The methodic used in this study is very specific, not popular and has been used last 10 years developed by Ocker and co-workers (1995) for in vivo pharmacological studies. In the beginning only one-barrel microcapillary was used to test the pharmacological action of many transmitters and substances.
These are the classical experiments carried out with this methodic and the problems touched in this chapter are not pointed to them. The problems have begun after the two- and in some cases three- chamber injection cappillaries have been used. Unfortunately the latter stimulations from two- or three-barrel capillary have shown many problems.
Since that time only a few control experiments were carried out but not systematically. Every methodic should have a specification and guide of use or at least a list with the possible problems, disadvantages and advantages. The first deepen work to study the setup itself, to describe the problems and to give criteria analyzing the results was Wenzel (2000) in her PhD Thesis, pp. 107 – 109. She has tried to describe and characterize the typical system errors, which
could give system errors and should been kept in mind by the interpretation of the received data.
Transition of injected substance into the other chamber
Already in the control experiments using DMSO as well as in the later experiments with potentially inhibitory substances, in single cases it was observed that already the first injection pulse following muscraine injection caused singing behavior. This could be explained if some of the muscarine transited /went over/ diffused into the other chamber of the injection capillaries.
In control experiments injecting into a drop of vaseline in fact after repeated injection of a colored solution the following drops of the originally clear solution was vaguely colored. This coloring was diminished with every on following drop and with the third drop not recognizable any more. To distinguish /declare any substance as initiating singing behavior, stricter criteria had top be met.
Consequently, only substances were regarded as song initiating that consistently initiated singing behavior in at least three consequently injections (Wenzel, 2000).
The pressure-injected solution seems therefore to be pressed into the neighboring capillaries well, or to diffuse into it. Obviously it is found there in a lower concentration. This is in accordance with the observation that some substances are able to induce stridulation unexpectedly, but this was of significantly shorter duration than the previously muscarine-induiced stridulation. On the other hand, the solvent in the second chamber could be diluted by such a process. In experiments with potentially inhibitory substances the “inhibitory” effect could be due to a dilution of the afterwards injected muscarine-solution. But as the shortening of the muscarine induced song was not observed for all substances tested which did not by themselves cause stridulation, and as not all inhibitory experiments did show this effect, the effect of any potential dilution can be neglected (Wenzel, 2000).
The volume injected from both chambers could not be determined
A further problem with the pressure injection was that despite the use of defined pressures and pulse durations in the application of optimally identical volumes of solution from both chambers of the double capillaries as well as from the capillaries used in different experiments. In the beginning of any
experiment the size of the resulting drop was adjusted to identical sizes by injection into the air, but which amount was released into the tissues could not be determined. The capillary can be blocked by haemolymph or by remains of tissue in part or in total. On the other hand, the end of the capillary might have been broken in the process of initial injection into the brain. Additionally to these small variations of applied volumes, the concentration of the substances used to get to the injected neuronal structures was significantly dependent on the distance as well as structure tissue between the spot of injection and the neurons. This is to caution that between different experiments as well as between two substances in one experiment the amount of active substances could not be presumed to be identical (Wenzel, 2000).
These problems have to be kept in mind then interpreting the results.
Direct quantitative comparison between different experiments was not possible due to the problem of unspecified concentrations at the place of injection (Wenzel, 2000).
Summarizing the control experiments it could been concluded that they are not sufficient to have a reliable guide and specification of the methodic – pharmacological stimulation into the brain of grasshoppers in vivo with two- or more chambers capillary.
One of the first aims of this study is to deepen the problematic described upper to test the methodic itself, to classify the problems, to find alternative protocols of injection a test substance preventing artificial results and misinterpretations.
For instance, to measure the concentration of the transitional substance in the next chamber after injection could be suitable the use of a capillary electrophoresis coupled to mass-spectrometry. In such manner the transition between the chambers could be quantified and control curves could be built for analyzing the results and this methodological disadvantage could be transformed to advantage and the method could become reliable and will reveal its potentialities.
The methodological problems mentioned here were basis of further investigations, described in section 3.3.
In several control experiments (data not shown) with proctolin in one chamber of the injection capillary and Ringer solution in the other, Ringer solution, applied after several injections proctolin, could also elicit stridulation that was of shorter duration when compared to the proctolin-initiated singing.
This means that a small portion of proctolin may have entered the chamber containing Ringer solution during previous ejections of proctolin and that was sufficient to induce stridulation in the pre-activated state of the control circuits.
One criteria has been involved by Wenzel (2000) that a test substance that stimulates stridulation in 3 consecutive injections, after initiating stridulation through another excitatory substance (muscarine), could be considered as a substance that can initiate and maintain stridulation. In the present study this criteria was not used after the mentioned above control experiments were carried out. Than and than only one substance can initiate and maintain stridulation if injecting by one-barrel capillary, is capable to do this. The potential stimulatory effect of the test substance can be also proved using two-barrel capillary if the second chamber contains Ringer solution. Control injections into vaseline, as done by Wenzel to measure the transition between both chambers, should be made more accurately using the entire stimulation protocol (for instance 3-1-4) by different openings of the capillary tip and different pulse parameters. Several control experiments, with dye-stained solutions under visual observation, could not be sufficient to generalize that 3 injections are sufficient to clear the transited substance, because lower concentrations of the dye could not be seen. Under physiological conditions a small amount of a test substance introduced to the next chamber, in µM concentrations, would be visually undetectable, may significantly affect the responses. Furthermore, the dilution of the control (excitatory) substance after pulses from the other chamber and not inhibitory pharmacological effect could alone decrease the duration of stridulation. The latter could lead to misinterpretations of the received data if the response (song parameters – latency and duration) is critically dependent on the concentration, or the volume under our stimulation conditions.
The results in section 3.2.4 showed that alterations in the limits of 20% for the durations and up to 50% increase of the latency could owe to dose effect. If these alterations are higher than this values it could be observed as pharmacological effect. Concerning the “all/none” responses received by smaller amount proctolin injected, the latter criteria should be not applicable if
no response occurs. In case of “completely inhibition” occurred along the experiment the entire stimulation protocol was repeated using 1,5X pulse duration. If the none-responses occurred again, than only the result was included in further evaluations.
In the present study another stimulation protocol 3-3-3 was suggested, that solves some of the problems described (section 3.3). Instead of filling the second chamber with a test substance (B) (Fig. 4-3, left) a mixture of the excitatory and the test substance (A+B) was included. Thus, the concentration of the excitatory substance (A) (either proctolin or muscarine), in both chambers is the same. In the case of transition between the two chambers only the concentration of the test substance would be altered with the repetitive injection from both chambers. Dilution and any alterations of the concentration of the excitatory substance resulting from injections from the other chamber is prevented througout the entire experiment.