Microdialysis is a progressive method for studying the neurochemical mechanisms of learning, allowing measure- ments of natural changes in transmitter and ...
Neuroscience and Behavioral Physiology, Vol. 36, No. 6, 2006
Microdialysis in Freely Moving Animals with Simultaneous Recording of Electrophysiological Processes at the Dialysate Collection Point V. A. Korshunov
Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 6, pp. 700–705, June, 2005. Original article submitted November 4, 2004.
a coaxial cannula in which the parts which are difficult to make are reusable and the disposable parts are easily replaced. The details of the instrument are diagrammed in Fig. 1. The cannula is made from segments of injection needles (diameters 0.4 and 0.8 mm) joined with solder (using 10–30% orthophosphoric acid as flux). The input tube 3 is connected to the collector 4. This tube was made by inserting a 0.4-mm diameter needle into a 0.8-mm diameter needle with fixation by soldering. The thicker needle reinforces the construction and functions as a holder 6. An opening is cut into this part, into which the curved recording tube is inserted as shown in Fig. 1, 5. This base assembly can be used repeatedly. A micropipette 2 was drawn from borosilicate glass using a microforge, with a tip length of 30–50 mm, and was inserted into the input tube 3 of the base assembly as far as the stop and was fixed with fused rosin 8 (it was sufficient to place a pellet of rosin on the edge of the input tube and apply a heated soldering iron). The micropipette was snapped off at the edge of the input tube and the end protruding below was cut at a distance of 0.3–0.5 mm. A length of dialysis tube (Spectrum OD × ID = 200 × 187) of length 1.6 mm 1 was placed downwards in the collecting tube of the base assembly 4 and attached with epoxy resin. The tip of the dialysis tube was filled with epoxy resin 7 to provide a hermetic seal. After the resin had set (24 h), the cannula was ready for use. The cannula could be used up to three times, after which the membrane and capillary needed to be replaced, as follows. Forceps were used to extract the dialysis tube. The upper part of the base assembly was brought into contact with a hot soldering iron and the capillary was extracted
Microdialysis is a progressive method for studying the neurochemical mechanisms of learning, allowing measurements of natural changes in transmitter and modulator concentrations in different brain states as well as (in the case of reverse dialysis) local application of substances which cannot be applied iontophoretically (for example, non-polar compounds). We describe here a method combining microdialysis with simultaneous recording of neurophysiological processes occurring at the point of dialysate collection in freely moving animals, allowing correlations between neurochemical and neurophysiological rearrangements in neural networks to be identified. The instrument described here combines microdialysis with recording of evoked potentials or neuron activity using stationary implanted macro- and semimicroelectrodes.
THE MICRODIALYSIS CANNULA Local dialysate collection and local application can be performed using various types of microdialysis cannulae [1]. Cannulae of the coaxial type have the smallest crosssectional area and produce a minimum of tissue damage, but they are difficult to make and traditional methods of attaching them to animals’ skulls (using dental cement) do not allow them to be reused. Employment of single-use cannulae is inappropriate, especially as only the two simplest parts are disposable – the semipermeable membrane and the capillary applicator. This article reports the construction of Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5a Butlerov Street, 117865 Moscow, Russia.
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Fig. 1. Cross-sectional diagram of cannula. 1) Dialysis tube (Spectrum OD × ID = 200 × 187); 2) glass capillary; 3) input tube; 4) collecting tube; 5) output tube; 6) support; 7) epoxy resin; 8) rosin; 9) silicone tubes. Sizes are given in mm. *The length depends on the depth of the structure being studied.
upwards using a fine wire. A 0.2-mm drill was used to remove residues of epoxy resin from the collecting tube and the base assembly was washed with alcohol (to remove traces of rosin) and then with ultraclean water (18 MΩ/cm). The base assembly was then ready for reuse.
CHRONIC RECORDING OF EVOKED POTENTIALS AND NEURON ACTIVITY Chronic recordings of evoked potentials were made using the construction shown in Fig. 2. Openings of diameter 0.8 mm were drilled into the Teflon platform 1 for the contact pins 6 and guide tube 2 in which the cannula was placed. M1 threads were cut into the bottom ends of the contact pins. The platform bore a step preventing the liquid from flowing from the guide tube to the contacts. Since dental cement does not provide reliable adhesion with Teflon, holes were drilled in the sides of the platform. Flowing through these, the cement mechanically attaches the platform to the animal’s skull. The guide tube was made of segments of injection needles of diameter 0.8 mm. The dis-
tance between the electrodes 7 and cannula 11 was decreased by grinding the guide tube on one side 2 and a groove was drilled for the fixer on the other side 3. The fixer consisted of 2–3 turns of wire (200 µm stainless steel) tightly wound around the guide tube at the groove. A ring made of 0.2-mm wire 5 was soldered to the guide tube to prevent vertical movement of the tube in the platform. The upper part of the guide tube was cut into a cone to facilitate attachment of the microdialysis cannula. Evoked potentials were recorded using electrodes (Nichrome 80–100 µm in factory insulation), which were inserted into the cleft between the ground side of the guide tube and the wall of the opening in the platform. Insulation was removed from the upper part of the electrodes, which were soldered to the contact pins on directly the platform, as Teflon is resistant to soldering iron temperatures. Depending on the orientation of the cells in the study area of the brain, the electrode tips could be located at different heights relative to the dialysis tube. The desired height in the guide tube was attained by inserting a prepared dialysis cannula to the stop and cutting the electrodes at the appropriate distance.
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Fig. 2. The platform apparatus. A) Plan view; B) lateral view with section through mandrel; C) lateral view with section through cannula. 1) Platform; 2) guide tube; 3) fixer; 4) electrode tube; 5) stop ring; 6) contact pins; 7) electrodes; 8) cement; 9) bone; 10) mandrel; 11) cannula.
Finer electrodes (25–30 µm) are needed for recording neurons. A separate guide tube 4 made of a segment of injection needle of diameter 0.3 mm was used to attach these. The needle was ground at the side and the flat surface was soldered to the flat side of the guide tube. Electrodes were attached, unsoldered, and cut as described above.
SURGERY An opening of diameter 1 mm was trepanned in the skull of an anesthetized, scalped animal in accord with the coordinates of the structure of interest. An injection needle of diameter 0.4 mm was inserted into the guide tube (Fig. 2, 2), the upper end of which was attached to a stereotaxic apparatus. The contact pins were connected to an amplifier. The electrode-bearing platform previously sterilized with alcohol was attached to the animals’ skull with physiological monitoring and fixed with dental cement. After the cement had hardened, the needle was withdrawn and replaced with a mandrel (Fig. 2, 10) covering the opening in the guide tube against contamination and ingress of unwanted particles.
EXPERIMENT Dialysis solution compositions have been described in the literature [1] and depend on the particular experimental aims. The dialysis solution is filtered to remove admixtures. The cannula is attached to a pump and filled with solution. The rat is placed on the floor of the experimental chamber; the mandrel is removed from the guide tube and replaced with the cannula, which is inserted to the stop. As shown by the experiment, the cannula insertion procedure had no effect on the position of the electrodes, on the amplitude of the evoked potentials recorded, or on the characteristics of the neurons being recorded. The rate of solution delivery was 1 µl/min. Dialysate is collected in a test tube suspended above the animal’s head on the silicone supply tube (Fig. 3, 1), allowing the tube to be changed without disturbing the animal. A segment of thick plastic tube 6 is placed around the two silicone tubes. A tube holder 7 is placed on the thick tube to allow rapid and easy exchange of test tubes 3. The fixer is formed from a brass plate of thickness 0.3 mm. Spilling of the collected dialysate when the animal shook
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Fig. 3. Test tube attachment. 1) Silicone supply tube; 2) cannula; 3) silicone delivery tube; 4) lid; 5) sleeve; 6) plastic tube; 7) test tube holder; 8) plastic test tube.
Fig. 4. Simultaneous recording of neurochemical and neurophysiological changes in hippocampal field CA1. The upper plot shows changes in serotonin concentrations in hippocampal field CA1; the lower plot shows changes in the amplitude of the population excitatory postsynaptic potential (EPSP) at the dialysate collection point in conditions of bipolar stimulation of Shäffer collaterals with single current impulses. Population EPSP were averaged for 10 impulse presentations. *Significant differences. Stimulation of the dorsal cervical nuclei (C) led to increases in the serotonin concentration in the hippocampus but had no effect on the amplitude of population EPSP. After tetanizing stimulation of Shäffer collaterals (T), potentiation of synapses tested for significant (p < 0.05) increases in population EPSP amplitude occurred on the background of an increasing serotonin concentration. This is evidence that with the stimulating electrodes in this position, the serotoninergic projections are within the area subject to the action of the stimulating current.
Microdialysis in Freely Moving Animals was avoided by covering the test tube with lid 4. The lid was cut from a standard plastic test tube and an opening was drilled into it of diameter 0.2–0.3 mm greater than the diameter of the silicone delivery tube 3. The silicone tube was passed through the opening. A sleeve 5 was applied to prevent the tube from coming out. Figure 4 shows changes in the serotonin concentration in hippocampal field CA1 in conditions of electrical stimulation of the cervical nuclei with simultaneous recording of evoked potentials at the dialysate collection point. After the experiment, the cannula is removed from the guide tube and replaced with a sterile mandrel. The cannula is washed and stored in ultraclean water (18 MΩ/cm)
587 until the next experiment. When studies with the individual animal are finished, the platform is removed from the skull, freed of cement, and used again after replacement of the electrodes. This study was supported by the Russian Foundation for Basic Research (Grant No. 04-04-48934). The author would like to thank V. N. Khokhlov and N. V. Shchegolevskii for assistance with the experiments. REFERENCE 1.
H. Beneviste and P. C. Huttemeier, “Microdialysis – theory and application,” Progr. Neurobiol., 35, 195–215 (1990).
