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A dual chamber for comparative studies using the brain slice preparation

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A dual linear-flow chamber for comparative studies using brain slices is described. Electrophysiological and ultrastructural analysis of rat hippocampal slices incubated in the chamber showed that its two compartments allows performance of reliable
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  Camp. Biochem. Physiol. Vol. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 2A, No. 3, pp. 701-704, 198s Printed zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA n Great Britain 0300-9629/8S 3.00 + 0.00 C 1985 Pergamon Press Ltd A DUAL CHAMBER FOR COMPARATIVE STUDIES USING THE BRAIN SLICE PREPARATION A. SCHURR, K. H. REID,? M. T. TSENG,~ H. L. EDMONDS, JR* and B. M. RIGOR* Laboratory of Cellular Neuroscience, Anesthesia and Critical Care Research Unit (ACCRU), Departments of *Anesthesiology, tPhysiology and IAnatomy, School of Medicine, University of Louisville, Louisville, Kentucky 40292, USA. Telephone: (502) 588-6544 zyxwvutsrqponmlkjih  Received 8 March 1985) Abstract l. A dual linear-flow chamber for comparative studies using brain slices is described. 2. Electrophysiological and ultrastructural analysis of rat hippocampal slices incubated in the chamber showed that its two compartments allows performance of reliable paired comparison studies in a highly efficient manner. INTRODUCTION The use of the brain slice preparation in electro- physiology and basic neuroscience has become rou- tine in recent years. The hippocampal slice prepara- tion is probably the single most investigated brain structure using this in vitro approach (Hatton, 1982). Maintaining a viable and active hippocampal slice requires the use of a special tissue chamber of which many designs have been described (Gibson and McIl- wain, 1975; Schwartzkroin, 1975; Spencer et al., 1976; Teyler, 1976; White et al., 1978; Haas et al., 1979). Although different in design, all tissue chambers provide for the five basic requirements of the brain slice (Teyler, 1980): (a) the proper chemical com- position for the fluid bathing the brain slice; (b) appropriate O2 and CO, levels; (c) the mechanical support for the slice; (d) illumination, and (e) ade- quate temperature. Multiple slices placed in the same chamber can be used for sequential analysis. How- ever, comparison of responses to changes in parame- ters such as gas mixture, bath media and temperature are difficult to do using a single chamber. Building two separate brain slice preparation sys- tems with two identical tissue chambers is expensive and complicated. The purpose of this paper is to report on the use and the advantages of a dual linear-flow chamber built in our laboratory which enables us to conduct comparative studies with rat hippocampal slices. MATERIALS AND METHODS The chamber design was based on a linear-flow design (Haas c’t al., 1979) and is shown in Fig. 1. Each compart- ment of the dual chamber has its own supply of artificial cerebrospinal fluid (ACSF) and gas. The temperature is controlled by a single device to produce an equal tem- perature in both compartments (34.5 + 0.5”C). Different temperatures can be produced by separating hose b (Fig. 1) and attaching a second temperature control device. Adult (200-600 g) male Sprague-Dawley rats were decap- itated and their brains removed, rapidly rinsed with cold ACSF and dissected. Isolated hippocampi were sliced trans- versly at 400 f 50 pm with a Mcllwain tissue chopper and the resulting slices were transferred to the dual linear-flow chamber with a pipette. Slices were supported on a nylon mesh (105 pm) and sub-perfused with ACSF (Schurr er al., 1984) at 0.5 ml/min. A humidified gas mixture of 95 0,/S CO, (1 l/min/compartment) was circulated above the slices. Oxy- gen concentration could be varied by replacing part or all of it with N,. Carbon dioxide concentration was held constant at 5 with all gas mixtures to ensure a pH value of the ACSF not higher than 7.4. High humidity in the chamber was achieved by passing the gas mixture through a bottle containing distilled water and by covering the chambers with removable covers. Oxygen and CO, tensions immediately above the slices were monitored using a mass spectrometer (Medspect II, Chemtron, St Louis, MO). Extracellular recordings from stratum pyramidale of the CA1 region were made using borosili&te micropipettes filled with ACSF (l-5 MR). A two-channel AC nreamolifier (x 100) and two‘ field-effect transistor (FET)’ headstages were used. The output was digitized and stored on a floppy disk for later analysis. Resolution was 256 points x 8 bits. The placement of the recording electrodes in the slice was controlled by Kopf hydraulic microdrives with a nominal resolution of I pm. Biopolar stimulation electrodes were made from two 75 pm diameter, teflon-coated, stainless steel wires inserted through a pre-pulled, double-barreled glass capillary (Fig. 2). The tips of the wires, bared from the teflon coat, were protruding 50~1000 pm from the tip of the glass capillary and were 2OOpm apart. Isolated stimulus pulses were 0.1 msec in duration and of an amplitude twice that required to elicit a minimal response. The threshold rarely exceeded 5 V. CA1 population responses were recorded automatically at predetermined intervals from one slice in each compartment of the dual chamber. A waveform analysis program was used to determine the amplitude and latency of the population spike (Schurr et al., 1984). Ultrastructural analysis was conducted in slices ob- tained after 6 hr incubation in the life span experiments. For the anoxia experiments samples were removed at the start and the end of recording periods. After initial fixation in paraformaldehyde-glutaraldehyde solution, a rectangular piece containing the region of CA1 studied was dissected, dehydrated and embedded in Araldite 502. After poly- merization, sections situated approx. 100pm beneath the surface of the slice were cut in a plane parallel to the long axis of the pyramidal cells. Sections were stained and examined in a Philips 300 electron microscope. RESULTS AND DISCUSSION Figure 3 summarizes the results of 13 experiments 701  A SCHURR er al f---- ui ‘i”___  Dual chamber for brain slice preparations 703 zyxwvuts ST BILITY ND VI BILITY OF HIPPOC MP L SLICES OMPARTMENT I I COMPARTMENT II I 2 3 4 5 6 7 8 9 IO II 12 13 EXPERIMENT NO Fig. 3. Stability and viability of rat hippocampal slices in the two compartments of the dual chamber. Each column represents the time measured from placement of slices in the chamber to failure of electrical activity (disappearance of population spike). Records were taken every IO min for the entire duration of each experiment. comparing the life span of hippocampal slices. For each experiment slices were prepared from one hippo- campus and placed in both compartments of the dual chamber. The duration of the electrical activity to its failure ranged from 6.3 to 25.9 hr (mean 12.7, SD 5.5, N = 26). There was no significant difference between the two compartments. The wide variability between the duration of the experiments may be due to intrinsic differences between rats. Based on these results the duration of our experimental protocol never exceeds 6 hr. No discernable difference in the cytoarchitecture of slices incubated in either chamber was found. A typical pyramidal cell after 6 hr incubation is shown in Fig. 4A. Note the finely dispersed chromatin, the ovoid shaped mitochondria and short segment of rough endoplasmic reticulum. The main use for the dual chamber is in compara- tive studies where the slices in one compartment are exposed to the “treatment”, while those in the other are used as “controls”. A “treatment” could be any change in the environmental conditions or an addi- tion of a drug in one compartment, while keeping the conditions in the other unchanged. Since no significant difference was found in the survival of the slices between the two compartments of the dual chamber, both can be used alternately, as the treat- ment compartment. A representative experiment in which the slices in one compartment were exposed to 10min of 95 N2/5 CO> (anoxia), while those in the other com- partment were kept under 95 0,/S CO, is shown in Fig. 5. The population spike amplitude decreased rapidly and totally disappeared within 3 min as a result of the change from 0, to N, atmosphere. The amplitude of the population spike in the control compartment did not change. Upon return to 95 0,/S:/ CO2 in the “treatment” compartment, the population spike recovered to its srcinal amplitude. While the slice which was exposed to IOmin anoxia exhibited full recovery of its electrical activity, it appeared to be somewhat damaged ultrastructurally (Fig. 4B); pyramidal cell nuclei appeared pycnotic with patches of condensed chromatin. In the watery cytoplasm there were scattered microtubular bundles, vacuoles and burst mitochondria. In the neuropil, there were darkly stained neurites mingled with swol- len dendrites, and there was an increase in the perivascular void space. The I10 experiments in which we used the dual chamber and the hundreds of slices which we tested electrically and mor- phologically have convinced us that comparative studies using slices from the same hippocampus are feasible and of great value. In conclusion, the dual chamber described here allows performance of reliable paired comparison Fig. 4. (A) A well preserved CA1 pyramidal cell after 6 hr incubation in the chamber. (B) Typical response of CA1 pyramidal cell to anoxia. This includes nuclear chromatin condensation, vacuole formation and watery cytoplasm. Magnification x 3080.  704 A. SCHURR et al. Fig. 5. The effect of a 10min anoxic episode (95 N2/50/;, CO,) on the population spike amplitude recorded from a hippocampal slice in one compartment of the dual chamber (a). The slice in the other compartment was continuously exposed to 95 0,/S CO, and its population spike ampli- tude is shown (0). Oxygen concentration in both compart- ments was measured (---). The anoxic episode started 30 min after the beginning of the recordings and terminated 10 min later. studies using brain slice preparations in a highly efficient manner. REFERE?JCES Gibson 1. M. and McIlwain H. (1975) Continuous recording of changes in membrane potential in mammalian cerebral tissue in vitro: Recovery after depolarization by added substances. .I. Physioi. 176 261-283. Haas H. L., Schaerer B. and Vomansky M. (1979) A simple perfusion chamber for the study of nervous tissue slices in oitro. J. Neurosri. Meth. 1 323-325. Hatton G. I. (1982) The brain in slices: new approaches to old problems. Fed. Proc. 42 2863-2864. Hatton G. I., Doran A. D., Salm A. K. and Tweedle C. D. I 980) Brain slice preparation: hypothalamus. Brain Res. Bull. 5, 405414. Schurr A., Reid K. H., Tseng M. 7’. and Edmonds H. L., Jr. (1984) The stability of the hippocampal slice prepara- tion: an electrophysiological and ultrastructural analysis. Brain Res. 297 357-362. Schwartzkroin P. A. (1975) Characteristics of CAI neurons recorded intracellularly in the hippocampal in aitro slice preparation, Brain Res. 85, 423-432. Spencer H. J.. Gribkoff V. K., Cotman C. W. and Lynch A. S. (1976) GDEE antagonism of iontophoretic amino acid excitations in the intact hippocampus and in the hippocampal slice preparation. Bruin Res. 105 47148 1. Teyler T. J. (1976) Plasticity in the hippocampus: a model systems approach. In Admnces in Ps~~~o~io~o~~: Neural Models zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ f Behaaiorol Pt’asticity (Edited by Riesen A. and Thompson R. F.), Vol. III, pp. 301-326. Wiley, New York. Teyler T. J. (1980) Brain slice preparation: hippocampus. Brain Res. Bull. 5 391-403. White W. F., Nadler J. V. and Cotman C. W. (1978) A perfusion chamber for the study of CNS physiology and pharmacology in oitro. Brain Res. 152 591-596.
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