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A dual chamber for comparative studies using the brain slice preparation
Camp. Biochem. Physiol. Vol. 82A, No. 3, pp. 701-704, 198s Printed in 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 (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 electrophysiology and basic neuroscience has become routine in recent years. The hippocampal slice preparation 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 McIlwain, 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 composition for the fluid bathing the brain slice; (b) appropriate O2 and CO, levels; (c) the mechanical support for the slice; (d) illumination, and (e) adequate temperature. Multiple slices placed in the same chamber can be used for sequential analysis. However, comparison of responses to changes in parameters such as gas mixture, bath media and temperature are difficult to do using a single chamber. Building two separate brain slice preparation systems 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 compartment 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 temperature 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 decapitated and their brains removed, rapidly rinsed with cold ACSF and dissected. Isolated hippocampi were sliced transversly 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. 701
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. Oxygen 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 obtained 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 polymerization, 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
A. SCHURR
er al.
f----
ui
‘i”___
Dual chamber for brain slice preparations STABILITY
I
AND
VIABILITY
OF HIPPOCAMPAL
q
COMPARTMENT
I
I
COMPARTMENT
II
2
3
4
5
6
7
EXPERIMENT
8
9
IO
II
12
SLICES
13
NO
703
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 swollen 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 morphologically 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. 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 hippocampus 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 comparative 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 addition 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 treatment 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 compartment 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 original amplitude. While the slice which was exposed to IOmin anoxia
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. SCHURRet al. 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 preparation: 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. Fig. 5. The effect of a 10min anoxic episode (95% N2/50/;, Spencer H. J.. Gribkoff V. K., Cotman C. W. and Lynch CO,) on the population spike amplitude recorded from a A. S. (1976) GDEE antagonism of iontophoretic amino hippocampal slice in one compartment of the dual chamber acid excitations in the intact hippocampus and in the (a). The slice in the other compartment was continuously hippocampal slice preparation. Bruin Res. 105, 47148 1. exposed to 95% 0,/S% CO, and its population spike ampliTeyler T. J. (1976) Plasticity in the hippocampus: a model tude is shown (0). Oxygen concentration in both compartsystems approach. In Admnces in Ps~~~o~io~o~~:Neural ments was measured (---). The anoxic episode started Models of Behaaiorol Pt’asticity (Edited by Riesen A. and 30 min after the beginning of the recordings and terminated Thompson R. F.), Vol. III, pp. 301-326. Wiley, New 10 min later. 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 studies using brain slice preparations in a highly perfusion chamber for the study of CNS physiology and efficient manner. pharmacology in oitro. Brain Res. 152, 591-596.
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 (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 electrophysiology and basic neuroscience has become routine in recent years. The hippocampal slice preparation 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 McIlwain, 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 composition for the fluid bathing the brain slice; (b) appropriate O2 and CO, levels; (c) the mechanical support for the slice; (d) illumination, and (e) adequate temperature. Multiple slices placed in the same chamber can be used for sequential analysis. However, comparison of responses to changes in parameters such as gas mixture, bath media and temperature are difficult to do using a single chamber. Building two separate brain slice preparation systems 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 compartment 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 temperature 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 decapitated and their brains removed, rapidly rinsed with cold ACSF and dissected. Isolated hippocampi were sliced transversly 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. 701
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. Oxygen 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 obtained 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 polymerization, 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
A. SCHURR
er al.
f----
ui
‘i”___
Dual chamber for brain slice preparations STABILITY
I
AND
VIABILITY
OF HIPPOCAMPAL
q
COMPARTMENT
I
I
COMPARTMENT
II
2
3
4
5
6
7
EXPERIMENT
8
9
IO
II
12
SLICES
13
NO
703
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 swollen 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 morphologically 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. 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 hippocampus 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 comparative 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 addition 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 treatment 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 compartment 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 original amplitude. While the slice which was exposed to IOmin anoxia
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. SCHURRet al. 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 preparation: 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. Fig. 5. The effect of a 10min anoxic episode (95% N2/50/;, Spencer H. J.. Gribkoff V. K., Cotman C. W. and Lynch CO,) on the population spike amplitude recorded from a A. S. (1976) GDEE antagonism of iontophoretic amino hippocampal slice in one compartment of the dual chamber acid excitations in the intact hippocampus and in the (a). The slice in the other compartment was continuously hippocampal slice preparation. Bruin Res. 105, 47148 1. exposed to 95% 0,/S% CO, and its population spike ampliTeyler T. J. (1976) Plasticity in the hippocampus: a model tude is shown (0). Oxygen concentration in both compartsystems approach. In Admnces in Ps~~~o~io~o~~:Neural ments was measured (---). The anoxic episode started Models of Behaaiorol Pt’asticity (Edited by Riesen A. and 30 min after the beginning of the recordings and terminated Thompson R. F.), Vol. III, pp. 301-326. Wiley, New 10 min later. 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 studies using brain slice preparations in a highly perfusion chamber for the study of CNS physiology and efficient manner. pharmacology in oitro. Brain Res. 152, 591-596.