In-vitro superfusion system (Release technique)
In-vitro superfusion system
The solutions of the in-vitro superfusion release technique result in a system which is easy to use and can be assembled in a mobile way. It enables the collection of neurotransmitters from the nerve tissues with electrical or chemical stimulation (for details see section How can it help?).
The two-chamber brain slice perfusion system is the unique combination of the wedge preparation technique, the in-vitro superfusion system and the radioactive neurotransmitter release technique. Its purpose is to evoke neurotransmitter release and biopotential responses in one part of the nervous tissue by selective stimulation of the other part, and to simultaneously measure the two responses.
System
Scheme of the system
Main components
- I. Central unit with superfusion chambers and pre-heater,
- II. Heat-stabilizer and buffer supply unit.
Central unit
I. Central unit with superfusion chambers and pre-heater
The elements of this component are placed on a specially designed stand in the following order, top to bottom:
- Incubation chambers with carbogen controller,
- Slice chambers with bubble trap and stimulating electrodes (superfusion unit),
- Heat exchanger (pre-heater).
1. Incubation chamber with carbogen controller
Carbogen controllers
Gas controller
The double-walled, 10 ml capacity chamber is connected to the thermo stabilizing unit through the liquid circulating in the jacket. The chamber is to keep the tissue slices prepared for the measurement alive, carbogenized, and at appropriate temperature, until the measurement starts. Continuous supply and appropriate condition of the gas necessary for incubation is provided by the buffer vessel, and the fine adjuster and atomizer unit.
The unit makes the process of measurement easier and faster.
2. Slice chambers with bubble trap and stimulating electrodes (superfusion unit)
Slice chamber 3
Slice chamber 2
Slice chamber 1
- The slice chambers are situated in the holder. The holders contain the stimulating electrodes. The electrodes are directly connected to the gold-plated banana sockets, which receive the cable of the electric stimulator.
- The slice chamber can be placed into the sealed slot in the inner surface of the holder. The holder can be closed with the mounting screws after the chamber is inserted. The bubble trap is located at the buffer inlet point in the lower closing holder.
- The buffer outlet can be found in the upper closing holder.
Screwdriver
Drawing of the measuring chamber
The slice chamber is the most important element of the system. Hence it was designed in a way enabling the tissue slice to be put into the chamber the fastest and safest way possible before measurement. It is also important that the (electrical or chemical) stimuli, initiating the process, reach the tissue slice with the best efficiency. The buffer liquid must be kept at stable temperature, and in continuous and bubble-free flow. To ensure these conditions, the chamber is made up from five easily attachable units.
Measuring space
Assembly of the measuring chamber
The lower closing lid, together with the mesh disk, can be fixed to the organ chamber with threads.
The organ slice is placed in the 98µL measuring space, which is closed by the mesh disk and the top closing lid.The lower closing lid, together with the mesh disk, can be fixed to the organ chamber with threads.
The closed chamber is fixed between the holders with screws. The stimulating electrodes reach into the measuring space above the mesh disk in a self-positioned way.
II. Thermal stabilizer and buffer conveyor unit
Thermal stabilizer
The buffer liquid is stored in the thermo stabilized tank of the circulation water bath. The inner temperature of the tank can be adjusted between 25-45°C with 0,1°C accuracy. The buffer liquid is delivered to the measuring space by the peristaltic pump. Heat loss during delivery is compensated by the heat exchangers with outer jacket. The jacket is connected to the water circulating circle, and heat loss of the buffer liquid is replaced while running through the spiral. The inner temperature of the heat exchanger is calibrated to the temperature required in the measuring space.
Accessories required for operation
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CWB-02 Circulation water bath
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PRO-IM-02 Peristaltic pump
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FC 203 B Fraction collector
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EXP-ST-04 Four-channel non-isolated stimulator with MCU controlling unit
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How can it help?
Examination and experimental influencing of neurotransmitter release is essential for studying information transfer between nerve cells and neural regulation of other organs. Neurotransmitter release under in-vitro circumstances can be evoked by electrical or chemical stimulation of nerve tissue preparations.
Scheme of the system
Given the particular sensitivity of the nerve cells, continuous stable supply of oxygen and nutrients must be provided for the preparation during the whole experiment. The closed-system superfusion device is used for this purpose. A peristaltic pump ensures continuous supply of pre-heated, carbogenized physiological solution for the nervous tissue preparation placed in the perfusion chamber of minimal dead space. The complete perfusion system includes the electric stimulator, the thermostat, carbogen (95% O2, 5%CO2) gas dosing unit, and the perfusion chambers. A great advantage of the perfusion chamber is that it is suitable for the examination of neurotransmitter release induced by electrical field stimulation or chemical stimuli. Electrical stimulation of the slice is performed by the platinum electrodes built into the chamber, while chemical stimulation can be achieved by substances dissolved in perfusion fluid, but the chemical stimulants can also be directly delivered to the perfusion chamber. The flow rate can be regulated, it can even be stopped for a given interval (e.g.: experimental ischemia). The effluent leaving the chambers contains the radioactive labeled neurotransmitter released from the preparation upon stimulation. This latter one can be virtually any neurotransmitter, or neuromodulator substance, of which an isotope labeled version is available, and which the nerve cells in the tissue are able to take up, store, and give off upon relevant stimuli into the intercellular space.
The most commonly used molecules in the experiments are tritium labeled, but 14C isotope labeling is also possible. Moreover, the two isotopes can be used simultaneously, which allows the parallel study of release of two different transmitters/modulators. The continuously circulating perfusion fluid washes away a part of the released radioactive neurotransmitter from the intercellular space, which thus appears in the effluent and can be measured. The major advantage of the radioactive neurotransmitter release technique is that the test results are available within a few hours.
Loading unit
In contrast to the conventional way isotope delivery to the tissue (soaking), we developed a more reliable, faster and safer method. For that, the LU -01 loading unit has been developed which can be organically integrated into the measuring system.
The essence of the LU-01 loading unit is that the tracers (e.g.: isotope) can be delivered to the tissue from a closed, heat-stabilized liquid chamber by stimulating impulses. The tissue is placed in the incubator vessel the same way as it is placed in the perfusion chamber of the measuring system. The same applies to the transmission of stimulus current (spatial stimulation). The outer jacket of the vessel is connected to the heating circle of the measuring system, however, carbogen supply and buffer solution input are independent.
Available models
You find details by clicking on the product code.
*The system is also available in one- or two-channel models, please, contact us for further details!
References
| Year | Author | Title | Source |
| 2004 | Bujdosó E. | Role of recently discovered opioid neuropeptides in the regulation of Open-Field Behaviour and the Hypothalamic-Pituitary-Adrenal axis | Department of Pathophysiology, Albert Szent-Györgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged |
| 2009 | Világi I, Dobó E, Borbély S, Czégé D, Molnár E, Mihály A. | Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex |
Exp Neurol. 2009 Sep;219(1):136-45. doi: 10.1016/j.expneurol.2009.05.005. Epub 2009 May 13. |
Note: experiments were performed with different types of isolated nerve tissue systems.
Media
Download the Isolatednerve tissue systems catalogue in pdf version!
