- Email: [email protected]
Primary culture of neurons derived from the adult mammalian brain
0361-9230/91 $3.00 + .OO
Brain Research Bulletin, Vol. 27, PP. 747-750. 0 Pergamon Press plc, 1991. Printed in the U.S.A
BRIEF COMMUNICATION
Primary Culture of Neurons Derived From the Adult Mammalian Brain NOBUKUNI Department
of Pharmacology,
OGATA
AND HIDEHARU
Faculty of Medicine,
TATEBAYASHI
Kyushu University, Fukuoka
Received 28 November
812, Japan
1990
OGATA, N. AND H. TATEBAYASHI. Primary culture of neurons derivedfrom the adult mammalian brain. BRAIN RES BULL 27(5) 747-750, 1991. -A method is described for the isolation and culture of neurons in the adult mammalian brain. The cell could be maintained in primary culture for more than several weeks. Whereas the neurons freshly dissociated from the adult brain did not respond to any of the neurotransmitter substances applied, the neurons regained the ability to respond to a variety of
neurotransmitters when cultured. The cultured neurons of the adult mammalian brain may be an excellent model for physiological as well as pharmacological investigations of the central nervous system. Isolation of brain neurons Ion channels
Culture
Brain slice
Patch-clamp
Striatum
Hippocampus
dissected out using fine iris knives and collected together in the tube for enzymatic treatment. The tissue fragments were then incubated in Krebs solution of the following composition (in mM) at 37°C: NaCl, 120; KCl, 5; CaCl,, 1.8; MgCl,, 1; piperazine-N,N’-bis[2-ethanesulphonic acid]-Na (PIPES-Na), 5 mM; glucose, 25 mM with 4-10 mg/ml trypsin (Type XI, Sigma) added in both cases of rats and guinea pigs. The pH of the solution was adjusted to 7.4 with NaOH. During the incubation, tissue fragments were continuously bubbled with 100% 0,. After about 1 hour of trypsination, the tissue fragment should be a little bit clumpy and the solution cloudy. After a l-l .5-hour incubation, the trypsin Krebs solution was removed and the tissue fragments were washed three times with normal Krebs solution. Then the tissue fragments were dissociated by gentle flushing through a fire-polished Pasteur pipette (inner tip diameter, 100 km). As to the enzyme, although we have not performed a systematic comparison, papain or pronase also produced good results, while collagenase was totally ineffective. A microscopic inspection of the tissue fragment produced by mild agitation was particularly useful in obtaining preliminary information relevant to subsequent primary culture, since it provided a good estimate of the cellular composition within the tissue. As shown in Fig. 2B and C, within the mildly agitated tissue fragment, individual cells are. visible, including the fine structure of the processes, yet the cellular architecture of the tissue is preserved. Due to the thickness of the preparation, only a small percentage of the cells was in focus. However, in actual observations, a sharp outline of most of the cells could be observed by bringing them into focus. Thus the neuron type constituting the nucleus, spatial distribution and relative proportion
THE patch-clamp studies on neurons from the mammalian brain have been performed using cultured cells derived from fetal or neonatal animals. Recently, neurons of the mammalian brain were acutely dissociated, and ionic currents were recorded from these neurons using the patch-clamp technique (7-9, 11, 12). These acutely dissociated brain neurons were proved to be an excellent preparation, particularly for the study of voltage-gated ion channels. We have developed a technique to culture neurons of the adult mammalian brain. These neurons allow the measurement not only of voltage-gated ionic currents but also of a variety of receptor-operated ionic currents. In this paper, we describe our method for culture of the adult mammalian brain neuron.
DISSOCIATION
experiment
OF BRAIN NEURONS
Adult guinea pigs (300-400 g) or rats (200-250 g) of either sex were used. Although the exact dates of birth of the animals were not checked, animals used in this report were all older than 2 months. Figure 1 illustrates the outline of the primary culture. Our technique to dissociate the brain neurons has been described in the previous papers (11,12). The method is basically similar to the methods reported by other investigators (7-9). The dissociation consists of four processes: 1) preparation of brain slices, 2) microdissection of the discrete area (4, 11, 12), 3) enzymatic treatment, and 4) mechanical agitation of the tissue fragments. All of these processes were performed under sterile conditions. Brain slices (about 400 Frn thick) were prepared as described previously (1). As shown in Fig. 2A, a discrete area of the brain within the slice can be precisely determined by applying the transillumination under binocular microscope. Each area was
747
748
OGATA AND TATEBAYASHI
f
Dissocibon
each well of the culture plate to cover the bottom of the well, and the culture plate was kept in the incubator for at least 6 hours before plating.
‘,
[ Preparation of brain slice 1
I-Krehssolution vibralwne
i
PRIMARY CULTURE
h4icrodiss8ctibn
1 Enzym3 freatrnenf 36-C Kmbs -+ water-baih
1
(
Culture disc coating )
9 + Tryp.sih
Ist day
I
(
Primary culture
Tmatnwnt with fibronectin
1
Plating S8mmantaining DMEM
4 Feeding
4
Serumanfaiii~
FIG. 1. Schematic neuron.
diagram
DMEM
for culture
of the adult mammalian
brain
in number of each neuron type and detailed morphological tures of the cells can be precisely checked.
fea-
CULTURE DISC COATING
General culture procedures are shown in the textbook by Freshney (5). Our culture method was basically similar to that reported previously (10). Since the glass coverslip has negative surface charges, which is unfavorable for cell attachment, its surface is usually treated by materials such as collagen or acidic polyaminoacids (e.g., polyomithine or poly-L-lysin). To further promote the attachment of the cell to the culture disc, we used ACLAR fluorohalocarbon film (Allied Fibers and Plastics, Morristown, NJ) instead of cover glass. With this procedure, the number of cells that survived during culture increased to about 3.5 times the control obtained with glass coverslips. Before use, the ACLAR disc was washed by chromic acid, sterilized in 70% ethanol, and dried in a sterile hood. The ACLAR film was treated by 20 p,g/ml poly-L-lysin (Sigma) and 20 pg/ml fibronectin (Boehringer Mannheim). Poly-L-lysine solution was added into each well of the 24-well culture plate and kept under ultraviolet light for at least 12 hours. Then the poly-L-lysine solution was discarded from the well, and the well was rinsed once with distilled water. Fibronectin solution (0.5 ml) was put into
The cell viability was checked under a phase-contrast microscope. Figure 2D shows a cell population after adequate trituration. Figure 2E-H illustrates typical examples of neurons obtained from hippocampal slices. If judged adequately dissociated, the cell suspension was gently pelleted at 140 x g for 3 min and suspended in 2 ml complete feeding medium of the following composition: Dulbecco’s modified Eagle’s medium (DMEM). 10% fetal calf serum (Gibco), 20 kg ascorbic acid and streptomycin-penicillin (Boehringer Mannheim, 100 pg/ml and 100 IU. respectively). Cells were then plated into the 24-well culture plate at a density of about 400 cells/mm’. The freshly plated cultures were incubated for 3-5 days before their first medium change. Afterward, the medium change was done twice a week. When the nonneuronal background cells became confluent during a maintained culture, cells were treated with cytosine arabinoside (10 FM) for 48 hours to suppress growth of nonneuronal cells. Cultures were allowed to mature for about 3 weeks prior to study. Cultures could routinely be maintained for periods exceeding 1 month. Figure 21-K shows cultured adult neurons. The yield of cell culture in adult animals was not so large as the yields in fetal or newborn animals. However, there were in most cases enough numbers of “healthy” neurons for the patch-clamp recording, which does not require so many neurons per one culture disc. The average number of viable neurons per dish (1.3 cm’) was 31.4k4.6 (n=50). We did not find obvious difference in culture between the rat and guinea pig. PATCH-CLAMP
EXPERIMENTS
Both freshly dissociated and cultured adult neurons allowed a stable recording of the membrane currents. Figure 3 illustrates typical examples of the voltage-gated (A) and receptor-operated (B) ionic current observed in striatal neurons. (A) shows a family of voltage-gated sodium currents observed in freshly dissociated neurons. Similarly, other voltage-gated ionic currents such as calcium or potassium currents could also be recorded (data not shown). Voltage-dependent currents were observed in both freshly dissociated and cultured adult neurons. On the contrary, the responses of the freshly dissociated neurons to exogenously applied neurotransmitters became progressively poorer as the more aged animals were used. For example, although most of the neurons derived from animals within a few weeks after birth responded to y-aminobutyric acid (GABA), about one-third of the neurons responded to GABA when the neurons were dispersed from animals 30-50 days after birth, and none of the neurons examined (n = 200) responded to GABA or several other neurotransmitter substances when the neurons were dispersed from animals older than 3 months. The only difference in procedure was the concentration of the proteolytic enzyme. The concentration was 0.5-l mg/ml for neonatal animals, l-2 mg/ml for animals 30-50 days after birth, and 4-10 mg/ml for animals older
FACING PAGE FIG. 2. Photomicrographs
obtained during a course of primary culture. (A) Brain slice prepared from the striatal region. Abbreviations: Cor. cortex; CC, corpus callosum; Fim, timbria; CP, caudate putamen (striatum); GP, glohus pallidurn; Th, thalamus. (B) and (C) Mildly dissociated caudate putamen and hippocampus, respectively. (D) Cellular aggregate of the freshly dissociated neurons prepared from the granule cell layer of the hippocampus. (EH) Freshly dissociated adult hippocampal neurons. (E) CA3 pyramidal cell; (F) CA1 pyramidal cell; (G) presumed basket cell; (H) granule cell. (I) and (J) Cultured striatal neurons. (K) Cultured CA1 hippocampal pyramidal cell. In (K), a patch electrode, the tip of which is in contact with the soma, is seen. All the photographs were taken through phase-contrast microscope except for (C). (C) was photographed through Nomarski interference microscope. (A), (B), (D-H), (J) and (K), guinea pig; (Cj and (I), rat.
749 CULTURE OF ADULT BRAIN NEURONS
20pm
750
OGATA AND TATEBAYASHI
A
than 3 months. neurotransmitters
heat -8Orn”
-
4
1nA -! 0.5 ms
B 1
DA
lOO~h4
--.I
0.1 nA
1 min GABA I-
10,UM
DA
100,CLM
0.1 nA lmin
FIG. 3. Patch-clamp recordings from freshly dissociated or cultured neurons of the adult guinea pig striatum. (A) A family of superimposed sodium currents observed in the freshly isolated neuron. Test pulses (V,,,,) to various depolarizing potentials were applied from a holding potential of -80 mV at a constant rate of 0.03 Hz. The numeral attached to each trace denotes V,=,,. (B) Responses of cultured neurons to bath-applied y-aminobutyric acid (GABA) and dopamine (DA). A holding potential was -80 mV in all three traces. Drugs were applied, at periods indicated by bars. Bl and B2 were recorded from different cells. The methods for electrical recording were similar to those described in previous papers (13). In short, a whole-cell variation of the patch-clamp technique (6) was used. Capacitative and leakage cunents were subtracted digitally by the P-P/4 procedure (3). The external solution (pH 7.4) contained (in mM): NaCl, 100; TEA-CI, 25; CaCl,, 1.8; KCl, 5; MgCI,, 1; glucose, 25; HEPES, 5; and CoCl,. The internal solution (pH 7.0) contained (in n&f): K-glutamate, 125; NaCl, 10; MgCl,, 2.5; HEPES, 5; EGTA, 5; and glucose, 5. Experiments were carried out at room temperature (22’C). Upward and downward deflections represent outward and inward currents, respectively.
Thus it is our impression that insensitivity to in aged animals may be due to impairment of
the receptor function by a high dose of enzyme in cell dispersion. Our concentration for adult animals (&lo mg/ml) was apparently much higher than the concentration used by other investigators (7,9) who found that freshly dissociated adult neurons responded to GABA. In contrast to the freshly dissociated neurons, all of the cultured adult neurons examined (n=30) responded to GABA. Rgure 3B illustrates responses of cultured sttiatal neurons to GABA or dopamine (DA), GABA induced an inward current, and DA produced both outward (Bl) and inward (B2) currents, depending on the cell examined. These responses to GABA and DA were similar to those observed for intact mammalian neurons (2,lO). In conclusion, the cultured neurons of the adult mammalian brain supplemented the limitation inherent to the freshly dissociated neurons. As described in this report, the technique to develop the cultured adult neurons is essentially the same as the technique employed for culture of the immature neurons. We thus believe that if the two independent techniques, one. dissociation of the brain neuron and the other, primary culture, are acquired, the primary culture of the adult mammalian neuron is not so difficult. The most important factor of a successful culture is undoubtedly proper trypsination followed by gentle but adequate ~turation. The concen~ation of the enzyme, the time of incubation and number of ~tura~ons should be empirically adjusted depending on various factors, such as species, age of the animal, area of the brain, and the batch of enzyme. The electrophysiological experiment using the freshly dissociated or cultured adult neurons can be done in parallel with the conventional brain slice electrophysiology, because the methods overlap. Patch-clamp recording from a single neuron is suitable for the study of ion channel activity, whereas it has no access to the synaptic potential. On the contrary, a conventional microelectrode experiment using brain slices is relevant to the study of the synaptic potential. Thus the cultured neurons of the adult mammalian brain may be an important model for integrated investigations of the central nervous system, although it should be borne in mind that it is not known whether the cultured adult neurons maintain their adult phenoty~. ACKNOWLEDGEMENTS
We thank Professor H. Kuriyama for his advice and Dr. M. Yoshii for providing the on-line data processing program and reading the manuscript. This study was supported by the Japanese Ministry of Education (Scientific Research 63570096).
REFERENCES 1. Abe, H.; Ogata, N. Ionic mechanism for the osmotically induced depolarization in neumnes of the guinea pig supraoptic nucleus in vitro. J. Physiol. 327:157-171; 1982. 2. Akaike, A.; Ohno, Y.; Sass, M.; Takaori, S. Excitatory and inhibitory effects of dopamine on neuronal activity of the caudate nucleus neurons in vitro. Brain Res. 418:262-272; 1987. 3. Bezanilla, F.; Armstrong, C. Inactivation of the sodium channel I. Sodium current experiments. J. Gen. Physiol. 70:549-566; 1977. 4. Cuello, A. C.; Carson, S. Microdissection of fresh rat brain tissue slices. In: Cuello, A. C., ed. Brain microdissection techniques. Chichester: John Wiley and Sons; 1983:37-125. 5. Freshney, R. I.; Culture of animal cells. A manual of basic technique. New York: Alan R. Liss; 1983. 6. Hamill, 0. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. PfIugers Arch. 391: 85-100; 1981. 7, Huguenard, J. R.; Alger, B. E. Whole-cell voltage-clamp study of the fading of GABA-activated currents in acutely dissociated hippo-
campal neurons. J. Neurophysiol. 56:1-18; 1986. 8. Kaneda, M.; Nakamura, H.; Akaike, N. Mechanical and enzymatic isolation of mammalian CNS neurons. Neurosci. Res. 5:299-315; 1988. 9. Kay, A. R.; Wang, R. K. S. fsoiation of neurons suitable for itch-cl~ping from adult mammalian central nervous systems. J. Neurosci. Methods 16:227-238; 1986. 10. Ogata, N.: Vogel, S. M.; Namhashi, T. Lindane but not deltamethrin blocks a component of GABA-activated chloride channels. FASEB J. 2:2895-2900; 1988. 11. Ogata, N.; Tatebayashi, H. Modulation of sodium current kinetics by chlorpromazine in freshly-isolated adult guinea pig striatal neurones. Br. J. Pharmacol. 98:1173-1184; 1989. 12. Ogata, N.; Tatebayashi, H. Sodium current kinetics in freshly-isolated adult guinea pig striatal neurones. Pflugers Arch. 416:594603; 1990. 13. Ogata, N.; Yoshii, M.; Narahashi T. Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells. J. Physiol. 420:165-183; 1990.
Brain Research Bulletin, Vol. 27, PP. 747-750. 0 Pergamon Press plc, 1991. Printed in the U.S.A
BRIEF COMMUNICATION
Primary Culture of Neurons Derived From the Adult Mammalian Brain NOBUKUNI Department
of Pharmacology,
OGATA
AND HIDEHARU
Faculty of Medicine,
TATEBAYASHI
Kyushu University, Fukuoka
Received 28 November
812, Japan
1990
OGATA, N. AND H. TATEBAYASHI. Primary culture of neurons derivedfrom the adult mammalian brain. BRAIN RES BULL 27(5) 747-750, 1991. -A method is described for the isolation and culture of neurons in the adult mammalian brain. The cell could be maintained in primary culture for more than several weeks. Whereas the neurons freshly dissociated from the adult brain did not respond to any of the neurotransmitter substances applied, the neurons regained the ability to respond to a variety of
neurotransmitters when cultured. The cultured neurons of the adult mammalian brain may be an excellent model for physiological as well as pharmacological investigations of the central nervous system. Isolation of brain neurons Ion channels
Culture
Brain slice
Patch-clamp
Striatum
Hippocampus
dissected out using fine iris knives and collected together in the tube for enzymatic treatment. The tissue fragments were then incubated in Krebs solution of the following composition (in mM) at 37°C: NaCl, 120; KCl, 5; CaCl,, 1.8; MgCl,, 1; piperazine-N,N’-bis[2-ethanesulphonic acid]-Na (PIPES-Na), 5 mM; glucose, 25 mM with 4-10 mg/ml trypsin (Type XI, Sigma) added in both cases of rats and guinea pigs. The pH of the solution was adjusted to 7.4 with NaOH. During the incubation, tissue fragments were continuously bubbled with 100% 0,. After about 1 hour of trypsination, the tissue fragment should be a little bit clumpy and the solution cloudy. After a l-l .5-hour incubation, the trypsin Krebs solution was removed and the tissue fragments were washed three times with normal Krebs solution. Then the tissue fragments were dissociated by gentle flushing through a fire-polished Pasteur pipette (inner tip diameter, 100 km). As to the enzyme, although we have not performed a systematic comparison, papain or pronase also produced good results, while collagenase was totally ineffective. A microscopic inspection of the tissue fragment produced by mild agitation was particularly useful in obtaining preliminary information relevant to subsequent primary culture, since it provided a good estimate of the cellular composition within the tissue. As shown in Fig. 2B and C, within the mildly agitated tissue fragment, individual cells are. visible, including the fine structure of the processes, yet the cellular architecture of the tissue is preserved. Due to the thickness of the preparation, only a small percentage of the cells was in focus. However, in actual observations, a sharp outline of most of the cells could be observed by bringing them into focus. Thus the neuron type constituting the nucleus, spatial distribution and relative proportion
THE patch-clamp studies on neurons from the mammalian brain have been performed using cultured cells derived from fetal or neonatal animals. Recently, neurons of the mammalian brain were acutely dissociated, and ionic currents were recorded from these neurons using the patch-clamp technique (7-9, 11, 12). These acutely dissociated brain neurons were proved to be an excellent preparation, particularly for the study of voltage-gated ion channels. We have developed a technique to culture neurons of the adult mammalian brain. These neurons allow the measurement not only of voltage-gated ionic currents but also of a variety of receptor-operated ionic currents. In this paper, we describe our method for culture of the adult mammalian brain neuron.
DISSOCIATION
experiment
OF BRAIN NEURONS
Adult guinea pigs (300-400 g) or rats (200-250 g) of either sex were used. Although the exact dates of birth of the animals were not checked, animals used in this report were all older than 2 months. Figure 1 illustrates the outline of the primary culture. Our technique to dissociate the brain neurons has been described in the previous papers (11,12). The method is basically similar to the methods reported by other investigators (7-9). The dissociation consists of four processes: 1) preparation of brain slices, 2) microdissection of the discrete area (4, 11, 12), 3) enzymatic treatment, and 4) mechanical agitation of the tissue fragments. All of these processes were performed under sterile conditions. Brain slices (about 400 Frn thick) were prepared as described previously (1). As shown in Fig. 2A, a discrete area of the brain within the slice can be precisely determined by applying the transillumination under binocular microscope. Each area was
747
748
OGATA AND TATEBAYASHI
f
Dissocibon
each well of the culture plate to cover the bottom of the well, and the culture plate was kept in the incubator for at least 6 hours before plating.
‘,
[ Preparation of brain slice 1
I-Krehssolution vibralwne
i
PRIMARY CULTURE
h4icrodiss8ctibn
1 Enzym3 freatrnenf 36-C Kmbs -+ water-baih
1
(
Culture disc coating )
9 + Tryp.sih
Ist day
I
(
Primary culture
Tmatnwnt with fibronectin
1
Plating S8mmantaining DMEM
4 Feeding
4
Serumanfaiii~
FIG. 1. Schematic neuron.
diagram
DMEM
for culture
of the adult mammalian
brain
in number of each neuron type and detailed morphological tures of the cells can be precisely checked.
fea-
CULTURE DISC COATING
General culture procedures are shown in the textbook by Freshney (5). Our culture method was basically similar to that reported previously (10). Since the glass coverslip has negative surface charges, which is unfavorable for cell attachment, its surface is usually treated by materials such as collagen or acidic polyaminoacids (e.g., polyomithine or poly-L-lysin). To further promote the attachment of the cell to the culture disc, we used ACLAR fluorohalocarbon film (Allied Fibers and Plastics, Morristown, NJ) instead of cover glass. With this procedure, the number of cells that survived during culture increased to about 3.5 times the control obtained with glass coverslips. Before use, the ACLAR disc was washed by chromic acid, sterilized in 70% ethanol, and dried in a sterile hood. The ACLAR film was treated by 20 p,g/ml poly-L-lysin (Sigma) and 20 pg/ml fibronectin (Boehringer Mannheim). Poly-L-lysine solution was added into each well of the 24-well culture plate and kept under ultraviolet light for at least 12 hours. Then the poly-L-lysine solution was discarded from the well, and the well was rinsed once with distilled water. Fibronectin solution (0.5 ml) was put into
The cell viability was checked under a phase-contrast microscope. Figure 2D shows a cell population after adequate trituration. Figure 2E-H illustrates typical examples of neurons obtained from hippocampal slices. If judged adequately dissociated, the cell suspension was gently pelleted at 140 x g for 3 min and suspended in 2 ml complete feeding medium of the following composition: Dulbecco’s modified Eagle’s medium (DMEM). 10% fetal calf serum (Gibco), 20 kg ascorbic acid and streptomycin-penicillin (Boehringer Mannheim, 100 pg/ml and 100 IU. respectively). Cells were then plated into the 24-well culture plate at a density of about 400 cells/mm’. The freshly plated cultures were incubated for 3-5 days before their first medium change. Afterward, the medium change was done twice a week. When the nonneuronal background cells became confluent during a maintained culture, cells were treated with cytosine arabinoside (10 FM) for 48 hours to suppress growth of nonneuronal cells. Cultures were allowed to mature for about 3 weeks prior to study. Cultures could routinely be maintained for periods exceeding 1 month. Figure 21-K shows cultured adult neurons. The yield of cell culture in adult animals was not so large as the yields in fetal or newborn animals. However, there were in most cases enough numbers of “healthy” neurons for the patch-clamp recording, which does not require so many neurons per one culture disc. The average number of viable neurons per dish (1.3 cm’) was 31.4k4.6 (n=50). We did not find obvious difference in culture between the rat and guinea pig. PATCH-CLAMP
EXPERIMENTS
Both freshly dissociated and cultured adult neurons allowed a stable recording of the membrane currents. Figure 3 illustrates typical examples of the voltage-gated (A) and receptor-operated (B) ionic current observed in striatal neurons. (A) shows a family of voltage-gated sodium currents observed in freshly dissociated neurons. Similarly, other voltage-gated ionic currents such as calcium or potassium currents could also be recorded (data not shown). Voltage-dependent currents were observed in both freshly dissociated and cultured adult neurons. On the contrary, the responses of the freshly dissociated neurons to exogenously applied neurotransmitters became progressively poorer as the more aged animals were used. For example, although most of the neurons derived from animals within a few weeks after birth responded to y-aminobutyric acid (GABA), about one-third of the neurons responded to GABA when the neurons were dispersed from animals 30-50 days after birth, and none of the neurons examined (n = 200) responded to GABA or several other neurotransmitter substances when the neurons were dispersed from animals older than 3 months. The only difference in procedure was the concentration of the proteolytic enzyme. The concentration was 0.5-l mg/ml for neonatal animals, l-2 mg/ml for animals 30-50 days after birth, and 4-10 mg/ml for animals older
FACING PAGE FIG. 2. Photomicrographs
obtained during a course of primary culture. (A) Brain slice prepared from the striatal region. Abbreviations: Cor. cortex; CC, corpus callosum; Fim, timbria; CP, caudate putamen (striatum); GP, glohus pallidurn; Th, thalamus. (B) and (C) Mildly dissociated caudate putamen and hippocampus, respectively. (D) Cellular aggregate of the freshly dissociated neurons prepared from the granule cell layer of the hippocampus. (EH) Freshly dissociated adult hippocampal neurons. (E) CA3 pyramidal cell; (F) CA1 pyramidal cell; (G) presumed basket cell; (H) granule cell. (I) and (J) Cultured striatal neurons. (K) Cultured CA1 hippocampal pyramidal cell. In (K), a patch electrode, the tip of which is in contact with the soma, is seen. All the photographs were taken through phase-contrast microscope except for (C). (C) was photographed through Nomarski interference microscope. (A), (B), (D-H), (J) and (K), guinea pig; (Cj and (I), rat.
749 CULTURE OF ADULT BRAIN NEURONS
20pm
750
OGATA AND TATEBAYASHI
A
than 3 months. neurotransmitters
heat -8Orn”
-
4
1nA -! 0.5 ms
B 1
DA
lOO~h4
--.I
0.1 nA
1 min GABA I-
10,UM
DA
100,CLM
0.1 nA lmin
FIG. 3. Patch-clamp recordings from freshly dissociated or cultured neurons of the adult guinea pig striatum. (A) A family of superimposed sodium currents observed in the freshly isolated neuron. Test pulses (V,,,,) to various depolarizing potentials were applied from a holding potential of -80 mV at a constant rate of 0.03 Hz. The numeral attached to each trace denotes V,=,,. (B) Responses of cultured neurons to bath-applied y-aminobutyric acid (GABA) and dopamine (DA). A holding potential was -80 mV in all three traces. Drugs were applied, at periods indicated by bars. Bl and B2 were recorded from different cells. The methods for electrical recording were similar to those described in previous papers (13). In short, a whole-cell variation of the patch-clamp technique (6) was used. Capacitative and leakage cunents were subtracted digitally by the P-P/4 procedure (3). The external solution (pH 7.4) contained (in mM): NaCl, 100; TEA-CI, 25; CaCl,, 1.8; KCl, 5; MgCI,, 1; glucose, 25; HEPES, 5; and CoCl,. The internal solution (pH 7.0) contained (in n&f): K-glutamate, 125; NaCl, 10; MgCl,, 2.5; HEPES, 5; EGTA, 5; and glucose, 5. Experiments were carried out at room temperature (22’C). Upward and downward deflections represent outward and inward currents, respectively.
Thus it is our impression that insensitivity to in aged animals may be due to impairment of
the receptor function by a high dose of enzyme in cell dispersion. Our concentration for adult animals (&lo mg/ml) was apparently much higher than the concentration used by other investigators (7,9) who found that freshly dissociated adult neurons responded to GABA. In contrast to the freshly dissociated neurons, all of the cultured adult neurons examined (n=30) responded to GABA. Rgure 3B illustrates responses of cultured sttiatal neurons to GABA or dopamine (DA), GABA induced an inward current, and DA produced both outward (Bl) and inward (B2) currents, depending on the cell examined. These responses to GABA and DA were similar to those observed for intact mammalian neurons (2,lO). In conclusion, the cultured neurons of the adult mammalian brain supplemented the limitation inherent to the freshly dissociated neurons. As described in this report, the technique to develop the cultured adult neurons is essentially the same as the technique employed for culture of the immature neurons. We thus believe that if the two independent techniques, one. dissociation of the brain neuron and the other, primary culture, are acquired, the primary culture of the adult mammalian neuron is not so difficult. The most important factor of a successful culture is undoubtedly proper trypsination followed by gentle but adequate ~turation. The concen~ation of the enzyme, the time of incubation and number of ~tura~ons should be empirically adjusted depending on various factors, such as species, age of the animal, area of the brain, and the batch of enzyme. The electrophysiological experiment using the freshly dissociated or cultured adult neurons can be done in parallel with the conventional brain slice electrophysiology, because the methods overlap. Patch-clamp recording from a single neuron is suitable for the study of ion channel activity, whereas it has no access to the synaptic potential. On the contrary, a conventional microelectrode experiment using brain slices is relevant to the study of the synaptic potential. Thus the cultured neurons of the adult mammalian brain may be an important model for integrated investigations of the central nervous system, although it should be borne in mind that it is not known whether the cultured adult neurons maintain their adult phenoty~. ACKNOWLEDGEMENTS
We thank Professor H. Kuriyama for his advice and Dr. M. Yoshii for providing the on-line data processing program and reading the manuscript. This study was supported by the Japanese Ministry of Education (Scientific Research 63570096).
REFERENCES 1. Abe, H.; Ogata, N. Ionic mechanism for the osmotically induced depolarization in neumnes of the guinea pig supraoptic nucleus in vitro. J. Physiol. 327:157-171; 1982. 2. Akaike, A.; Ohno, Y.; Sass, M.; Takaori, S. Excitatory and inhibitory effects of dopamine on neuronal activity of the caudate nucleus neurons in vitro. Brain Res. 418:262-272; 1987. 3. Bezanilla, F.; Armstrong, C. Inactivation of the sodium channel I. Sodium current experiments. J. Gen. Physiol. 70:549-566; 1977. 4. Cuello, A. C.; Carson, S. Microdissection of fresh rat brain tissue slices. In: Cuello, A. C., ed. Brain microdissection techniques. Chichester: John Wiley and Sons; 1983:37-125. 5. Freshney, R. I.; Culture of animal cells. A manual of basic technique. New York: Alan R. Liss; 1983. 6. Hamill, 0. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. PfIugers Arch. 391: 85-100; 1981. 7, Huguenard, J. R.; Alger, B. E. Whole-cell voltage-clamp study of the fading of GABA-activated currents in acutely dissociated hippo-
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