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Using field potentials from the dentate gyrus

Ultimately we aimed to see how, over time, pyrethroid injections changed the phase of inhibition which underlies the paried-pulse inhibtion phenomenon in the dentate gyrus. Specifically we wanted to know if two pyrethroids in combination would produce effects like one, the other, or something inbetween.

To use the data from each experiment, first raw waveforms have to be interpreted, and the data processed, then finally the trends can be compared across each preparation. Hopefully one sees a consistent effect can draw some kind of clear conclusion.... hopefully. The raw data was aquired from recording electrodes in the dentate gyrus; they are measured as electrical potentials aquired when paired pulses are applied to the afferent perforant path. From the potentials waveforms can be drawn, as shown below. From these waveforms points of interest were marked by hand.

Two typical hippocampal records showing normal and deltamethrin-evoked spike inhibition. The upper record (a) shows the response to two stimuli delivered 20 ms apart in the normal preparation. Asterisks 1-3 mark the positions of the pre-spike baseline, the (downward) spike, and the post-spike baseline respectively. Asterisk 3 also marks the peak of the EPSP. The second spike demonstrates normal recurrent inhibition, its amplitude being 51% of the first spike. After deltamethrin (b) the response to the first stimulus is unchanged, but the response to the second stimulus given now at 200 ms (note the break in the x-axis) shows a similar degree of spike inhibition to that earlier seen at 20 ms. The y-axis shows response amplitude (mV), and the x-axis time since the first stimulus (ms). from Ray & Fry, 2005

Each waveform will have a pair of 'wiggles' (as above) each being the potnentials seen following stimulation to the perforant path. For each waveform one can derive the amplitude of both peaks producing two values in mV, one for each of the pair of pulses applied to the perforant path, called P1 and P2. One can express P2 as a percentage of P1 giving a measure of the evoked inhibition at the given interval. For instance in the figure above, the upper trace's P2 is about 51% of P1, at a delay of 20 miliseconds. Incidentally, this would mean that the IC50 is approximately 20msec.

The peak from the neuron is complicated by the general change in field potnetial. It is possible to extract the spike's amplitude by hand marking the beggining, peak and end of the spike. The response's amplitude is found where a vertical line from point 2 meets a diagonal line passing through 1 and 3.

Preliminary setup examples

In order to use effective stimulus voltages and interpulse intervals it is necessary to do some preliminary experiments to assess how one can expect the preration to respond. These were performed in the absence of any pyrethroid, but with every other aspect of the preparation including anaesthesia, to assess how a normal preparation will respond. To induce a good inhibitory response one needs to know the voltage at which to stimulate the perforant path. A basal, control plot of 100*(P2/P1) will also help us to orientate how pyrethroids are shifting the inhibition.

Here the amplitude of each spike is plotted against the voltage used to stimulate the perforant path. Note that P2 is bigger than P1 for low voltages, due to summation of EPSPs and little recurrent inhibition. Voltages around 20-50V produce substantial inhibition.

The evoked inhibition is dependent on the initial stimulus. It can be seen that inhibition will be optimal as long as P1 is > 0.4mV, which can be achieved with stimulus voltages of > 20V (previous diagram).

Here a +50V stimulus was used, producing optimal inhibiton, in the absence of pyrethroid. It can seen that inhibition is detectable for the first 35 ms, after which paired-pulse potentiation overcomes the effect, and P2 rises above 100% of P1. The IC50 is approximately 20ms.