Hardware and Software

The hardware used for the experimental setup consisted of an experimental chamber which had its insides, as well as its door, covered with sound-absorbing foam. Inside this chamber, an air-suspended platform (Newport Corporation, Irvine CA, USA) carried an animal holder, as well as two micromanipulators and the headstage of the intracellular amplifier. The air-suspended platform isolates the animal holder and the two microma-nipulators from mechanical vibrations in the environment.

One micromanipulator, a standard model from Leica (Leica Microsystems, Bensheim, Germany) held the electrode by means of an electrode holder (PPH-1P-BNC90, 90^◦ angle with Ag wire, npi electronic, Tamm, Germany). The other micromanipulator (MM33, Märzhäuser, Wetzlar, Germany), held a modified pair of tweezers that were used to stabilize the auditory nerve. The tweezers were modified such that they ended in rings,

tweeter

electrode-holder

tweezers

micro-manipulator

electrode

animal holder

Figure 4.1: The experimental setup used to perform the intracellular recordings onLocusta migratoria.

The picture shows the micromanipulator holding the electrode holder with the electrode, on top of the animal holder in front of one of the two tweeters. To the right are the tweezers used to to stabilise the auditory nerve. For the purpose of the picture, the sound absorbing foam which is used extensively in the chamber is removed on the front of the micromanipulator.

rather than tips, in order to gently clamp the auditory nerve and thus exert counter pressure to the electrode (Figure 4.1). The headstage of the intracellular amplifier was also placed on the air suspended platform in order to keep the length of cable from the electrode to the headstage as short as possible, as the changes in membrane potential recorded from the receptor neurons are very weak and thus susceptible to interference from the environment.

Electrodes used for the experiments were 1.5 mm outer diameter/0.84 mm inner diam-eter borosilicate glass (World Precision Instruments, Sarasota FL, USA). The electrodes were pulled with a P-87 electrode puller (Sutter Instrument Company, Novato CA, USA).

The electrodes were filled with either 3 M or 1 M KCL solution. To prevent the prepara-tion from drying out, Ringer solution was used. The solution was applied with a pipette directly into the thorax. See Appendix for further details on the electrodes and the Ringer solution.

The experiments took place at room temperature (approximately 22^◦ Celsius). Usu-ally, these types of preparations are directly heated via the animal holder with a Peltier element to keep the temperature constant. However, this causes increased evaporation of the Ringer solution. Normally, this is not a problem, as this can be compensated by adding fresh Ringer solution after a recording – adding Ringer solution during a recording usually causes the electrode to lose contact with the neuron. In our experiments however, the stimuli presented to the locust varied a high number of parameter values (more on this in Section 4.3), meaning that long intracellular recordings of 50 minutes and longer were necessary.

For stimulus generation, a data acquisition board (PCI-MIO-16E-1, National Instru-ments, Austin TX, USA) with a sampling rate of 100 kHz was used. The stimulus was sent via an interface (model 2090, National Instruments, Austin TX, USA) to an atten-uator (produced in-house) which attenuated the stimulus in the digital domain in order

computer

oscilloscope

power amplifier

headstage

attenuator

electrode experimental chamber

locust Hi-Fi amplifier

monitoring speaker

filter element intracellular

interface

data acquisition

tweeter board

amplifier

Figure 4.2: Schematic diagram of the experimental setup. Stimulus generation: Using the OEL pro-gramming environment on the computer, the stimulus was produced by the data acquisition board and sent via the interface to an attenuator, which attenuated the stimulus in the digital domain in order to avoid loss of fidelity. The attenuator sent the stimulus to a power amplifier, which was used to drive the speakers (two tweeters) in the experimental chamber. Data acquisition: In the experimental chamber, the membrane voltage was recorded from an auditory receptor neuron of a locust by an electrode con-nected via a headstage to an intracellular amplifier. The amplified membrane potential was monitored visually on an oscilloscope and acoustically on a monitoring loudspeaker driven by a Hi-Fi amplifier. A filter element, receiving the membrane potential signal from the intracellular amplifier, band-pass filtered it and sent it via an interface, which digitized the membrane potential signal, to the data acquisition board in the computer.

to avoid loss of fidelity. The attenuator sent the stimulus to a power amplifier (DCA450, Denon Electronic, Tokyo, Japan) which was used to drive the speakers in the experimen-tal chamber. The speakers were two tweeters (Esotec D-260, Dynaudio, Skanderborg, Denmark), facing each other, with the prepared animal in between, positioned at a dis-tance of 60 cm from each tweeter. The stimulus was emitted by the tweeter ipsilateral to the ear from which an intracellular recording took place.

In order to record membrane potential changes, electrodes were connected via a head-stage to an intracellular amplifier (BRAMP 01, npi electronic, Tamm, Germany) which amplified the signal from the electrode. The intracellular amplifier was connected to a filter element (DPA 2F, npi electronic, Tamm, Germany), both components were housed in an EPMS 07 housing (also made by npi electronic) which provided power and earthing.

The filter element was used to filter out electrical noise present in the setup. The settings were 30 Hz for the high-pass filter and 10 kHz for the low-pass filter. The filter element was connected via the 2090 interface to the PCI-MIO-16E-1 data acquisition board in the computer. We used a sampling rate of 20 kHz for data acquisition. A schematic of the setup is shown in Figure 4.2.

The software used for stimulus generation and data acquisition was OEL, the Online Electrophysiology Lab programming environment, written in C++ by Jan Benda and Christian Machens (Figure 4.3). OEL runs on the Linux operating system and provides the user with various possibilities regarding the management of stimulus presentation,

Figure 4.3: Screenshot of the Online Electrophysiology Lab software used for stimulus generation and data acquisition. The top right section shows the membrane potential. Individual spikes are identified by OEL; and the respective spike times are written to a file. The bottom right section shows the sensitivity of the receptor neuron in an intensity vs. frequency plot (left) and its response curve in a firing rate vs.

intensity plot (right).

including online data analysis. OEL analyzes the incoming membrane potential signal and identifies individual spikes by employing an algorithm presented by Todd and Andrews (1999), and writes the spike times to a file. Further data analysis and processing was done using MATLAB (The MathWorks, Natick MA, USA).

An oscilloscope (TDS 224, Tektronix Inc., Beaverton OR, USA) was used to monitor the spike size. A Hi-Fi amplifier (A-109, Pioneer Electronics Inc., Long Beach CA, USA) connected to the intracellular amplifier, and driving a small monitoring speaker (Maxi300, Quadral, Hannover, Germany) was used to receive acoustic feedback while searching for an axon inside the auditory nerve.