A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, ‘non-invasive’ (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.• A versatile and unique refraction-based optical recording system with a Java application was developed.• The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).• The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.


Method name:
In vitro pharmacological method based on a rapid, easy, cheap, and effective procedure and isolated heart preparations Keywords: Pharmacology Optical recording system Isolated heart preparation The great pond snail Lymnaea stagnalis a b s t r a c t In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, 'non-invasive' (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.
• A versatile and unique refraction-based optical recording system with a Java application was developed. • The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail ( Lymnaea stagnalis ). • The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

Specifications table
Subject area: Pharmacology, Toxicology and Pharmaceutical Science More specific subject area: Physiology Method name: In vitro pharmacological method based on a rapid, easy, cheap, and effective procedure and isolated heart preparations Name and reference of original method: Resource availability: Software availability: https://www.blki.hu/en/research#digirec

Background
In the field of neuroscience and ecotoxicology, there is a great need to investigate the effects of chemicals at different levels. Since the 1970s, the great pond snail ( Lymnaea stagnalis ) has been a widely used invertebrate model in ecotoxicology for testing the effects of heavy metals, pesticides, and pharmacologically active compounds (reviewed by [1 , 9 , 12] ) as well as in neuroscience for studying the functioning of the nervous system including chemical modulation (reviewed by [2 , 5 , 9 , 11 , 13 , 14] . The heart of L. stagnalis is an excellent organ for carrying out pharmacological experiments, including the study of (neuro)chemical modulation (reviewed by [2] ). It can be said that such investigations on isolated heart preparations, not only in the case of L. stagnalis but also in general, utilize classical mechanical force transducer-based approaches. The aim of the present work was to develop a rapid, easy, cheap, digital, and in vitro pharmacological method based on an effective and non-invasive refraction-based optical recording approach and isolated heart preparations.

Heart preparations and animals
For this study, isolated heart preparations ( Fig. 1 ) were obtained from five-month-old L. stagnalis specimens which has been cultured in our laboratories in Brighton and Tihany. The animals were maintained in large plastic holding tanks (stocking density: 100 individuals/tank) containing 10L oxygenated artificial snail water with low copper content at a constant temperature of 20°C ( ± 1°C) on a light:dark regime of 12 h:12 h. Snails were fed on lettuce ad libitum three times a week.
The physiology of the two-chambered heart of L. stagnalis is well-known: while heartbeat is generated by a muscle pacemaker located in the heart (i.e. myogenic), its regulation is basically implemented by many neurotransmitters and neuropeptides (e.g., serotonin, dopamine, and FMRFa) which are released by several types of motoneurons into the heart [3 , 4 , 6-8 , 17-20] .
Individual hearts were dissected from the animals and individually pinned out without cannulation (i.e. both auricle and ventricle could contract) on Sylgard-coated dishes ( Fig. 2 ) containing Lymnaea physiological saline. Pinning out serves a fundamental purpose, as the action of stretching the hearts stimulates the myogenic heartbeat [10 , 15 , 16] . Using a three-way tap (the perfusates can be switched quickly with only minimum mixing), pressure heads, and vacuum for wash out, the heart preparations can be easily maintained and perfused with physiological saline or the test chemical(s) in the dish.

Refraction-based optical recording system
The optical recording system developed in our study is a novel and extremely unique method by which sensitive recordings of the heart muscle (or any contractile tissue) contractions can be made and subsequently analysed. In contrast to the classic mechanical force transducer set-up (muscle is fixed to a transducer, contractions are recorded and converted to digital format by an analogueto-digital converter, amplified, displayed on an oscilloscope and/or computer, and analysed using a specific software), our system provides a rapid, easy, cheap, digital and 'non-invasive' recording.
The system utilizes the inbuilt optical sensor and signal converter of an optical/laser computer mouse that sensitively measures motions of the mouse dots per inch (DPI). This information is transmitted into a computer through a USB port (or even via WiFi or Bluetooth connection) and subsequently converted into cursor movements (graphs) on the monitor through a specialised software named DigiRec (detailed in the next subsection). In the present study, the optical sensor and the converter were taken out of the mouse (1600 DPI, 0.02 mm resolution) to be used for recording the movements made by the contracting heart. Importantly, the greater the DPI of the mouse applied, the greater the sensitivity. A heart preparation pinned out along either an X or Y axis is placed directly above the optical sensor ( Fig. 3 ).

DiciRec
The DigiRec application used for the recording was written in Java programming language and can be run under the Linux operation system. The program monitors the changes of the refraction caused by muscle contraction above the optical sensor and not the movements of the cursor on the screen, allowing a more accurate recording. The individual records are stored in the own file format of the software but the data can be exported in different well-known file formats (CSV, XLS) for further data processing.
The graphical interface ( Fig. 4 ) allows the user to start and stop recording, display graphs, and replay the events. The first slider manipulates the width of the graph, while the second one manipulates the resolution in milliseconds. The time is displayed at the bottom along with information about the movements of the heart using the following coordinates: Max X, Total X, Max Y, and Total Y. The first values are movements of the heart in DPI but the software also displays the values in mm. Importantly, in order to display the appropriate mm values, one needs to enter the DPI value of the applied mouse into the "gui.props " utility program. The optical sensor records the changes of the refraction as movements in a positive or negative direction along either axis ( Fig. 5 ).   5. A representative record from an isolated heart preparation ( A ). The lower panel of the interface shows the muscle activity which corresponds to the coordinates (Max X, Total X, Max Y, and Total Y) along which the heart is pinned out (along the Y axis in this instance ( B )). Blue columns contain the delta which is the amount of displacement within the given time interval (represented by a bar) detected by the sensor, i.e. the sensor gives no specific coordinates but displacement values. The red line, which changes according to the blue bars, represents the actual position. The delta is always added to the red value. During the phases of the heart contraction the auricle (A) contraction is followed by the ventricle (V) contraction ( C ). A representative auricle and ventricle contraction event recorded by DigiRec is shown on panel D . Average 'total distance' measurements for the control and DA recordings. Each bar represents mean ± SD. The white column shows the control data (before dopamine treatment) while the grey column represents the data after the dopamine treatment. Significance of differences to the control group is marked by asterisks ( * * * P ≤ 0.001, paired t-test).

Method validation and suitability of the recording system
To assess the suitability of the developed recording system in pharmacologic experiments, we tested if we were able to record a known pharmacological effect on the heart preparations. The substance tested was dopamine which is known to have a cardioexcitatory effect in a variety of molluscan species including L. stagnalis [7] . Specifically, dopamine has been shown to increase both heart rate and contraction amplitude in the heart of L. stagnalis with slightly increasing the tone level [7] .
During the experiments, we tested the effects of a 10 μM dopamine solution on individual heart preparations (n = 6). The two parameters investigated, amplitude and frequency, are together referred to as total distance. For each heart preparation, the original heart contractions were recorded as control followed by recording the heart contractions after treatment with dopamine. This always was a continuous recording. Compared to the control recordings, our results demonstrated that dopamine has significantly increased the amplitude and frequency of the contractions (P < 0.001; paired t-test) ( Fig. 6 ). Our results confirm dopamine as a cardioactive neurotransmitter in L. stagnalis and highlight that the optical recording system is entirely suitable for pharmacological experiments.

Perspectives
Nowadays, there is a great need to test the physiological effects of different chemicals. The development and optimization of new methods are crucial to move forward such investigations. The refraction-based recording system illustrates the progression of technology from the mechanical force transducer system used not only for L. stagnalis research but also for higher organisms. Our system offers a highly suitable tool in ecotoxicology (e.g., testing the effects of pharmacologically active compounds found in the ecosystem) or neuroscience (e.g., testing the effects of different neurotransmitters and neuropeptides).

Ethics statements
All procedures on animals were performed according to the protocols approved by the Scientific Committee of Animal Experimentation of the Balaton Limnological Research Institute (VE-I-001/01890-10/2013).