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Interpreting the graphs and figures

Initial comparison of each treatment group reveals some obvious trends. Deltamethrin produced increases in the IC50 as high as 350ms, while esbiolethrin was more modest with a peak value at 60ms, and the dual-treatment was somewhere in between with a maximal IC50 of around 275ms.

The timescale over which each agent acted was also obviously different; deltamethrin took between 35 and 80 minutes to reach full effect, and was still noticeably influencing the IC50 at 150 minutes. In comparison esbiol reached full effect in under 10 minutes, and its effect had largely dissipated by 30 minutes. As would be expected the dual-treatment group had a timescale almost identical to deltamethrin - as esbiol was applied in the middle of the time span, and rapidly wore off, therefore the onset and outset of effects were due to deltamethrin only.

Looking for interactions

The graph comparing dual-treatment with theoretical additive effects of deltamethrin and esbiol depicts the combined effect as less than is predicted by addition. Using stats to support this was not wholly successful, with only one of the three points tested showing significance. This was mainly due to the large standard deviations in the deltamethrin group. Still the results make it unconvincing that any additive effect was occurring between deltamethrin and esbiolethrin.

Next, the graph plotted from deltamethrin and the dual treatment group is quite revealing. It appears that the dual treatment is slightly less effective at extending the inhibitory phase than deltamethrin alone. Statistical analysis once again showed a significant difference only one of three time points, at 55mins. At this point the difference is quite significant (p < 0.001), however leaning on one result while ignoring the other values would be insensate. Thus, while tempting as it is to concur with the previous in vitro findings that Type I and Type II pyrethroids competed, one must conclude that there is inconclusive data.

Additive, Competitive... Inconclusive

From a purely visual perspective, it appears that the dual esbiolethrin and deltamethrin treatment group experienced less inhibition that would be expected if the pyrethroids were showing additive effects. It also looked like the dual-treatment group produced less effect than would be expected from deltamethrin alone, particularly at the 55mins time bin.

However using these data we cannot make solid conclusions about what is occurring between the pyrethroids. The differences found when comparing each treatment group were too small to be certain of their importance. Ambiguity leaves us only able to remark on the apparent trends, speculating until more compelling evidence is found.

Revisiting the hypothesis

It was assumed in conducting this experiment that the competitive effects seen in sodium channels might transfer to the hippocampal preparation, and that would be reflected in this in vivo model. Yet this appears not to be the case. It was reasonable to assume a tight relationship between the effect on sodium channels, toxicity and the extension of paired pulse inhibition. However it appears that the link may be more complex than was originally thought.

While an action on sodium channels hasn't been proved to be necessary in extending the paired pulse inhibition effect, there has been no evidence to suggest otherwise [Ray & Fry, 2006]. How then to reconcile these in vivo findings with the previous in vitro ones?

Perhaps actions on other ion channels, or other targets, also contribute to the effect of extending paired pulse inhibition. Indeed perhaps pyrethroids not only act, but also interact at other sites than just the sodium channel, producing complex pharmacodynamics. For instance some mild antagonism has been observed between cispermethrin and Type II pyrethroids at the maxi chloride channel [Burr & Ray, 2004].

Voltage gated calcium channels and voltage gated chloride channels have been implicated as targets for pyrethroids, and so have GABA gated chloride channels at higher concentrations. Each of these targets has been shown to be unessential for toxic effects, however they may still contribute to extension of inhibition in the dentate gyrus. Living biological systems are so complex that a seemingly minor component might be playing other, as yet unseen, roles.

Back to first principles

Taking another step back it's time to apply some skepticism to Song and Narahashi’s observations in cell and patch clamp preparations. In those experiments ultra-high doses of pyrethroid were used, in which the chemical is typically suspended in water with another solvent to aid solubility. While practical for in vitro experiments, these conditions are far from how pyrethroids would realistically enter an organism, which may have affected the responses of their preparations. Perhaps the effects they saw simply wouldn't precipitate in vivo at doses around 1.5 x LD50.

Why competition after all?

Furthermore, doubt can be shed on the underlying explanation for why competition might occur between pyrethroids. Due to the density of sodium channels in excitable cells, merely 1% need to be modified to induce a hyperexcitable state. Even at the high concentrations of pyrethroid used by in vitro, it is unlikely that all the channels were simultaneously occupied by the first pyrethroid.
Yet when the second pyrethroid was administered, the total response changed, as if every affected channel were solely under the influence of the second agent. This was obvious because the effect of the Type I pyrethroid replaced that of the Type II pyrethroid (from very long to quite long kinetics).

Why then would the second pyrethroid specifically compete for the places occupied by the first, when other channel proteins were available for binding? This can be explained as a consequence of pyrethroids' greater affinity for open channels; thus deltamethrin modified channels, which are more often open (greater Po), will be higher affinity targets than unmodified channels [Motomura et al, 2001].

It has also been suggested that the pyrethroids bind with sodium channels at different sites, but that these sites have allosteric interactions, and thus two different pyrethroids can simultaneously be affecting a channel. Such explanations are yet to be thoroughly tested, and remain speculative.

The way in which pyrethroids interact is still an open question. However they do interact, it is not dramatic or obvious. It owuld be interesting to do experiments with progressive dosing levels to reveal more specifically how and when interactions are occurring. It will also be interesting to know how the original in vitro work (which showed competitive effects) would have turned out using more moderate doses, or even a variety of doses.