Are there salt water equivalents of leeches?

Importance of blood components for the function of the central nervous system of the leech

Table of Contents

1 Introduction
1.1 Anatomy and physiology of the leech
1.2 Hirudo's nervous system
1.3 Blood composition of Hirudo
1.4 Effect of storage media on isolated segmental ganglia
1.5 question

2 Material and Methods
2.1 Obtaining and keeping the leeches
2.2 Preparation and storage of the segmental ganglia
2.3 Storage media
2.4 test solutions
2.5 microelectrodes
2.6 Bath earthing
2.7 Measuring apparatus and experimental set-up for electrophysiology
2.8 Electrophysiological test protocols
2.9 microscopy
2.10 Data evaluation

3 results
3.1 Electrophysiological experiments
3.2 Morphological changes in the segmental ganglia

4 discussion
4.1 Objective of the work & summary of the test results
4.2 Material uptake and energy metabolism in the segmental ganglia of the leech
4.3 The hypothesis of anaplerotic uptake of organic acid residues

5 Summary

6 Bibliography and sources

7 Appendix

8 Declaration

9 Acknowledgments

1 Introduction

1.1 Anatomy and physiology of the leech

The leeches of the genus Hirudo are originally parasitic annelids native to Eurasia and Africa. The natural habitat is shallow, nutrient-poor bodies of water such as the bank areas of rivers and lakes, marshland and other humid land biotopes, rarely also brackish water zones in the estuary of rivers.

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Figure 1: Habitus of Hirudo verbana. (Photo from deserts 2003).

The body of the leech is bristle-free, 3 to 5 cm long in the unstretched state and up to 15 cm long in the stretched state (Fig. 1). Adult leeches can reach up to six inches in length when stretched. The body is segmented, whereby the outer rings (annuli) do not depict the actual segmentation. The coelom is secondarily reduced and fused with connective tissue. The skin muscle tube consists of ring, transverse, longitudinal and dorsoventral muscles and is strong. At the front and rear ends of the body, Hirudo has two muscular suction cups that are used to move and attach to surfaces. The front suction cup (oral suction cup) has three strong, tooth-reinforced jaws that can be used to bite through the skin of host animals. Leeches parasitize on all available vertebrates and usually take in several ml of blood per meal. Hirudo is hermaphrodite and egg-laying and becomes sexually mature at around 3 years of age. Leeches can live up to 20 years. (Sawyer 1986, Mehlhorn & Piekarski 2002)

The leech has a closed blood vessel system with four large main vessels (dorsal and ventral vessels as well as two lateral vessels), which run through the animal in the longitudinal direction and are connected to one another by numerous smaller vessels. The lateral vessels, encased by muscles, form the heart tubes of the leech, which ensure a continuous flow of blood through rhythmic contractions (Kristan et al. 2005).

The most prominent species of the genus Hirudo is the medicinal leech Hirudo medicinalis. The Hungarian leech has long been regarded as its subspecies Hirudo medicinalis var. Verbana. According to recent molecular genetic studies, however, it is now regarded as a separate species of Hirudo verbana (Trontelj et al. 2004, Siddall et al. 2007). Regardless of the taxonomic status, the term Hirudo verbana is used in this work.

1.2 Hirudo's nervous system

The leech's central nervous system (CNS) is segmented and is considered a variant of the rope ladder nervous system. It consists of 34 ganglia, the first 6 of which are fused to form a head ganglion and the last 7 to form an anal ganglion. The remaining 21 ganglia form the segmental ganglia, the abdominal marrow, which is located in the animal's ventral blood vessel. The segmental ganglia are connected to one another by paired connectors and the Faivre's nerve, as well as to their effector organs by 2 pairs of lateral roots. Each segmental ganglion can be assigned to a body segment. The nervous system is unvascularized, but due to its location, blood is constantly flowing around it (Sawyer 1986).

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Figure 2: Central nervous system of the leech. Anatomy and location in the body.

left: Ventral view with the position of the head and anal ganglion as well as the 21 segmental ganglia (after Gillon & Wallace 1984). Right: Elevation drawing with skin muscle tube, digestive tract, the large blood vessels and segmental ganglia chain in the ventral vessel (from Nicholls & van Essen 1974)

The individual segmental ganglia have a cross-section of ~ 500 gm and are stereotyped.

The ganglion as a whole is surrounded by a connective tissue capsule. The ~ 400 outer neuron cell bodies are arranged in 6 packages, each of which contains neurons, a large, highly branched packet glial cell and a large number of microglial cells. The neuropil inside the ganglion contains axons and dendrites of the neurons, which form synaptic contacts here, as well as two large, branched neuropil glial cells. Only the fifth and sixth segmental ganglion, which innervate the genital organs, deviate from this schematic structure with a total of ~ 700 neurons.

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Figure 3: Segmental ganglion of the leech.

left: transmitted light from the ventral side (from Nicholls & Baylor 1968); anterior above, posterior below. The contours of the neuron cell bodies are clearly recognizable. The packet boundaries appear as lighter, cell-free areas. The connective and the Faivre nerve connect the segmental ganglia with one another, the paired lateral roots connect the segmental ganglion with the periphery. Right: Cross-section schematic drawing (Nicholls & van Essen 1974, after Coggeshall & Fawcett 1964). The neuropil in the center contains axons and dendrites of the outer neuron cell bodies. The connective tissue capsule surrounds the ganglion.

In the context of this work, two types of nerve cells are of particular importance. The centrally located, paired Retzius neurons with a soma 50 - 80 pm in diameter are the largest neurons of the leech ("colossal ganglion cell", Retzius 1891). They are electrically coupled to one another, spontaneously fire action potentials and are involved in coordinating swimming movements, controlling muscle tone and mucus secretion (Carretta 1988). The Leydig neurons are significantly smaller and lie peripherally in front of the bifurcation of the lateral roots. They too spontaneously fire action potentials and are involved in modulating the heartbeat by controlling the contraction of the lateral vessels (Arbas & Calabrese 1990).

1.3 Blood composition of Hirudo

The composition of Hirudo's blood has been studied several times. The existing analyzes are inconsistent; Housing conditions, blood collection methods and analysis results vary greatly in some cases.

Nicholls & Kuffler (1964) determined the Na + content to be ~ 130 mM and that of K + to be ~ 4 mM, but made no statement about the anions present. Boroffka (1968) determined the ionic composition of the blood with 125 mM Na + and 36 mM Cl ". Thus, there was a deficit of ~ 90 mM negative charges, which was attributed to the possible presence of polyvalent anions Compounds carried out which showed that organic acid residues are the most important anions in the blood of Hirudo medicinalis (Zerbst-Boroffka 1970), according to which it contains citrate, lactate, fumarate and succinate, each in concentrations between 5 and 15 mM The anion deficit could thus be regarded as clarified within the scope of the measurement uncertainty.

Table 1: Resting Hirudo blood composition. Literature data, abbreviated. A complete list can be found in Tab. 16 in the appendix.

Zebeetal. (1981) examined the metabolic processes of the leech in environments with different oxygen levels. They came to the conclusion that under normoxic

Conditions ~ 9 mM malate is present as the only organic anion in relevant concentration. Thus there was again a strong anion deficit. This result was largely confirmed by Hildebrandt & Oeschger (1987). Wenning & Hoeger (1987) again confirmed malate as the most important organic cation, but found a total concentration of organic acid residues equivalent to ~ 83 mM single negative charges, whereby the anion deficit was again considered to be closed. Hoeger et al. (1989) confirmed this result, but found that the blood composition is apparently strongly influenced by the method of blood sampling. Experiments on energy metabolism by Hildebrandt & Zerbst-Boroffka (1992) and Nieczaj & Zerbst-Boroffka (1993) essentially confirmed these data and also found that the composition of the blood depends on the salinity of the water and the CO2 content of the Air in which the leeches were kept. The data collected by Zerbst-Boroffka (1970) resemble the blood composition after strong muscle activity or after several hours in salt water. The mentioned literature data are summarized in abbreviated form in Table 1 and in full in Table 16 in the appendix. See comment on p. 56 regarding areas highlighted in orange. In summary, based on the current data on the composition of leech blood, it can be stated that malate is the most common anion with ~ 15 mM, Na + is the most common cation with ~ 130 mM.

1.4 Effect of storage media on isolated segmental ganglia

In Ringer's solution (“normal salt solution”) adapted to the leech, the life of isolated segmental ganglia is about one day (Lucht 1997, Falkenberg 2009). Isolated neurons survive ~ 2 days in Ringer's solution (Nicholls 1987).

The cultivation of isolated leech segmental ganglia or neurons is an established working technique. Leibovitz medium L-15 was first used as a culture medium by Miyazaki et al. (1975) used for this purpose. L-15 medium is a tissue culture medium which is suitable for the cultivation of neuronal tissue, but also of invertebrate cell lines (Morton 1970). It contains inorganic salts (Na +, Cl-, K +, Mg2 +, H2POz), 17 proteinogenic amino acids (no aspartate, glutamate, or proline), vitamins from the B group, galactose, 5 mM pyruvate and other components in concentrations in the gM- Area. (Leibovitz 1963).

Usually 2 - 10% fetal calf serum (FBS), as well as antibiotics (e.g. penicillin, ampicillin, streptomycin) and antimicotics (e.g. nystatin, amphotericin) are added to the medium. In most cases, glucose is also added, almost always at a concentration of 30 mM.

In this culture medium, the electrophysiological properties of the neurons of isolated segmental ganglia remain unchanged for ~ 3 weeks, only the connective tissue capsule becomes slightly cloudy (Miyazaki & Nicholls 1976, Ready & Nicholls 1979). Isolated neurons can retain in situ properties for several weeks even without glial cells (Fuchs et al. 1981). Even without the addition of glucose, the cells have been reported to have a shelf life of up to 3 weeks (Nicholls et al. 1990).

Independent of the Leibovitz culture medium, various blood substitute solutions with different proportions of organic acid residues were proposed based on the existing analyzes. Falkenberg (2009) investigated the influence of Zerbst-Boroffka blood substitute solution (5 mM citrate, 15 mM succinate, 10 mM fumarate, 10 mM lactate; cf. Appendix, Tab. 17) on the electrophysiological behavior of the Retzius neurons in isolated segmental ganglia. The following results were obtained:

The resting membrane potential (rest Em) of the Retzius neurons was independent of whether they were stored in normal salt solution (NSL) or blood substitute solution (BEL) on the day of preparation. The resting Em practically collapsed within three days in NSL, while it remained stable in BEL. In BEL, spontaneous action potentials are formed over a period of at least 3 days, in NSL only for one day. The Cl - equilibrium potential (Ea) of the Retzius neurons was permanently more negative in BEL than in NSL. The mechanical stability and transparency of the connective tissue capsule of the segmental ganglia remained in BEL for at least 3 days, while ganglia stored in NSL quickly became cloudy and brittle.

Zokoll (2010) found that reduced BELs containing only citrate, fumarate or succinate are similarly suitable for extending the shelf life of isolated ganglia as complete BELs with all components. A reduced BEL, which only contained lactate as an organic acid residue, on the other hand, had no effect in this direction. The anaplerotic addition of citrate, fumarate and succinate to the citric acid cycle was cited as a possible explanation for the extended shelf life of the ganglia in the presence of organic acids.

1.5 question

The position of the leech's central nervous system in the ventral vessel suggests that the nervous system is supplied with energy via the animal's blood (see 1.2). The studies by Falkenberg (2009) and Zokoll (2010), which are based on an analysis of the blood composition by Zerbst-Boroffka (see 1.4), have shown that the lifespan of isolated segmental ganglia was significantly extended in the presence of citrate, succinate or fumarate, while lactate did not have this effect. This finding suggests that the segmental ganglia are able to take up and metabolize these organic acid residues.

However, later investigations showed that malate is the primary organic anion in leech blood (see 1.3). Therefore it was examined here whether the presence of malate extends the life span of isolated segmental organisms similarly to citrate, succinate or fumarate.

There is also a large number of studies in which isolated segmental ganglia or individual cells were cultured for up to 3 weeks. The culture medium used throughout was Leibovitz-15 medium (see 1.4) that contains, inter alia, 5 mM pyruvate. Therefore it was also examined here whether pyruvate is able to extend the life span of isolated segmental ganglia.

To nourish segmental ganglia in culture, glucose was often added to the medium, usually 10 mM in Ringer's solution (Nicholls & Baylor 1968) and 30 mM in Leibovitz medium. However, it is known that cells of the leech CNS can be cultivated in Leibovitz medium for up to 3 weeks without the addition of glucose. The question arises as to whether glucose is actually suitable for feeding isolated segmental ganglia. That is why it was investigated here whether glucose is able to extend the life span of isolated segmental ganglia.

In addition, the question of whether malate, pyruvate or glucose change the electrophysiological properties of the cells of isolated segmental ganglia was investigated.

2 Material and Methods

2.1 Obtaining and keeping the leeches

Animals caught in the wild by the importer for 32 weeks and sold under the trade name “Medicinal leeches: Hirudo medicinalis / verbana / orientalis” were used in Serbia and near Turkey. All test animals were identified as H verbana on the basis of their abdominal and back drawings (cf. Siddall et al. 2007). The animals were not fed during the interim storage (M. Aurich, Biebertaler Leegelzucht, personal communication from March 8, 2011). The keeping in the laboratory took place in air-permeable closing

Plastic dishes that were ~ 2.5 cm high filled with tap water mixed with water treatment agent, at 10 - 12 ° C in a refrigerator. At the time of the experiment, the leeches were adult, unstretched 3-5 cm and stretched 9-13 cm long. The stomach was filled with blood in each case during the preparation.

2.2 Preparation and storage of the segmental ganglia

The leeches were pinned to the abdomen and head suction cups with pins in a bowl with a wax base and stretched. It was killed by making a deep incision behind the oral suction cup and severing the connector between the 1st and 2nd segmental ganglion. The animal was cut open along the dorsal median line. The opening of the body cavity and dissection of the CNS was carried out using eye scissors under a reflected light stereomicroscope (Lucht 1998). At the individual segmental ganglia, a ~ 2 mm connective dorsal and ventral side roots were left, and ~ 1 mm lateral roots were left in each case. The isolated segmental ganglia were transferred to glass dishes with ~ 1.8 ml storage solution each, covered with watch glasses so that they were permeable to air and stored at 10-12 ° C in the refrigerator. To reduce the bacterial load, the glucose-containing medium (see 2.3) was changed daily, all other media at least every 3 days.

2.3 Storage media

All solutions that were brought into contact with the interior of the body cavity and the isolated CNS were physiological solutions with pH 7, buffered with HEPES (2- (4- (2-hydroxyethyl) -1-piperazinyl) ethanesulfonic acid) , 4. All storage media also served as superfusion solution on the measuring apparatus in the experiments.

The following stock solutions were used to prepare the storage media:

1M NaCl, 1 M KCl, 1 CaCh, 500mM MgCh, 1M HEPES, 100mM Na pyruvate, 100mMNa2 malate. The pH was adjusted with 1M NaOH. A complete list of the chemicals used can be found in Tab. 13 in the appendix.

Normal salt solution (NSL) is a modified Ringer's solution for leech preparations (Lohr 1998, cf. Nicholls & Baylor 1968).In addition to storing ganglia, it was also used as an irrigation solution during dissection. The solution was stored in the refrigerator and made up weekly.

Table 2: Composition of the storage media used. Concentrations in mmol / l, osmolarity in mosm / l

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To prepare normal salt solution with glucose (glucose NSL), 0.49 g of glucose monohydrate were weighed in, dissolved in 250 ml NSL and the solution was then sterile-filtered using a syringe filter with a pore diameter of 0.22 μm. The solution was stored in the refrigerator and made up every three days.

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Table 3: Substances in the storage media

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In the blood substitute solutions (BEL) used, the concentrations of cations and HEPES of the NSL were retained, Cl- was proportionally replaced by organic acid residues.

5 mM pyruvate blood substitute solution (pyruvate BEL) is based on the pyruvate content of Leibovitz medium. The solution contains 5 mM pyruvate, with which the Cl content was reduced by 5 mM compared to NSL. The solution was stored in the refrigerator and made up weekly.

15 mM malate blood substitute solution (Malat-BEL) is based on the analyzes of leech blood by Zebe et al. (1981) and Hildebrandt & Zerbst-Boroffka (1992). The solution contains 15 mM malate. Since malate is a divalent anion, the Cl content was reduced by 30 mM compared to NSL. The solution was stored in the refrigerator and made up weekly.

2.4 test solutions

For the electrophysiological tests, test solutions with increased K + concentration, serotonin (5-hydroxytryptamine, 5-HT) and kainate were used. A complete list of the chemicals used can be found in Tab. 13 in the appendix.

40 mM K + test solutions were prepared analogously to the storage media in 2.3, 36 mM NaCl being replaced by 36 mM KCl in each case. The pH was adjusted with 1 M KOH instead of NaOH. The solutions were stored in the refrigerator and made up weekly

To prepare the serotonin test solution, 1 mM serotonin-creatinine-sulfate complex was weighed into the appropriate storage medium. The solutions were stored in the refrigerator and made up every 3 days.

To prepare kainate test solutions at 1, 3, 10 and 30 pM, the appropriate amount of 25 mM kainic acid stock solution was pipetted into the respective storage medium. The solutions were stored in the refrigerator and made up weekly

2.5 microelectrodes

Electrolyte-filled single-channel glass microelectrodes were used. To produce the electrodes, borosilicate glass capillaries (outer diameter 1.5 mm, inner diameter 0.86 mm, length 7.5 cm) were drawn out sharply with a vertical puller. These raw electrodes were filled with electrolyte without air bubbles using a disposable syringe with a 23-gauge cannula (0.5 M K2SO4 / 20 mM KCl in aqueous solution). A chlorinated silver wire (0 0.5 mm) was inserted into the electrode shaft so deep that the front end of the wire was ~ 1 mm before the start of the taper. The electrodes were sealed airtight at the rear end with hard adhesive wax. The electrode resistance was ~ 60 MD.

A compilation of material and equipment can be found in Tab. 13 and Tab. 14 in the appendix.

2.6 Bath earthing

The test bath was grounded via an agar bridge. To create the bridge, pieces of plastic tubing (0 outside 3 mm, 0 inside 2 mm, length 4 cm) were filled bubble-free with 3% agar in 3 mM KCl solution, a chlorinated silver wire was inserted up to ~ 8 mm from the end of the tube and the electrodes at the rear end with it Hard adhesive wax sealed airtight.

2.7 Measuring apparatus and experimental set-up for electrophysiology

The isolated segmental ganglia were fixed in an experimental chamber. For this purpose, the bottom of a flow-through chamber made of Plexiglas was filled with two-component silicone rubber and the connective and lateral roots left on the ganglia were pinned into them with minutiae (fine insect needles) while pulling gently. The superfusion solutions were fed in according to the principle of a siphon, with the tubes (0 inside 1 mm) being led through a mixer tap fitted with clamps in order to be able to change the solution quickly. The solution was sucked out of the chamber through a capillary connected to a roller pump. The chamber volume was ~ 0.13 ml and the superfusion rate was ~ 14 pl / s; thus the solution in the chamber was changed approximately every 9 seconds.

The bath electrode was led through a hole into the test chamber. The bore was located opposite the solution inflow in the vicinity of the suction capillary, so that contact of the preparation with the KCl diffusing from the agar bridge was excluded. The microelectrode was fixed in the holder of a mechanical micromanipulator. Both electrodes were connected to a measuring head via copper cables with crocodile clips, which in turn was connected to an electrode amplifier via a low-resistance cable. Starting from the electrode amplifier, the signal was displayed on an oscilloscope and recorded in analog form using a paper pen. At the same time, the signal was digitized with an analog-to-digital converter (digitizer) and transmitted via a serial connection to a PC, where it was recorded with a data recording program at a recording frequency of 5 kHz.

The experiments were carried out for optical control under a stereo microscope with a magnification of 17.5 to 112.5 times. For this purpose it was possible to illuminate the chamber from below.

To reduce noise and interference, the measuring chamber, suction device, micromanipulator, electrodes and measuring head of the electrode amplifier were located on a vibration-damped steel plate that was mounted on pneumatic shock absorbers. This part of the experimental setup was housed within a Faraday cage made of sheet steel. The individual components (with the exception of the microelectrode) were connected to one another in an electrically conductive manner and earthed via the earthing connection of the oscilloscope.

A list of the devices used with manufacturer and type designations can be found in Tab. 14 in the appendix.

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Figure 4: Experimental setup for electrophysiology.

Simplified scheme of the measuring apparatus.

The cells examined were identified based on their location and their electrophysiological behavior. After the electrode had been punctured, it was waited for a while until the measured membrane potential remained stable in the range of ± 1 mV over a period of at least 1 min. This healing time was between 2 and 30 minutes, usually around 10 minutes (see Figs. 10, 13, 16). After the healing, the respective test was carried out. After completing the experiment and pulling the electrode out of the cell, the recording continued for about 1 min in order to be able to determine any drift in the reference potential. All solutions used were examined in bath controls for their influence on the electrode potential. Only changing the superfusion solution from NSL to Malat-BEL led to a change in the electrode potential of -4 mV. This effect was taken into account in the data analysis. All experiments were carried out at room temperature from 18 to 25 ° C.

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