouting, the degree of mossy fiber sprouting in the model was set at 50% of the maximal sprouting observed in the pilocarpine model of epilepsy (Buckmaster et al. 2002), which exhibits a very high-density of sprouting. Hilar cell loss was modeled by randomly removing 50% of hilar interneurons (HIPP cells) and 50% of mossy cells, which is the approximate degree of cell loss observed after FPI in our hands (Toth et al. 1997). Additional details of the model can be found in Dyhrfjeld-Johnsen et al. (2006).
Basic input-output curves of mossy cells are unchanged after injury
To examine whether mossy cells exhibit any major changes in their intrinsic properties, the mean firing rate was measured in response to 500-ms depolarizing pulses, when cells were held at −60 mV (representative traces in Fig. 1A). As shown in Fig. 1B, there was a trend toward increased firing rates in FPI cells, but this increase was not significant despite a large number of cells in both groups (control: n = 20; FPI: n = 20). Several additional parameters of the firing patterns were measured to explore whether more subtle alterations occurred. There were no differences between FPI and control mossy cells in the mean interspike interval, the first interspike interval, the adaptation of the interspike interval, or the latency to the first spike (Fig. 1, C–E). Next, the current-voltage relationship of mossy cells was measured in response to 500-ms hyperpolarizing pulses when cells were held at −60 mV. The responses of FPI cells to these pulses were not different from the responses of control cells (measurements taken at the end of the pulse; control: n = 20; FPI: n = 20; Fig. 1F). Accordingly, the apparent input resistance around and below −60 mV was not changed between control and FPI cells at any current amplitude tested. However, the resting membrane potential (Vm) of mossy cells was significantly depolarized after FPI (Vm in control: −66.6 ± 1.4 mV, n = 20; in FPI: −63.2 ± 0.8 mV, n = 20; Fig. 1F, inset). Therefore after FPI, mossy cells have maintained their target firing rate and input resistance. This result suggests two possible interpretations. It is possible that the intrinsic properties of mossy cells simply have not changed. Alternatively, several intrinsic properties have may have changed concurrently, in a coordinated manner, resulting in the unaltered I-F and I-V relationships.
Alterations in action potential threshold and waveform
To explore whether subtle changes occur to mossy cells' intrinsic properties after FPI that cannot be detected in the current-voltage or current-firing relationships, several parameters of the mossy cell action potential (AP) in control and FPI mossy cells were measured. AP threshold can be regulated in an activity-dependent manner (Henze and Buzsaki 2001), and an altered AP threshold has been predicted in a genetic form of epilepsy (Spampanato et al. 2004). As there is no single accepted method of determining the AP threshold (Sekerli et al. 2004), three different methods were used to calculate this value (see methods for detailed explanation and Fig. 2A for illustration). The AP threshold of mossy cells was significantly depolarized after FPI, when single APs were evoked with 20-ms current pulses (Fig. 2B; change in 1st derivative: control = −34.7 ± 0.3 mV, FPI = −32.8 ± 0.4 mV; 1st derivative threshold: control = −34.8 ± 0.3 mV, FPI = −32.8 ± 0.3 mV; maximum of 2nd derivative: control = −32.1 ± 0.3 mV, FPI = −30.6 ± 0.7 mV; control, n = 12; FPI, n = 19). Note that both the change in first derivative and the first derivative threshold measures gave nearly identical values. The AP threshold was also depolarized after FPI when a train of APs was evoked by a 500-ms, 200-pA current injection, for AP numbers 2–10 (Fig. 2C; control: n = 7, FPI: n = 16).
In theory, if the AP threshold was depolarized without additional concurrent changes, mossy cells should fire fewer APs after FPI in response to the same amount of current injection, and yet the firing rates remain similar to control values (Fig. 1B). An increase in input resistance could offset the depolarized AP threshold and allow the FPI cells to fire at the same rate as the control cells. Therefore the positive current injection experiments were repeated in the presence of TTX to block APs. The FPI cells responded to positive current injections with larger voltage deflections than did the control cells (significant increase from 40 to 320 pA; n = 10 in control, n = 9 in FPI; Fig. 2D). The input resistance of mossy cells was increased enough after FPI to counteract the observed shift in AP threshold (input resistance in response to 120 pA current injection in control: 125.8 ± 7.1 MΩ; in FPI: 154.6 ± 4.8 MΩ) because the membrane potential reached in response to a 240-pA current injection increased by 5 mV, whereas the AP threshold only was depolarized by 1.5–2.0 mV (voltage resulting from 240-pA injection: control: −34.5 ± 1.4 mV; FPI: −29.5 ± 1.0 mV).
Because differences in a cell's AP waveform can impact neurotransmitter release from its boutons, the AP waveform was closely examined in mossy cells after FPI (Fig. 3A). There was no difference in AP height between control and FPI mossy cells (Fig. 3B). In the same cells, the AP width was analyzed at one-half of the maximum height. Although there was a trend for APs from FPI cells to be wider, the difference was not significant (Fig. 3C). However, in FPI mossy cells, there was a significant increase in the AP width at one-third of maximum height (Kamal et al. 2006) (Fig. 3D). This difference suggests that the widening of the AP after FPI only occurs at the later phases of the AP.
Therefore when examining the detailed intrinsic properties of mossy cells after FPI, the following alterations were seen: a depolarized AP threshold, an increased input resistance around AP threshold, and an increased AP width. Next, the intrinsic conductances of mossy cells were examined to find the mechanisms underlying these alterations.
Shift in activation curve of sodium current
To uncover the origin of the depolarized AP threshold observed after FPI (Fig. 2, B and C), the fast sodium current (INa), which contributes to the action potential threshold, was measured. To obtain accurate voltage-clamp measurements of sodium currents, nucleated outside-out patches (Martina and Jonas 1997) were obtained from mossy cells. To measure the activation curve, patches were held at −90 mV, with a 50-ms prepulse to −120 mV, and 20-ms test pulses to command potentials between −80 and +40 mV (representative traces shown in Fig. 4A) were applied. The capacitance of the patches was not different between control and FPI cells (capacitance in control: 2.4 ± 0.3 pF, n = 8, in FPI: 2.3 ± 0.6 pF, n = 4). The maximum current amplitude was also similar between control and FPI (maximum amplitude in control: 0.39 ± 0.08 mV; in FPI: 0.32 ± 0.04 mV, current-voltage dependence of INa shown in Fig. 4B). After FPI, the voltage of half-maximal activation of INa was significantly depolarized (V1/2 in control: −31.1 ± 1.17 mV; in FPI: −25.3 ± 1.28, P < 0.05; Fig. 4C). Therefore after FPI, the activation curve of INa is shifted by 5.8 mV in the positive direction, which is consistent with the observed depolarization of the AP threshold.
Downregulation of a delayed-rectifier potassium current
A downregulated potassium current could lead to the wider AP observed in mossy cells after FPI because potassium currents contribute to the repolarizing phase of the AP. Three potassium currents were isolated in control and FPI mossy cells: an A-type current (IA) blocked by 3 mM of 4-aminopyridine (4-AP), a delayed-rectifier blocked by 25 mM of TEA (ITEA), and a residual current insensitive to both 4-AP and TEA (IRES; representative traces shown in Fig. 5A). The amplitude of ITEA was decreased in FPI cells at test potentials from −30 to 0 mV (at 0 mV: 1,876 ± 155 pA in control, n = 11; 1,329 ± 171 pA in FPI, n = 10; Fig. 5B). However, the activation curve of this current was not shifted (Fig. 5B, inset). The decrease in amplitude was specific to ITEA, as the amplitudes of IA and of IRES were similar between control and FPI cells (amplitude of IA at 0 mV—in control: 2,264.1 ± 158 pA, n = 14; in FPI: 2,392 ± 184 pA, n = 12; amplitude of IRES at 0 mV—in control: 2,152 ± 258 pA, n = 9; in FPI: 2,305 ± 168 pA, n = 10; Fig. 5, C and D).
To determine whether the downregulation of ITEA could lead to the observed broadening of APs, this current was pharmacologically decreased in control mossy cells by adding a low concentration (2.5 mM) of TEA to the bath and measuring AP width before and after the addition of the drug. The results show a significant widening of the AP with the addition of TEA (representative traces in Fig. 6A, summary plot in B; n = 3). This effect is similar to the broadening seen after FPI (compare Fig. 6B with Fig. 3D). The addition of 2.5 mM TEA to control mossy cells did not affect their AP height (Fig. 6C). Because this current is slowly inactivating, the decreased ITEA could contribute to the increased input resistance shown in Fig. 2D at voltages above −30 mV. Thus a delayed-rectifier potassium current is downregulated in mossy cells after FPI, and this may be a cause of the observed AP broadening.
Functional effects of decreased potassium current expression
Does a wider AP have an effect downstream of the mossy cell? Previous studies have shown that a wider somatic AP can lead to increased calcium entry in the bouton and a corresponding increase in transmitter release (Bollmann and Sakmann 2005; Borst and Sakmann 1999; Geiger and Jonas 2000). To examine whether a wider AP in mossy cells would lead to increased glutamate release onto postsynaptic granule cells, whole cell recordings were made from dentate granule cells in slices from control animals, and EPSCs were evoked by low-intensity stimulation in the inner molecular layer. As mossy cell axons are the only glutamatergic fibers in this area in control animals, these evoked EPSCs originate from mossy cell axons. TEA (2.5 mM) was then added to the bath, the concentration that leads to an AP width in control mossy cells similar to the AP width in FPI cells (Figs. 3D and 6B). Note that granule cells were recorded with a cesium-containing intracellular solution to block potassium channels to rule out postsynaptic effects of TEA. The granule cell EPSC amplitude increased significantly within 5 min of TEA addition (Fig. 6D: increase in amplitude: 51.5 ± 13%, n = 5). Therefore the decreased expression of a TEA-sensitive delayed rectifier (in the absence of other compensatory alterations at the synapse; see discussion) is expected to lead to a significantly increased mossy cell to granule cell EPSC.
Computational modeling of modifications to intrinsic properties of mossy cells
So far we have uncovered several alterations in mossy cell physiology. Could these alterations, if they occurred separately, significantly affect the excitability of the dentate network? Experimentally, it is difficult to separate the effects of isolated posttraumatic changes in mossy cells on the dentate network. Therefore computational modeling studies were performed to examine whether any of the altered parameters seen in mossy cells after FPI could modify network activity. The simulations were run on a 52,100-cell anatomically and physiologically realistic model of the dentate gyrus (Dyhrfjeld-Johnsen et al. 2006). The excitability of the network was tested with a single perforant path stimulation (see methods). Reflecting the behavior of the biological dentate (Ratzliff et al. 2004; Santhakumar et al. 2001, 2005), the simulated "control" network never spread activity to the entire network (data not shown) (see Dyhrfjeld-Johnsen et al. 2006), and the firing rate of granule and mossy cells was low (average granule cell firing: 0.2 Hz; mossy cell firing: 0.3 Hz). However, because mossy cells after FPI exist in an altered anatomical environment, simulated mossy fiber sprouting and hilar cell loss were added, to create an "FPI-only" network (i.e., without intrinsic changes; see methods). With these anatomical changes added, the same single stimulation led to the activation of the entire network (latency to full network activation in FPI-only network: 99.3 ± 2.0 ms, n = 3 trials; Fig. 7, A and G). In addition, the average granule cell and mossy cell firing rates over the entire 500-ms simulation were significantly increased (granule cell firing: 28.5 ± 0.6 Hz; mossy cell firing: 7.5 ± 0.1 Hz; n = 3 trials, Fig. 7, A, E, and F). The remaining simulations, which tested the effect of the altered intrinsic properties of mossy cells after FPI, were performed by modifying the physiological parameters of this FPI-only network. Specifically, we tested the effects of the depolarized Vm, altered mossy cell to granule cell EPSC resulting from the increased glutamate release due to the downregulation of ITEA and the depolarizing shift in the INa activation curve.
First, we tested whether the significant shift of Vm shown in Fig. 1F could lead to increased firing in response to the same perforant path stimulation. When a 3-mV shift in Vm was added to the mossy cells in the network, the network's excitability increased significantly (FPI plus 3-mV shift of MC Vm: latency to full network activity: 39.9 ± 0.49 ms; granule cell firing: 65.1 ± 0.5 Hz; mossy cell firing: 25.9 ± 0.2 Hz; Fig. 7, B and E–G; n = 3 trials).
Second, we tested the effect of the downregulation of ITEA. Because the decrease of this conductance was shown to increase the AP width and therefore also increase the amplitude of the mossy cell to granule cell EPSC, we modeled this change by increasing the synaptic weight of the mossy cell to granule cell EPSC by 50% (from 0.3 to 0.45 nS), corresponding to the experimental data shown previously (Fig. 6D). Due to the recurrent excitatory loop formed by granule cells and mossy cells, we predicted that the increased mossy cell to granule cell EPSC caused by the increased AP width of mossy cells could have significant effects on network behavior. Accordingly, with the increased EPSC size incorporated into the network, the spread of activity through the dentate network occurred significantly faster than the FPI-only network after perforant path stimulation (FPI plus increased MC-GC synaptic weight: latency to full network activity: 74.7 ± 0.09 ms; n = 3 trials; Fig. 7, C and G). Additionally, both granule cells and mossy cells fired significantly more (granule cell firing: 45.8 ± 0.3 Hz; mossy cell firing: 12.3 ± 0.1 Hz; Fig. 7, C, E, and F).
Finally, a 5-mV shift in the sodium current activation curve was added to the mossy cells in the FPI-only dentate model. Predictably, the mossy cells' firing rate decreased significantly (FPI plus shift in activation of INa: mossy cell firing: 0.003 ± 0.0001 Hz; n = 3 trials; Fig. 7, D and F). The sodium current activation shift also led to a significantly lower granule cell firing rate and slower activation of the full network (FPI plus shift in activation of INa: granule cell firing: 16.3 ± 0.03 Hz; latency to full network activity: 175.4 ± 0.9 ms; Fig. 7, D, E, and G).
In summary, experimental data showed that despite the fact that the firing patterns were not different between control and FPI mossy cells, the Vm was depolarized, a potassium conductance was decreased, and the sodium current's activation curve was shifted. Computational modeling indicated that each of these isolated perturbations can lead to significant alterations in dentate network activity. Note that because several additional known and unknown factors (see following text) may have changed in these posttraumatic mossy cells, it is currently not possible to test the net combined effect of all posttraumatic alterations in mossy cells on the simulated dentate network. Thus the only purpose of the modeling experiments was to show that, individually, subtle changes in the physiology of one cell type can lead to significant changes in network activity.
Increase in the effect of the Ih blocker ZD7288 after FPI
Because the current-firing plots hid several, mutually opposing (i.e., increased Vm, increased input resistance around AP threshold, depolarizing shift in INa, and a downregulated delayed rectifier potassium current) alterations in mossy cells after FPI, it is possible that the current-voltage relationships at and around −60 mV also concealed some interesting changes. As mentioned in the preceding text, mossy cells showed a significantly depolarized Vm after FPI (Vm in control: −66.6 ± 1.4 mV, n = 20; in FPI: −63.2 ± 0.8 mV, n = 20; Fig. 8A, left). One possible cause of a depolarized Vm is an increase in the hyperpolarization-activated cation current, Ih (Beaumont and Zucker 2000; Chen et al. 2001; Maccaferri and McBain 1996; Pape 1996; Santoro and Tibbs 1999; Siegelbaum 2000). In agreement with this possibility, when mossy cells were incubated in the Ih blocker ZD7288 (ZD; 10 μM), Vm was hyperpolarized in both control and FPI mossy cells, and the difference between the two groups was abolished (control Vm in ZD: −70.0 ± 2.3 mV, n = 16; in FPI: −69.0 ± 1.6 mV, n = 19; Fig. 8A). To further examine whether Ih might be upregulated, we examined the effects of Ih on voltage changes during and after a hyperpolarizing current pulse: a slow afterdepolarization due to deactivation of the current after the pulse and a slow depolarizing sag due to activation of the current during the pulse (Fig. 8B, inset). Both the afterdepolarization and the sag were increased in mossy cells after FPI (Fig. 8, B and C; control: n = 20; FPI: n = 20). To demonstrate that Ih is the current underlying the afterdepolarization and the sag in mossy cells, these experiments were repeated after incubation of the slices in 10 μM ZD, which allows for a complete block of Ih. The afterdepolarization was nearly abolished when Ih was blocked, and there were no differences between control and FPI cells in the presence of ZD (Fig. 8D1; control: n = 12; FPI: n = 13), and the sag was completely abolished in ZD (Fig. 8D2).
If Ih is the only conductance that has been altered in this voltage range, the input resistance of mossy cells should be decreased. However, as shown in Fig. 1F, the input resistance at around and below −60 mV is similar between control and FPI mossy cells. This would appear to contradict our suggestion that Ih is upregulated. To investigate this apparent discrepancy, the input resistance of individual mossy cells was measured in control ACSF and after a switch of the perfusate to ACSF containing a high concentration (100 μM) of ZD. The input resistance was larger in FPI mossy cells at most current amplitudes after the switch to the ZD-containing solution, indicating that blockade of Ih reveals a difference in input resistance (Fig. 8E; control: n = 9; FPI: n = 10). Additionally, the change in input resistance caused by the perfusion of ZD was significantly greater in FPI at most current amplitudes (Fig. 8F) (Shah et al. 2004). In light of the unchanged input resistance at hyperpolarized voltages (Fig. 1F), these data suggest that there are two opposing forces at work: an increased Ih to decrease the input resistance and the decrease of one or more unknown conductances, such as a leak potassium conductance, to increase the input resistance back to control values.
Alterations of synaptic inputs to mossy cells after FPI
Do the properties of synaptic inputs to mossy cells change after head injury, and what are the common features of these posttraumatic synaptic alterations? Modifications to synaptic properties of other cell types have been shown to occur in the dentate gyrus after FPI (Santhakumar et al. 2000, 2001; Toth et al. 1997). Specifically, the frequency of mIPSCs (AP-independent events recorded in the presence of TTX) in granule cells is decreased after FPI and the frequency of sIPSCs (recorded in the absence of TTX) is increased in granule cells after FPI. Although alternative interpretations are possible, the most parsimonious explanation for the decreased mIPSC frequency and the increased sIPSC frequency (Santhakumar et al. 2001; Toth et al. 1997) is that the death of hilar interneurons leads to fewer GABAergic boutons available for release, yet the surviving interneurons fire more frequently as has been experimentally demonstrated (Ross and Soltesz 2000). The population of inhibitory cells that innervates granule cells is thought to be similar to the population that innervates mossy cells (Acsady et al. 2000). Therefore it was expected that the changes in frequency of mIPSCs and sIPSCs would parallel those seen in granule cells. Indeed, when mIPSCs were compared from control and FPI mossy cells, the following parameters were changed significantly: the interevent interval was increased (in control: 110.4 ± 2.0 ms, n = 13; in FPI: 181.5 ± 4.2 ms, n = 10; Fig. 9C), the amplitude was increased (in control: 59.0 ± 1.0 pA; in FPI: 65.6 ± 1.3 pA; Fig. 9E), and the 10–90% rise time was shortened (in control: 0.87 ± 0.01 ms; in FPI: 0.82 ± 0.01 ms; Fig. 9G). When sIPSCs were recorded, the same three properties were changed significantly after FPI but in the opposite direction. The interevent interval of sIPSCs was decreased (in control: 128.4 ± 6.5 ms, n = 14; in FPI: 103.8 ± 2.8 ms, n = 12; Fig. 9D), the amplitude was decreased (in control: 127.5 ± 3.3 pA; in FPI: 97.6 ± 2.7 pA; Fig. 9F), and the 10–90% rise time was lengthened significantly (in control: 0.72 ± 0.01 ms; in FPI: 0.75 ± 0.02 ms; Fig. 9H). Note that the significant difference between interevent intervals of sIPSCs is due to the presence of very large events in control but not FPI mossy cells. The decay time constant was not changed for either mIPSCs (in control: 7.12 ± 0.3 ms; in FPI 7.6 ± 0.2 ms) or sIPSCs (in control 7.42 ± 0.2 ms; in FPI 7.53 ± 0.3 ms).
Excitatory inputs to mossy cells include those from mossy fibers (granule cell axons), those from other mossy cells, and those from CA3 pyramidal cells (Scharfman 1994). After FPI, and in many other models of hyperexcitability, mossy fibers sprout (Cavazos et al. 1991; Cronin et al. 1992; Okazaki et al. 1995; Santhakumar et al. 2001) and mossy cell axons are lost due to death of mossy cells (∼50% after moderate FPI) (Toth et al. 1997). Therefore it is reasonable to suppose that glutamatergic synaptic events might also change in mossy cells after FPI. Recordings of excitatory synaptic events were made in the presence of picrotoxin to block fast GABAA-mediated inhibitory events. The following significant changes were seen in mEPSC properties after FPI: the interevent interval of mEPSCs was increased (in control: 202.7 ± 6.0 ms, n = 10; in FPI: 416 ± 5.3 ms, n = 9; Fig. 10C), the amplitude was decreased (in control: 71.4 ± 1.6 pA; in FPI: 50.5 ± 1.6 pA; Fig. 10E), and the 10–90% rise time was shortened (in control: 0.83±.0.1 ms; in FPI: 0.77 ± 0.01 ms; Fig. 10G). As was the case with the inhibitory events, the direction of the changes were reversed between mEPSCs and sEPSCs and accordingly the following three significant alterations were seen in sEPSCs after FPI: the interevent interval was decreased (in control: 118.5 ± 3.6 ms, n = 12; in FPI: 70.4 ± 1.3 ms, n = 12; Fig. 10D), the amplitude was increased (in control: 77.8 ± 1.5 pA; in FPI: 116.5 ± 4.3 pA; Fig. 10F), and the 10–90% rise time was decreased (in control: 0.78 ± 0.01 ms; in FPI: 0.83 ± 0.01 ms; Fig. 10H). There were no changes in the decay time constant for either mEPSCs (in control: 4.32 ± 0.2 ms; in FPI: 3.89 ± 0.3 ms) or sEPSCs (in control: 4.34 ± 0.2 ms; in FPI: 4.26 ± 0.2 ms). In summary, synaptic events in posttraumatic mossy cells are significantly modified in an apparently highly systematic manner as evidenced, for example, by the alterations in miniature events that are invariably opposite to those seen in spontaneous events.
Endocannabinoid signaling in mossy cells is present but unchanged after FPI
As noted in the preceding text, opposing alterations of m- and sIPSCs occur in mossy cells after FPI. In light of these wide-spread but apparently highly specific changes in synaptic inputs, we next tested if the short-term plasticity of sIPSCs also underwent significant alterations after FPI or if they stayed the same in spite of the altered m- and sIPSC properties. Although the exact source of sIPSCs is unknown, many of these events originate from perisomatically projecting interneurons, such as basket cells expressing the cannabinoid type-1 (CB1) receptors, which are known to project to mossy cells (Acsady et al. 2000). Cannabinoids can modulate inhibitory synapses through a process termed DSI (Kreitzer and Regehr 2001; Llano et al. 1991; Ohno-Shosaku et al. 2001; Pitler and Alger 1992; Wilson and Nicoll 2001). DSI takes place when the postsynaptic cell is depolarized and releases endocannabinoids that are thought to diffuse to the presynaptic membrane. These cannabinoids bind to the CB1 receptor, and transiently suppress GABA release via the inhibition of voltage-gated Ca2+ channels. Recently, the cannabinoid signaling system has been shown to exhibit long-term plasticity in different models of epilepsy (Bernard et al. 2005; Chen et al. 2003; Wallace et al. 2003), and cannabinoids have been shown to have neuroprotective and antiepileptic effects (Bernard et al. 2005; Blair et al. 2006; Marsicano et al. 2003; Shafaroodi et al. 2004). Therefore to determine whether DSI undergoes alterations after trauma, mossy cells were examined for the presence of DSI in the control condition and for persistent plasticity of this cannabinoid-mediated signaling after FPI.
Because DSI has been demonstrated in mossy cells only recently (Hofmann et al. 2006), we first carried out experiments to characterize DSI in these neurons under our conditions. On depolarization of control mossy cells from −60 to 0 mV for 100 or 500 ms, a transient decrease in the sIPSC charge transfer was observed (Fig. 11, A, C, E, and F: percentage baseline charge transfer for 3-s postdepolarization, with respect to the prepulse period, in response to a 100-ms pulse: 79.9 ± 6.2%, n = 15; in response to a 500-ms pulse: 69.4 ± 8.4%, n = 16). The decrease in charge transfer was abolished in the presence of the CB1 receptor antagonist AM251, indicating that this plasticity is endocannabinoid-mediated DSI (Fig. 11, C, E, and F). Additionally, DSI in mossy cells was similar to DSI recorded in CA1 pyramidal cells from control animals under the same conditions (percentage baseline charge transfer for 3-s postdepolarization with a 100-ms pulse: 72.9 ± 5.4%, n = 7; with a 500-ms pulse: 68.7 ± 6.9%, n = 5), indicating that cannabinoids have comparable effects on the GABAergic inputs arising from CB1-containing interneurons to both mossy cells and CA1 pyramidal cells.
Next, DSI in mossy cells was examined after FPI. The mean amplitude of DSI was not changed after FPI (Fig. 11, B and D–F: percentage of baseline charge transfer for 3-s postdepolarization with a 100-ms pulse: 81.2 ± 5.1%, n = 17; 500-ms pulse: 73.4 ± 7.2%, n = 16). Additionally, there was no alteration in the percentage of cells expressing DSI (percentage of DSI-expressing cells in control: 66.7% of 15 cells for 100 ms, 68.75% of 16 cells for 500 ms; in FPI: 64.7% of 17 cells for 100 ms, and 62.5% of 16 cells for 500 ms). DSI in FPI mossy cells was also completely blocked in the presence of AM251 (Fig. 11, D–F). In summary, mossy cells exhibit cannabinoid-mediated inhibition of spontaneous inhibitory events, but this form of short-term plasticity is not changed after head trauma, in spite of the extensive alterations in intrinsic and synaptic properties to posttraumatic mossy cells.
In this paper, we examined the plasticity of intrinsic and synaptic properties in single mossy cells after traumatic brain injury. The experiments were performed 1 wk after head injury because previous research has established the presence of robust hyperexcitability at this time point (Santhakumar et al. 2001; Toth et al. 1997) and also because this time point is after the immediate posttraumatic period characterized by hilar cell loss and transient changes in single-cell properties (Ross and Soltesz 2000; Toth et al. 1997). Our current data show that mossy cells display a large number of significant alterations in their properties after traumatic brain injury. The individual alterations take place in an apparently coordinated manner that suggests that certain regulatory mechanisms are likely to underlie the observed intrinsic and synaptic modifications. Concerning the intrinsic properties, we found extensive modifications in various ion channels that occur without changes in either I-F or I-V curves. In the depolarizing direction, where there is no change in the I-F relationship (Fig. 1B), a depolarized AP threshold, due to the depolarizing shift in INa, is apparently opposed by a depolarized Vm and an increased input resistance. In the hyperpolarizing direction, where there is no change in the I-V relationship (Fig. 1F), an increase in Ih is apparently opposed by the downregulation of an unidentified conductance. Regarding the synaptic events, the presence of increased sEPSC frequency and amplitude indicate that the mossy cells experienced sustained increases in incoming excitation in the posttraumatic dentate gyrus. Furthermore, the identified posttraumatic alterations in mEPSC and mIPSC amplitudes are exactly as could be predicted from culture experiments on homeostatic responses after the artificial elevation of network activity (Turrigiano and Nelson 2004; Turrigiano et al. 1998). Similarly, the decreased frequency of miniature IPSCs and EPSCs is apparently opposed by an increased frequency of spontaneous I/EPSCs. In the subsequent sections, we first discuss how modifications in intrinsic and synaptic properties in mossy cells described in this paper may play a role in dentate network hyperexcitability, and we integrate these new findings in the context of current theories of mossy cell functions in seizure-propagation. Second, we discuss how the current data on intrinsic and synaptic changes in posttraumatic mossy cells can be considered as evidence for homeostatic regulatory mechanisms shaping the plasticity of single-cell properties in hyperexcitable networks.
Role of surviving mossy cells in the hyperexcitable dentate gyrus
Mossy cells are one of the most vulnerable cell types in the mammalian brain. Indeed, mossy cells are lost under a variety of pathological conditions, including epilepsy, trauma and ischemia (Buckmaster and Jongen-Relo 1999; Ratzliff et al. 2002; Sutula et al. 2003). The precise nature of the contributions of mossy cells to hyperexcitability in the dentate gyrus is not fully understood. One theory (the dormant basket cell hypothesis) (Sloviter et al. 2003) focused on the effects of mossy cell loss, arguing that the loss of mossy cells deprives GABAergic interneurons of their excitatory input, resulting in a hyperexcitable dentate gyrus through the hypoexcitation of basket cells and hypoinhibition of granule cells. An alternative theory (the irritable mossy cell hypothesis) (Ratzliff et al. 2002; Shantakumar et al. 2000) proposed that mossy cell loss itself leads to hypoexcitability in granule cells, and that it is the surviving mossy cells that play key roles in seizure propagation. The basic tenet of the irritable mossy cell hypothesis, i.e., that mossy cells contribute an overall pro-excitatory influence onto granule cells, is supported by single-cell deletion data from both experiments and computational network models showing that the removal of mossy cells invariably leads to decreased granule cell excitability (Ratzliff et al. 2004; Santakumar et al. 2005). According to the irritable mossy cell hypothesis, mossy cells either passively distribute granule cell hyperexcitability arising from other pro-excitatory modifications such as mossy fiber sprouting or they are intrinsically hyperexcitable, in which case they could actively amplify incoming excitatory signals. Therefore it is important from this respect that our current data demonstrate that mossy cell intrinsic cellular excitability does not exhibit significant alterations after trauma as determined by the unchanged I-F and I-V curves. Although these results may be at first regarded as evidence that mossy cells do not actively amplify incoming excitation, several caveats should be noted. First, it is possible that EPSPs generated on dendrites may be differentially amplified after trauma because our current data indicate significant alterations in Ih in posttraumatic mossy cells, and seizure-induced changes in Ih have been shown to exert major effects on the processing of both inhibitory and excitatory synaptic inputs (Chen et al. 2001; Shah et al. 2004). Second, posttraumatic mossy cells may amplify incoming signals by translating each action potential to larger EPSCs in their postsynaptic target cells due to the wider action potentials resulting from the downregulated delayed-rectifier potassium current. Although our results show that a decreased delayed rectifier current enhances the mossy cell to granule cell EPSCs, it is possible that additional compensatory mechanisms may have taken place at the mossy cell to granule cell synapse. The mossy cell to granule cell EPSC amplitude, and the effect of TEA on these EPSCs were not measured from FPI animals because stimulation of the inner molecular layer after trauma evokes responses from both the sprouted mossy fibers and mossy cell axons. Therefore we cannot rule out the possibility that counteracting mechanisms have taken place to decrease the amplitude of the mossy cell to granule cell synapse to adjust for the increased AP width in mossy cells. For example, calcium channels could be downregulated in mossy cell boutons to decrease transmitter release presynaptically or AMPA receptors could be removed from the granule cell membrane to decrease the EPSC amplitude postsynaptically. Thus the absence of alterations in I-F and I-V curves cannot be considered as final evidence against the idea that mossy cells actively contribute to dentate hyperexcitability. In any case, however, it should be emphasized that it has already been established using computational modeling techniques that surviving mossy cells play critical roles in long-range synchronization of the dentate gyrus even without changes in their intrinsic properties (Dyhrfjeld-Johnsen et al. 2006; Santhakumar et al. 2005). Indeed in the hyperexcitable FPI-only baseline model used in this paper, the intrinsic and synaptic properties of mossy cells were exactly as in control networks that do not demonstrate hyperexcitability, and the removal of these mossy cells clearly decreases the perforant path-evoked hyperexcitable responses in granule cells (Dyhrfjeld-Johnsen et al. 2006; Santhakumar et al. 2005), indicating their overall pro-excitatory network effects in dentate hyperexcitability.
Does homeostasis occur in mossy cells after trauma?
Recent studies have shown that neurons demonstrate the ability to powerfully regulate themselves to maintain a target level of activity or excitability through homeostatic regulation of synaptic and intrinsic cellular parameters (Desai et al. 1999; MacLean et al. 2005; Niven et al. 2003; Pulver et al. 2005; Turrigiano and Nelson 2004). In this context, homeostatic plasticity refers to changes in synaptic or intrinsic properties that allow a neuron to maintain its firing within a target range. Under healthy normal conditions, both synaptic scaling and regulation of intrinsic conductances have been demonstrated to be involved in homeostatic plasticity (Lissin et al. 1998; O'Brien et al. 1998; Turrigiano et al. 1998; Watt et al. 2000; Wierenga et al. 2005). The key observations from such studies on homeostatic responses that are especially relevant to our situation are that after induction of increased network activity, intrinsic excitability and mEPSC amplitude decrease (Turrigiano et al. 1998), whereas mIPSC amplitude should theoretically increase (Kilman et al. 2002; Turrigiano and Nelson 2004). The overall interpretation of these results is that cells respond to increased incoming excitation by decreasing their intrinsic excitability and the postsynaptic responses to glutamate, and increase their postsynaptic responses to GABA, in an apparent attempt to restore their original firing rates.
The existence of hyperexcitable states, such as epilepsy, clearly shows that networks under pathological conditions can persistently stay outside of the normal, "target" range of activity. This presents an interesting paradox: why do some changes in activity levels lead to neuronal homeostasis, and yet others lead to pathology? There are least three distinct possible solutions to this paradox. First, it is possible that in hyperexcitable states, homeostatic mechanisms have not been enlisted. Because the triggers for homeostatic plasticity have not been fully characterized, it is possible that the pathological changes occurring in the hyperexcitable brain do not elicit the homeostatic response. For example, although significant advances are beginning to emerge (Desai et al. 2002; Echegoyen et al. 2005), little is known about the presence of homeostatic regulation in adult mammalian systems in vivo. A second possible solution to the paradox is that homeostatic plasticity has been engaged in hyperexcitable states, but the initial insult was so severe that the result is an imperfect homeostasis. For example, acute deafferentation of the crustacean somatogastric ganglion leads to the loss of oscillatory behavior, but remarkably, the neurons in this ganglion "self-tune" their conductances to re-establish oscillations within a few hours. However, this tuning is not ideal, as both the period of the oscillations and the phase relationships between neurons have changed (Luther et al. 2003; Mizrahi et al. 2001). A third possible solution to the paradox of homeostasis in single cells within hyperexcitable networks is that homeostasis occurs, but hyperexcitability persists for other reasons. Indeed, as mentioned in the preceding text, computational modeling techniques using anatomically and biophysically realistic, large-scale models of the dentate gyrus clearly demonstrated that robust hyperexcitability can arise in the dentate gyrus even without changes in intrinsic properties of any of the cell types, purely due to modifications in network architecture arising from mossy fiber sprouting and hilar cell loss (Dyhrfjeld-Johnsen et al. 2006; Santhakumar et al. 2005).
Our current data are most consistent with the third of these alternative solutions, i.e., that the homeostatic regulatory mechanisms resulted in unchanged intrinsic excitability in mossy cells that reside within the hyperexcitable, posttraumatic dentate gyrus. The best evidence in favor of homeostasis in mossy cells is that the I-F and I-V curves are unchanged after trauma, but numerous changes in distinct membrane currents suggest that significant reconfiguration of these currents took place after trauma. Several observations are reminiscent of homeostatic plasticity: the increased voltage response to positive current pulses is counterbalanced by a depolarized AP threshold, and the possible increase in Ih is counterbalanced by the downregulation of an unidentified conductance (as evidenced by the increased input resistance in the presence of ZD). Therefore although it is not possible to provide incontrovertible evidence that these intrinsic alterations are indeed produced as a result of homeostatic plasticity, the seemingly finely tuned, mutually opposing alterations in distinct ion channels are certainly consistent with homeostatic processes set in motion by the insult. The observations from our simulation experiments that the relatively small alterations in the conductances of a single cell type can have robust effects on the excitability of the network as a whole further emphasize the importance of coordination among the individual plasticity events affecting distinct ion channel subtypes. It is interesting to note in this regard that recent computational and experimental studies have shown that there are many solutions to the problem of how a cell can preserve its proper firing pattern with different combinations of intrinsic conductances (Marder and Goaillard 2006; Marder and Prinz 2003). For example, the expression levels of IA and Ih are co-regulated in both lobster and crab somatogastric ganglion cells to ensure the proper rhythmic firing of these neurons (MacLean et al. 2003, 2005; Schulz et al. 2006). In addition, in cultured neocortical cells, the blockade of activity with TTX leads to homeostatic responses through alterations of INa and ITEA (Desai et al. 1999), two currents that were also shown to be altered in our system.
Are the data concerning the alterations in miniature and spontaneous I/EPSCs consistent with homeostasis? Based on results from experiments using externally imposed changes in network activity in cultures (Kilman et al. 2002; Turrigiano and Nelson 2004; Turrigiano et al. 1998), homeostatic responses in single cells to network hyperexcitability are expected to include decreases in mEPSC amplitudes and increases in mIPSC amplitudes. Consistent with hyperexcitable responses from both granule and mossy cells to perforant path stimulation (Santhakumar et al. 2000, 2001; Toth et al. 1997), the presence of increased sEPSC frequency and amplitude indicates that posttraumatic mossy cells experience sustained increases in the levels of excitatory inputs from other neurons in the posttraumatic dentate gyrus network. Accordingly, the posttraumatic mossy cells showed decreased mEPSC amplitude and increased mIPSC amplitude as would be expected from homeostatic responses in single cells to network hyperexcitablity. Regarding the additional alterations in synaptic inputs that our data revealed in posttraumatic mossy cells, the following interpretations may be considered. Both the mEPSC and the mIPSC frequencies were decreased (interevent intervals increased), which is consistent with the loss of release sites after the death of presynaptic neurons after the trauma (although alternative explanations, e.g., changes in release probabilities, cannot be excluded). In spite of the decreased mEPSC and mIPSC frequencies, the sEPSC and sIPSC frequencies were both increased. The most parsimonious explanation for the opposing changes in miniature and spontaneous event frequencies is that the surviving cells that are presynaptic to mossy cells increased their firing rates. The exact mechanism underlying this apparent compensatory alteration is not known although the additional excitatory drive arising from the sprouting of mossy fibers onto granule cells and directly or indirectly onto GABAergic neurons is likely to be a major factor.
Summary and outlook
Mossy cells give rise to the associational and commissural pathway of the dentate gyrus, innervating ∼30,000 granule cells along the septo-temporal axis on the ipsilateral side (Buckmaster et al. 1996; Wenzel et al. 1997). Based on this extensive connectivity and their high vulnerability, these cells are considered to be a key element in the dentate circuit under both healthy (Lisman 1999) and pathological conditions (Ratzliff et al. 2002, 2004). The current study revealed important information concerning a number of mossy cell properties from control animals, including resting membrane potential, input resistance, action potential accommodation properties, Na+ current parameters, amplitude and kinetic measures of miniature excitatory and inhibitory events, and the properties of DSI. These parameters from control mossy cells will be incorporated into the large-scale anatomically and biophysically realistic network models that are being developed and constantly updated (Dyhrfjeld-Johnsen et al. 2006; Santhakumar et al. 2005). In addition, our data identified a large number of posttraumatic alterations in mossy cell properties after head injury, yielding important information concerning the roles of mossy cells in hyperexcitable responses in the dentate gyrus. Finally, when taken together, our results indicate that mossy cells after trauma undergo a series of changes in a manner consistent with homeostatic mechanisms as evidenced by the functionally opposing changes in membrane currents and synaptic events. Although we do not fully understand the nature of the underlying regulatory processes, it is likely that such homeostatic and compensatory processes play considerable roles in shaping the intrinsic and synaptic plasticity phenomena in hyperexcitable states.
This work was supported by National Institutes of Health Grant NS-35915 to I. Soltesz, by University of California Irvine Medical Scientist Training Program funding to A. L. Howard, R. J. Morgan, and J. C. Echegoyan, and by Deutsche Forschungsgemeinschaft Grant NE1185/1–1 to A. Neu.
We thank R. Zhu for technical assistance.
↵1 The online version of this article contains supplemental data.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in acc23ordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society


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text:
So I chatted up this girl online for a date. I felt a really good connection, so I said let's go skating and grab some food. There is this cool roller skating rink where they play 80s music, it's cheesy but loads of fun.
Anyway, we meet up before hand, and she shows up dressed in like a turtleneck under a fur lined hoodie, and this big coat, hat, scarf, some kind of warm pant. Bundled.
I'm confused, it's warm out, we're going to a warm roller skating arena. Then it hit me... can you guess? She thought ice skating lol...
Ok so whatever laugh it off, but she just felt and looked so awkward the whole time. I held her coat while we roller skated, but she was still way overdressed and her cheeks were pretty red. She didn't complain, and one point told me she was wearing long johns under, so I asked her if she wanted to change and she just said no.
I tried to play it cool, but even over dinner she looked like she was feeling awkward. Did I blow the date?
Most Helpful Girl
When you asked her out did you just say "let's go skating" or did you mention the roller rink? If you didn't specifically mention roller rink why on earth would she assume anything other than ice skating? No one calls roller skating "skating" ... NO ONE.
Honestly if I was her I'd have beenough pretty annoyed and would have expected you to change plans and go ice skating once you saw how I was dressed but probably would have still just went with it like she did. If I liked the guy enough I'd give him another chance but I won't lie to ya - this is a pretty poor first impression.95|40|0
Actually my friend calls ot skating. For her birthfay she invited me and told me wed be skating. So i dressed warm showed up and turns out. Yeah, no. Not ice skating.
- Show AllShow Less
I would assume skateboarding lmao.
Who on earth calls it roller skating? Everyone I know calls it skating.
Most Helpful Guy
No man you did nothing wrong (besides non specify it wasn't ice skating of course).
I think you should invite her again and take her to Ice skating that would be funny. It is normal she felt weird because of her mistake but you should invite her again so she will not believe that you think she is an idiot. :V


text:
Looking for a way to secure financing to develop its twin-turboprop FF-1080 Freight Feeder, American Utilicraft (AMUC) has signed a memorandum of understanding with Averitt Express, a Tennessee trucking company. The MOU is the first step in AMUC's plan to provide air-cargo capacity to Averitt, which wants to supplement its logistic network, now based primarily on ground transport, with more airborne freight capacity. Under the proposed capacity services agreement, which the two companies hope to define in the next few months, AMUC would build, own and operate up to 25 aircraft under a Part 121 operating certificate obtained by acquiring a current operator.
Jim Carey, AMUC executive v-p, told AIN, "I can't talk about money yet, but I can say that we have initial support from a major New York bank that is interested in working with us. We're comfortable with the financing and just need to nail down the details." Explaining AMUC's rationale for the plan, he said, "If we sell 25 aircraft, we would get a net profit of about $1 million per aircraft. But if we operate 25 aircraft, we may be able to get $2 million to $3 million profit per aircraft per year. It is simply a better business plan for AMUC."
As reported previously in this column, AMUC, based in Lawrenceville, Ga., is now trading on the Pink Sheet Exchange following its delisting from the Over-the-Counter Bulletin Board. Last month, its stock was selling for about $1.20 per share, down from about $1.70 per share in May.


text:
The People of Tulane Cancer Center Research
Yan Dong, Ph.D.
Contact Information
Research Interests
Selected Publications
Y Zhan, G Zhang, X Wang, Y Qi, S Bai, D Li, T Ma, O Sartor, EK Flemington, H Zhang, P Lee, and Y Dong. (2017) Interplay between cytoplasmic and nuclear androgen receptor splice variants mediate castration resistance. Molecular Cancer Research, 15, 59-68. PMID: 27671337. Featured in the "Molecular Cancer Research Highlights: Selected Articles from This Issue" with a figure.
S Cao, Y Zhan, and Y Dong. (2016) Emerging data on androgen receptor splice variants in prostate cancer. Endocrine-Related Cancer, Invited review. 23:T199-T210. PMCID:PMC5107136. Featured in "Uro Today".
D Xu, Y Zhan, Y Qi, B Cao, S Bai, W Xu, SS Gambhir, P Lee, O Sartor, EK Flemington, H Zhang, CD Hu, and Y Dong. (2015) Androgen receptor splice variants dimerize to transactivate target genes. Cancer Research, 75, 3663-3671. PMCID:PMC4558376. Featured in "MDLinx", "Just Cancer News", "BioWorld from Thomson Reuters". Editorial: Emmanuel S. Antonarakis and Jun Luo. (2015) AR splice variant dimerization - clinical implications Nature Reviews Urology, 12, 431-433.
B Cao, Y Qi, G Zhang, D Xu, Y Zhan, X Alvarez, Z Guo, X Fu, SR Plymate, O Sartor, H Zhang, and Y Dong. (2014) Androgen Receptor Splice Variants Activating the Full-Length Receptor in Mediating Resistance to Androgen-Directed Therapy. Oncotarget, 5, 1646-56. PMCID:PMC4039237.
Y Zhan, B Cao, Y Qi, S Liu, Q Zhang, W Zhou, D Xu, H Lu, O Sartor, W Kong, H Zhang, and Y Dong. (2013) Methylselenol Prodrug Enhances MDV3100 Efficacy for Treatment of Castration-Resistant Prostate Cancer. International Journal of Cancer, 133, 2225-33. PMCID:PMC3750963. Featured in "MDLinx".
B Cao, X Liu, J Li, S Liu, Y Qi, Z Xiong, A Zhang, T Wiese, X Fu, J Gu, PS Rennie, O Sartor, BR Lee, C Ip, L Zhao, H Zhang, and Y Dong. (2013) 20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor. International Journal of Cancer, 132, 1277–1287. PMCID:PMC3509250.
1430 Tulane Ave, New Orleans, LA 70112 email@example.com


text:
tand6 tragedyEdit
I am back after sooooo long sorry about that my dear fans I kinda forgot stuff about the story but itll be fine i am sure
true assassins never diiiiiiiiiiiiie! chap 6
Everyone was fighting! me and derek and bella and jace and alec were fighting against all the evil people.
James and Victoria and Laurent and Angela had come tru the ceiling!
"lol wtf are you guyz doing?" said angela rain bennet. She was a fucking stupid-ass gothic everybody hated. "wear out of here." And then they left.
"Nooooo fucking gothic bitch" I said "ur not even a real goff like the cool ones like milena and chella!"
"heeeeeeeeeheeeeeeheeeeee" wolfbloodrei screeched (cuz shes a demon wereman) "now you will DYE!"
Alec's pov
"NOOOOOO ALEX!" I murmured. All the evil folks were laughin and then bamfed away because they knew they had killed our only hope. Our only light. The life of the party. My one true love
"Its okay lol" derek said.
"WHAT HOW CAN YOU SAY YOU LOVE HIM AND THEN SAY ITS OKAY THAT HES DED?????" I disillusionated.
"Where both assassins and true assassins never die" (A/N TITLE DROP!!! it's a clever thing that only good authors do!) derek explained.
"But we can't bring back the dead" bella said.
"Yeah dats stupid" jace acquiesced.
"OMG you get me!" Bella said. "This must be TRUE LOVE." and they started to make out. I knew Clary was still in the infirmary pregnant with Jaces baby but wtf straight sex is stupid and useless anyway.
"Anywayyyyy" derek sighed "we just need to get him to a quest bed so we can get him back from the dead." (A/N see i told you there would be more homestuck later on!)
"But" bella said pulling herself away from jace for a moment "that means we need to do it be4 his dream self dies and we arent even on the right planet!"
"yeah usually but I said were assassins you stupid bitch." derek said nicely. "We don't follow you stupid rules."
"Okay then what are the rules?" bella asked lasciviously.
"The quest bed is just any bed where Alex has made love! And he has infinite dream selves so we have all the time we need to find one."
"That wont be necessary" I said sexily. "we just need to go to the greenhouse."
"Olol thats right you two screwed up there" jace laughed.
"Is that true?" Derek said almost crying.
"Yeah sorry it was a squirt of the moment thing."
"lol okay maybe we should get to know each other then" derek winked.
"Maybe later?" I said blushing. I wasnt sure cuz I was kind of a romantic guy and thought alex was my true love though. "But right now we have a task ahead of ourSELVES" (geddit cuz dream selves are an important thing in homestuck!)
And so derek and me carried alex's sexah body upstairs to the greenhouse while the two horndoggies bella and jace were making out passionately in the bloodied hallway. Lol straight weirdoes. We placed Alex wear we had been sexing it up earlier today and...
an find out next time
- Alex
- Derek
- Bella
- Jace
- Alec
- James
- Victoria
- Laurent
- Angela
- Milena (mentioned)
- Chella (mentioned)
- Clary (mentioned)
|True assassins never die! chapters
|Previous chapter
|All chapters
|Next chapter
|1 • 2 • 3 • 4 • 5 • 6 • 7


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This gorgeous ivory & pearl beaded clutch purse is the perfect accessory for a bride to be or the perfect evening bag. Open the classic silver clasp frame to an ivory satin lining. This ivory handbag will have plenty of room for all of your essentials and includes a chain for a convenient hands free option. This clutch is made to order and will ship in 2-3 weeks from your order. (Contact me prior to order if you need expedited production/shipping)
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2-3 weeks
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Just contact me within: 14 days of delivery
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Buyers are responsible for return shipping costs. If the item is not returned in its original condition, the buyer is responsible for any loss in value.
I will only use your shipping and billing address, and contact information
We get requests for custom multiple same item clutch purse orders for gifts, bridal parties, etc. We currently offer the following discounts:
2-3 clutches - 10% discount
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For retail inquiries, please contact me for further information.
If you are worried about a perfect match or prefer to see a fabric sample we do offer free fabric samples to those in the United States on most of our products as long as the fabric is in stock. When contacting us, please specify which item/fabrics you are requesting to see fabric samples of along with your name and mailing address. If you would like to see something that is long listed, just let me know what you are looking for as I do have many options that are currently not listed.
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Custom orders are an option! We have worked locally with some brides to use a piece of their mother's wedding dress, etc. These details can make for a very special keepsake accessory! Contact me with the following: 1) What style clutch you want made, 2) What type of fabric you have, 3) How much fabric you have to work with (yardage is good to know or measure by inches H x L)
PLEASE NOTE: Customers are responsible for the mailing cost of the fabric to and from us (we will return any remnants not used) and it is understood that fabric will not return in same condition as cuts will be made to create your clutch. Bonne Amie Boutique is not responsible for damages to your fabric caused by shipping, etc.


text:
Quaternium-15, Use Test
The recruitment status of this study is unknown. The completion date has passed and the status has not been verified in more than two years.
Verified April 2006 by Mekos Laboratories AS.
Recruitment status was: Not yet recruiting
Recruitment status was: Not yet recruiting
Mekos Laboratories AS
Information provided by:
Mekos Laboratories AS
ClinicalTrials.gov Identifier:
First received: April 4, 2006
Last updated: NA
Last verified: April 2006
History: No changes posted
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
No Study Results Posted on ClinicalTrials.gov for this Study
|Study Status:
|This study is not yet open for participant recruitment.
|Study Completion Date:
|No date given
|Primary Completion Date:
|No date given


text:
Founder's Colony is a startup accelerator with a passion for getting startups off the ground. Make your business plan a reality with capital and partner services provided to each of our accepted applicants.
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For the second Startup Weekend in a row, Founders Colony will be a sponsor.


text:
Before the Revolving Line of CreditEdit
Announcer: "Tonight on Supernanny...Jo meets the Forrest Family."
Nathan: "Poopy-head."
Submission ReelEdit
Family IssuesEdit
Parent MeetingEdit
House RulesEdit
Alex's Birthday PartyEdit
Jo: "The next day, Alex had a Beyblade-themed birthday party in the Forrest family backyard."
Alex: "Grant, Bronson, Ian, Kevin, Bryan, Nate, Carlos, Miguel, hi! Come on in! The party's starting!"
Nicole: "Alex was dressed up as his favorite Beyblade character, Gingka. All of his friends were also dressed up as their favorite Beyblade characters, such as Ryuga or Gingka. It was great."
[The kids are having fun]
Daniel: "We had everything; the favors, the piñata, the music, the decorations, the balloons, the games, the activities, and an all-day tournament."
Jo: "There were pastries, rice krispie treats, cupcakes, cookies, chocolate, and a Beyblade Birthday Cake."
[Nicole brings out a large round cake that looks like an oversized Beyblade]
Daniel: "Favors, such as flat bottlecap zipper pulls, ball chain necklaces, spinning tops, keychains, sticker sheets, and all sorts of things were being given out to each child."
[The kids are having playing games]
Alex: "We played all sorts of games, such as 'Hot Beyblade,' and 'Pin the Beyblade in the Beystadium.' We also had a one-on-one Beyblade Tournament."
Nicole: "Okay, who's ready to sing 'Happy Birthday'?"
Nicole: "One, two, three..."
Nicole/Daniel/Jo/Guests: "Happy birthday to you, happy birthday to you, happy birthday dear Alex, happy birthday to you..."
Jo: "Make a wish, Alex!"
[Alex blows out the candles, but the candles re-light themselves]
Guests: "Huh?"
Alex: "Huh? what the--?"
Nicole: "How weird, they're not supposed to re-light themselves."
[Alex blows out the candles again]
Naughty PillowEdit
Jo: "Later on, Nathan was refused a snack because dinner was being cooked. And he didn't like it."
Nathan: (whining) "Why can't I have a snack?"
Nicole: "Because we are having dinner."
[Nathan hits Nicole across the leg]
Nicole: "Nathan, you do not hit mommy. That is not nice! If you do that again, you will go on the Naughty Pillow. Do you understand?"
Nathan: "NO!" (hits Nicole)
Nathan: "You are a horrible person!"


text:
Our favorite giveaway tool, Rafflecopter, is having their own giveaway!
There will be 2 winners of 16GB iPad Mini WiFi and 25 Premium Rafflecopter accounts.
How To Join
Well, go to the contest URL and enter via Rafflecopter of course hehehe
This contest ends on December 31, 2012. Open worldwide. Shipping of prizes outside US & Can will be shouldered by the winner.
Contest URL: http://blog.rafflecopter.com/2012/11/rafflecopter-premium-launch-giveaway/
[do action="disclaimer"/]


text:
Grateful Dead
Hey Folks:
Looking for deadheads in NZ for a catch up on old times. Also, I do a little bit of Dead-inspired music when I have spare time with two upcoming shows in Wellington and Masterton (have a look at richardleschen.com).
If you can't make it to a show thats cool, maybe we can meet for a coffee while I'm on the road...
see ya, R
Here's a tip for my fellow Melbourne souls:
Vegetable pakoras, $5; Madhumoti, 95 Irving Street, Footscray.
Most often the pakoras I find in Melbourne - especially at the cheaper end of the spectrum that Bennie and I favour - are variously greasy, too spicy and tough as old boots.
Not these.
These are heavenly
For your five bucks you get four good-sized patties deep fried just right, non-greasy but crunchy and full of juicy veges, with sweet onion to the fore. Yummo!
I'm still finding my way through the longish menu for the other good hits at this newish, clean friendly, cheap eatery that is on the corner oppositie the Dancing Dog Cafe.
Then again, maybe we could meet at: Motorhead @ The Forum, October 19.
OK - a question for my fellow Melbournites: Anyone you can tip as playing around Melbourne these days who does GD-type thing?
For the past couple of years, after three decades of incessant gig-going, I've been laying pretty low and doing other stuff. And the decade before that was mostly jazz at Bennetts Lane and elsewhere, so I'm singularly ill-informed.
The Paul Williamson Hammond Combo plays every Monday night (free) at the Rainbow Hotel in Fitzroy and are fantastic. Straighahead jazz/blues/R&B in their own fashion, but they groove as hard as anybody. And every week they have sit-in guest which always makes it interesting.
I think the clearest GD trip I've ever experienced regularly came froma now defunct outfit called Diana Kiss, which - like the Hammond Combo - had a long-running residency at the Espy. Lineup: Ross Hannaford (gtr), Peter Jones (dms), Jex Saarelaht (keys), Russell Smith (keys/tpt), Stu Speed (bass). Monster grooves all 'round and a Dead-like way with cover versions, same as with the Hammond Combo. And I reckon it could even be said that Hannaford is Australia's version of J Garcia.
Then there's a band called Checkerboard Blues Lounge - i suspect they, too, inhabit a jammy world, although they don't play too often.
Any suggestions?
BTW, I reckon a Monday night at the Rainbow would be a great time/place for a first-up Melbourne Deadhead gathering. Any Monday, all Mondays!
Hey all, happy to see other Melbourne Deadheads, I knew you guys were out there! A Deadheads Club as mentioned or any other contact sounds great.
greetings fellow deadheads from down under been on the bus for 40 very odd years and ain't
getting off till the end.
cheers from oz!
Fascinating - thanks for filling me on that bit of Dead lore.
Personally, I can relate to "Those damn hippies couldn't concentrate too long on anything much" more than other explanations.
"didn't work too well" is probably overstating the case for the sceptics. From the report on the experiments in the Fortean Times:
The Dream Laboratory's report on the experiment noted: "The average evaluation of the two judges was computed for each pair of dream transcripts and target pictures. If coincidence, rather than ESP, had been operating, the judges' evaluation of the correct transcript/target pairs would have been higher than all other pairs one time out of six. For Miss Parise, one correct pair obtained the highest rating. In the case of Mr Bessent, the judges gave the highest score to the correct pairs four times out of six… Thus, for Mr Bessent, the ESP hypothesis is supported. Further, some support is given to the position that the agents must know who the target is to be transmitted to and where he is located for telepathy to occur."
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The run of 6 shows at Port Chester from Feb 18 through Feb 24, 1971 were dubbed the ESP Experiment. Various pictures were projected onto a screen behind the band and the audience was supposed to focus on these images and try to 'transmit' them psychically to a bunch of subjects in a nearby lab. Apparently it didn't work to well. Those damn hippies couldn't concentrate too long on anything much.
These shows were also among the first "Betty Boards" to surface into the mainstream. (Another useful fact to be gleaned from the Deadhead's Taping Compendium Vol 1)


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Discussion in 'Classifieds' started by JohnHan, Aug 2, 2015.
Trade you a kraken 130 octo heavy lifter for it. Wookong, supreme18" props, 330 kv motors, 4- gen ace 5500 6 cells, imersion uhf receiver, retracts, Arris gimble, sony nex camera, 5 ft in diameter!!! Arms fold down for easy transport. Flown once! 30 min flight at 5280 above sea leavel!
Sorry. Not interested in a trade.


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It strains credulity, but these shatter-brained distinctive featured Pokemon protagonists are down with much about evil-living shags, they are not as clean-handed as you anticipated when you get a load of them over TV… Take a slant at the amatorious widget hottie getting swung with her frabjous corky puppies shampooed with Cupid's toothpaste and her incommodious Boris which is eased and poked and liquid hairdressing of… The most responsible part for cock-loving cuties from Pokemon is to be engaged in wild banging parties with some of the best endowed dudes and provide their tight fuck holes to be completely messed up by the stiffest dicks.
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