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Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures
Neuroscience Letters, 22 ( 1981) 5 1 - 5 6 (~ Elsevier/North-Holland Scientific Publishers Ltd.
5l
C H E M I C A L L Y D E F I N E D M E D I U M E N H A N C E S BIOELECTRIC ACTIVITY IN M O U S E S P I N A L C O R D - D O R S A L ROOT G A N G L I O N C U L T U R E S
A L P H O N S M . M . C . HABETS, ROBERT E. BAKER, ELI B R E N N E R and H E R M S J. R O M I J N
Netherlands Institute for Brain Research, IJdijk 28, 1095 KJ Amsterdam (The Netherlands) (Received October 20th, 1980; Revised version received November 17th, 1980; Accepted November 17lh,
1980)
Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of c o r d - D R G explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and D R G - s p i n a l cord interconnectivity were well sustained in CDM.
The inherent variability and undefined nature of sera makes the use of serumsupplemented media less than desirable in experiments where precise control of the environmental factors is required. Recent success in culturing several neural cell types in chemically defined medium (CDM) [1, 4-7] led us to adopt C D M in order to study bioelectric activity and interconnectivity of organotypic mouse spinal c o r d - d o r s a l root ganglion (DRG) cultures. Cross-sections of spinal cord with attached DRGs (0.5-1.0 m m thick) were cut from 13-14-day mouse embryos and cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 20% horse serum (HS) in Nunc culture dishes (32 m m diameter with air vents) on a collagen substrate. After 0 - 7 days in vitro, 5fluorodeoxyuridine (FDU) was added to the medium at a concentration of 10 5 M for 5 - 1 0 days in order to suppress fibroblast and glial overgrowth. Cultures were maintained thereafter in MEM plus 20°70 HS for periods of up to 50 days. Parallel cultures were grown in CDM initially as reported by Bottenstein and Sato [1], later as modified by Romijn et al. [6]. A preincubation (overnight for 18-20 h) in CDM plus 20% HS was followed by two rinses with CDM alone. Cultures were then maintained only in the presence of the serum-free CDM. Ten culture dishes (5 M E M - H S and 5 CDM) were housed in a stainless steel box, set on grids over sterile distilled water at 37°C in a constant environment of 99% air and 1070 CO2. Twenty-four hours after plating the c o r d - D R G explants in MEM-HS, the DRG capsule disintegrated and the ganglion cells began to form an extensive monolayer. The cord began to flatten and by 3 - 4 days after explantation the entire explant had
52 b e c o m e a n ill-defined, thin mass o f cells a n d neurites. However, the characteristic shape o f the f o r m e r grey m a t t e r areas in vivo could still be recognized. After two weeks in vitro, the cultures had a s s u m e d the a p p e a r a n c e s h o w n in Fig. 1A. The D R G cells h a d migrated v a r y i n g distances f r o m the cord, a n d usually served as a h u b for thick fibre t r u n k s b o t h r a d i a t i n g to the periphery a n d l i n k i n g the cord with the D R G . Large n e u r o n e s , frequently aligned in rows, characterize these D R G areas (see inset, Fig. 1A).
lmrrl
Fig. 1. Spinal cord-dorsal root ganglion (DRG) explants from 13-day foetal mouse in living unstained cultures. A: example of cord-DRG explant grown 12 days in HS-supplemented medium. This culture was exposed to 10 5 M FDU from day 2 to 7 in vitro, Inset: 580 × magnification of DRG showing the typical row-like alignment of the sensory neurones. B: example of cord-DRG explants grown 29 days in vitro in Romijn modified medium. Note the homogeneously dispersed glial background. Inset: 580 × magnification of DRG showing well-fasciculated fibre tracts and a more compact grouping of the sensory neurone. Scale = 1 ram.
53 CDM cultures mature in a rather different fashion. Little observable flattening of the cord of DRG occurs. Cord explants retain their characteristic shape as seen in vivo, while the DRG remain globe-like (Fig. 1B and inset). An individual ganglion may remain attached to the cord, forming a bulbous extension of the cord with no visible fibre connections, or may migrate away from the cord explant (Fig. 1B). The Bottenstein and Sato CDM suppressed fibroblast growth and retarded the growth of glial elements. Romijn's modification of the CDM allowed a clear-cut increase in background glial elements and appeared to enhance the formation of nerve bundles (Fig. 1B). Fibres migrating from the uppermost surfaces of the cord, either singly or in groups, frequently connected with the collagen surface only at their growing tips. A major portion of the fibre trunk or fibre thus remains unattached to any substratum and freely moves in the medium. As a consequence, these explants were anchored somewhat less firmly to the collagen substratum than were explants cultured in serum-conditioned medium, and more care had to be exercised during fixation and staining procedures so as not to loosen the explants from the plate surfaces. Bioelectric activity within the spinal cord explants was recorded at 2 - 4 weeks in vitro using saline-filled glass micropipettes (tip size 5-15 ~m; 0.6-3 M~). The electrodes were positioned under visual control using an inverted (phase contrast) microscope mounted on a vibration-free table. Cultures grown in both MEM-HS and CDM were recorded from 16 sites, evenly spaced across the entire extent of each cord explant. During recording, cultures were kept at 37°C with a continuous flow of medium through the culture dish, the reservoir of medium being constantly gassed with CO2. All cultures (n = 29) displayed spontaneous neuronal activity which was comparable with that reported by Crain and co-workers [2, 8]. Among individual cultures, and sometimes within the same culture or even at the same recording site, the firing patterns varied from continuous (sometimes very regular) to strongly phasic firing. In addition, fluctuations in the overall firing level with an irregular time-course were observed (Fig. 2Ai,2, Bi,2). The number of sites from which spontaneous electric activity was recorded in cultures grown in the three different media is summarized in Table I. To test whether or not bioelectric activity is differentially affected by culture in the various media, the data were subjected to a one-way analysis of variance [9]. This test confirmed that the three growth media indeed were associated with different spontaneous electric activities: F(2,26) = 3.84, P < 0.05. Subsequent analysis showed that Romijn's modification of the CDM influences electrical activity more favourably compared with the combined data of cultures grown in MEM-HS and CDM (Bottenstein and Sato), /7(1,26) = 7.13, P < 0.05. Single unit data from 3 MEM-HS and 2 CDM (Romijn's modification) cultures suggested a tendency for MEM-HS cultures to fire relatively randomly, whereas the CDM cultures possessed a more temporally organized firing pattern, viz. either more phasic activity or more regular tonic firing. Furthermore, the occurrence of spike-associated slow wave activity was sometimes observed in
54
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Fig. 2. Bioelectric activity in spinal cord explants cultured in MEM-HS (A) and in Romijn modified medium (B). AI and BI: spike frequency plot representing the firing frequency of a single unit in the course of a 14-min recording period. Spikes (left part of A2 and B2) were amplitude-discriminated and counted every second. Discrimination of spikes is indicated by dots below the CRO traces (right part of Az and B2). A3 and B3: spike activity evoked by stimulation (arrow) of the DRG; strength and durations of the stimuli were - 2 5 V, 0.1 msec and - 18 V, 0,2 msec, respectively. Age in vitro of the various cultures was 23 days (Ah A2), 20 days (A3), 18 days (B], B2) and 22 days (B3). Amplitude and time scales as indicated.
CDM cultures but never in MEM-HS cultures. Recordings made before perfusing with pure MEM at the start of the experiment established that both CDM and HS cultures expressed their spontaneous bioelectric activity in the original culturing medium, and thus were not induced by the change of medium. Stimulating the DRG with a glass micropipette (tip 3-5 /~m; 3-8 Mfl), with monopolar cathodal pulses (duration 0.1-0.2 msec, strength 4-40 V, frequency 1
55
TABI.E l N U M B E R OF ACTIVE R E C O R D I N G SITES ( O L T OF 16 SITES RECORDED) PER C U L T U R E Medium MEM-HS CDM (Bottenstein and Sato) CDM(Romijn'smodification)
Mean ± S.D. n = 13 n = 7 n 9
8.38 ± 4.19 6.00 ± 3.96 11.78 ± 4.13
per 10 sec), evoked spike activity at one or more of the 16 recording sites in most of the cultures (in 11 out of 13 M E M - H S cultures (see also ref. 3), 4 out of 7 Bottenstein and Sato C D M cultures and 8 out of 9 CDM (Romijn's modification) cultures). Thus, the DRG proved capable of establishing functional connexions with spinal cord explants in either type of medium. From these data it is concluded tha( culturing c o r d - D R G explants in CDM affords enhanced preservation of their organotypic architecture over that seen in HS-supplemented medium. CDM also sustains synapse formation in dissociated fetal rat cerebral cortex tissue [6]. Bioelectric activity was well sustained in both MEM~HS and CDM. The more temporally organized spike trains and the more frequent occurrence of slow wave activity in CDM cultures, as compared with M E M - H S cultures, suggest that C D M actually favours the development of neuronal interconnections within spinal cord explants. This would be in line with the observation of relatively extensive neurite formation in the neuroblastoma B-104 cell line when grown in CDM [11.
The authors thank M.A. Corner for critical reading of the manuscript and H.G. van Oyen for his advice in the statistical analysis. They further acknowledge the accurate type-work of J. Sels and conscientious art-work of A.T. Potjer and H. Stoffels.
1 2 3
4 5
Bottenstein, J.E. and Sato, G . H . , Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 514-517. Corner, M.A. and Crain, S.M., Patterns of spontaneous bioelectric activity during maturation in culture of fetal rodent medulla and spinal cord tissues, J. Neurobiol., 3 (1972) 25-45. Crain, S.M. and Peterson, E.R., Enhanced afferent synaptic functions in fetal mouse spinal cordsensory ganglion explants following NGF-induced ganglion hypertrophy, Brain Res., 79 (1974) 145 152. Honegger, P., Lenoir, D. and Favrod, P., Growth and differentiation of aggregating fetal brain cells in a serum-free defined medium, Nature (Lond.), 282 (1979) 305-308. Messer, A., Maskin, P. and Mazurkiewicz, J.E., Effects of using a chemically defined medium for primary rat monolayer cerebellar cultures: morphology, G A B A uptake and kainic acid sensitivity, Brain Res., 184 (1980) 243-247.
56 6 7 8
9
Romijn, H.J., Mud, M., Wolters, P. and Habets, A.M.M.C., Outgrowth and synaptogenesis ~ dissociated fetal rat cerebral cortex ill serum-free defined medium, submitted. Snyder, E.Y. and Kim, S.U., Hormonal requirements for neuronal survival in culture, Neurosc~ Lett., 13 (1979) 225-230. Tarrade, T. and Crain, S.M., Regional localization of patterned spontaneous discharges during maturation in culture of fetal mouse medulla and spinal cord explants, Develop. Neurosci., I (1978) 119-132. Winer, B.J., Statistical Principles in Experimental Design, McGraw-Hill, Kogakuska Ltd., Tokyo, 1971.
5l
C H E M I C A L L Y D E F I N E D M E D I U M E N H A N C E S BIOELECTRIC ACTIVITY IN M O U S E S P I N A L C O R D - D O R S A L ROOT G A N G L I O N C U L T U R E S
A L P H O N S M . M . C . HABETS, ROBERT E. BAKER, ELI B R E N N E R and H E R M S J. R O M I J N
Netherlands Institute for Brain Research, IJdijk 28, 1095 KJ Amsterdam (The Netherlands) (Received October 20th, 1980; Revised version received November 17th, 1980; Accepted November 17lh,
1980)
Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of c o r d - D R G explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and D R G - s p i n a l cord interconnectivity were well sustained in CDM.
The inherent variability and undefined nature of sera makes the use of serumsupplemented media less than desirable in experiments where precise control of the environmental factors is required. Recent success in culturing several neural cell types in chemically defined medium (CDM) [1, 4-7] led us to adopt C D M in order to study bioelectric activity and interconnectivity of organotypic mouse spinal c o r d - d o r s a l root ganglion (DRG) cultures. Cross-sections of spinal cord with attached DRGs (0.5-1.0 m m thick) were cut from 13-14-day mouse embryos and cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 20% horse serum (HS) in Nunc culture dishes (32 m m diameter with air vents) on a collagen substrate. After 0 - 7 days in vitro, 5fluorodeoxyuridine (FDU) was added to the medium at a concentration of 10 5 M for 5 - 1 0 days in order to suppress fibroblast and glial overgrowth. Cultures were maintained thereafter in MEM plus 20°70 HS for periods of up to 50 days. Parallel cultures were grown in CDM initially as reported by Bottenstein and Sato [1], later as modified by Romijn et al. [6]. A preincubation (overnight for 18-20 h) in CDM plus 20% HS was followed by two rinses with CDM alone. Cultures were then maintained only in the presence of the serum-free CDM. Ten culture dishes (5 M E M - H S and 5 CDM) were housed in a stainless steel box, set on grids over sterile distilled water at 37°C in a constant environment of 99% air and 1070 CO2. Twenty-four hours after plating the c o r d - D R G explants in MEM-HS, the DRG capsule disintegrated and the ganglion cells began to form an extensive monolayer. The cord began to flatten and by 3 - 4 days after explantation the entire explant had
52 b e c o m e a n ill-defined, thin mass o f cells a n d neurites. However, the characteristic shape o f the f o r m e r grey m a t t e r areas in vivo could still be recognized. After two weeks in vitro, the cultures had a s s u m e d the a p p e a r a n c e s h o w n in Fig. 1A. The D R G cells h a d migrated v a r y i n g distances f r o m the cord, a n d usually served as a h u b for thick fibre t r u n k s b o t h r a d i a t i n g to the periphery a n d l i n k i n g the cord with the D R G . Large n e u r o n e s , frequently aligned in rows, characterize these D R G areas (see inset, Fig. 1A).
lmrrl
Fig. 1. Spinal cord-dorsal root ganglion (DRG) explants from 13-day foetal mouse in living unstained cultures. A: example of cord-DRG explant grown 12 days in HS-supplemented medium. This culture was exposed to 10 5 M FDU from day 2 to 7 in vitro, Inset: 580 × magnification of DRG showing the typical row-like alignment of the sensory neurones. B: example of cord-DRG explants grown 29 days in vitro in Romijn modified medium. Note the homogeneously dispersed glial background. Inset: 580 × magnification of DRG showing well-fasciculated fibre tracts and a more compact grouping of the sensory neurone. Scale = 1 ram.
53 CDM cultures mature in a rather different fashion. Little observable flattening of the cord of DRG occurs. Cord explants retain their characteristic shape as seen in vivo, while the DRG remain globe-like (Fig. 1B and inset). An individual ganglion may remain attached to the cord, forming a bulbous extension of the cord with no visible fibre connections, or may migrate away from the cord explant (Fig. 1B). The Bottenstein and Sato CDM suppressed fibroblast growth and retarded the growth of glial elements. Romijn's modification of the CDM allowed a clear-cut increase in background glial elements and appeared to enhance the formation of nerve bundles (Fig. 1B). Fibres migrating from the uppermost surfaces of the cord, either singly or in groups, frequently connected with the collagen surface only at their growing tips. A major portion of the fibre trunk or fibre thus remains unattached to any substratum and freely moves in the medium. As a consequence, these explants were anchored somewhat less firmly to the collagen substratum than were explants cultured in serum-conditioned medium, and more care had to be exercised during fixation and staining procedures so as not to loosen the explants from the plate surfaces. Bioelectric activity within the spinal cord explants was recorded at 2 - 4 weeks in vitro using saline-filled glass micropipettes (tip size 5-15 ~m; 0.6-3 M~). The electrodes were positioned under visual control using an inverted (phase contrast) microscope mounted on a vibration-free table. Cultures grown in both MEM-HS and CDM were recorded from 16 sites, evenly spaced across the entire extent of each cord explant. During recording, cultures were kept at 37°C with a continuous flow of medium through the culture dish, the reservoir of medium being constantly gassed with CO2. All cultures (n = 29) displayed spontaneous neuronal activity which was comparable with that reported by Crain and co-workers [2, 8]. Among individual cultures, and sometimes within the same culture or even at the same recording site, the firing patterns varied from continuous (sometimes very regular) to strongly phasic firing. In addition, fluctuations in the overall firing level with an irregular time-course were observed (Fig. 2Ai,2, Bi,2). The number of sites from which spontaneous electric activity was recorded in cultures grown in the three different media is summarized in Table I. To test whether or not bioelectric activity is differentially affected by culture in the various media, the data were subjected to a one-way analysis of variance [9]. This test confirmed that the three growth media indeed were associated with different spontaneous electric activities: F(2,26) = 3.84, P < 0.05. Subsequent analysis showed that Romijn's modification of the CDM influences electrical activity more favourably compared with the combined data of cultures grown in MEM-HS and CDM (Bottenstein and Sato), /7(1,26) = 7.13, P < 0.05. Single unit data from 3 MEM-HS and 2 CDM (Romijn's modification) cultures suggested a tendency for MEM-HS cultures to fire relatively randomly, whereas the CDM cultures possessed a more temporally organized firing pattern, viz. either more phasic activity or more regular tonic firing. Furthermore, the occurrence of spike-associated slow wave activity was sometimes observed in
54
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Fig. 2. Bioelectric activity in spinal cord explants cultured in MEM-HS (A) and in Romijn modified medium (B). AI and BI: spike frequency plot representing the firing frequency of a single unit in the course of a 14-min recording period. Spikes (left part of A2 and B2) were amplitude-discriminated and counted every second. Discrimination of spikes is indicated by dots below the CRO traces (right part of Az and B2). A3 and B3: spike activity evoked by stimulation (arrow) of the DRG; strength and durations of the stimuli were - 2 5 V, 0.1 msec and - 18 V, 0,2 msec, respectively. Age in vitro of the various cultures was 23 days (Ah A2), 20 days (A3), 18 days (B], B2) and 22 days (B3). Amplitude and time scales as indicated.
CDM cultures but never in MEM-HS cultures. Recordings made before perfusing with pure MEM at the start of the experiment established that both CDM and HS cultures expressed their spontaneous bioelectric activity in the original culturing medium, and thus were not induced by the change of medium. Stimulating the DRG with a glass micropipette (tip 3-5 /~m; 3-8 Mfl), with monopolar cathodal pulses (duration 0.1-0.2 msec, strength 4-40 V, frequency 1
55
TABI.E l N U M B E R OF ACTIVE R E C O R D I N G SITES ( O L T OF 16 SITES RECORDED) PER C U L T U R E Medium MEM-HS CDM (Bottenstein and Sato) CDM(Romijn'smodification)
Mean ± S.D. n = 13 n = 7 n 9
8.38 ± 4.19 6.00 ± 3.96 11.78 ± 4.13
per 10 sec), evoked spike activity at one or more of the 16 recording sites in most of the cultures (in 11 out of 13 M E M - H S cultures (see also ref. 3), 4 out of 7 Bottenstein and Sato C D M cultures and 8 out of 9 CDM (Romijn's modification) cultures). Thus, the DRG proved capable of establishing functional connexions with spinal cord explants in either type of medium. From these data it is concluded tha( culturing c o r d - D R G explants in CDM affords enhanced preservation of their organotypic architecture over that seen in HS-supplemented medium. CDM also sustains synapse formation in dissociated fetal rat cerebral cortex tissue [6]. Bioelectric activity was well sustained in both MEM~HS and CDM. The more temporally organized spike trains and the more frequent occurrence of slow wave activity in CDM cultures, as compared with M E M - H S cultures, suggest that C D M actually favours the development of neuronal interconnections within spinal cord explants. This would be in line with the observation of relatively extensive neurite formation in the neuroblastoma B-104 cell line when grown in CDM [11.
The authors thank M.A. Corner for critical reading of the manuscript and H.G. van Oyen for his advice in the statistical analysis. They further acknowledge the accurate type-work of J. Sels and conscientious art-work of A.T. Potjer and H. Stoffels.
1 2 3
4 5
Bottenstein, J.E. and Sato, G . H . , Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 514-517. Corner, M.A. and Crain, S.M., Patterns of spontaneous bioelectric activity during maturation in culture of fetal rodent medulla and spinal cord tissues, J. Neurobiol., 3 (1972) 25-45. Crain, S.M. and Peterson, E.R., Enhanced afferent synaptic functions in fetal mouse spinal cordsensory ganglion explants following NGF-induced ganglion hypertrophy, Brain Res., 79 (1974) 145 152. Honegger, P., Lenoir, D. and Favrod, P., Growth and differentiation of aggregating fetal brain cells in a serum-free defined medium, Nature (Lond.), 282 (1979) 305-308. Messer, A., Maskin, P. and Mazurkiewicz, J.E., Effects of using a chemically defined medium for primary rat monolayer cerebellar cultures: morphology, G A B A uptake and kainic acid sensitivity, Brain Res., 184 (1980) 243-247.
56 6 7 8
9
Romijn, H.J., Mud, M., Wolters, P. and Habets, A.M.M.C., Outgrowth and synaptogenesis ~ dissociated fetal rat cerebral cortex ill serum-free defined medium, submitted. Snyder, E.Y. and Kim, S.U., Hormonal requirements for neuronal survival in culture, Neurosc~ Lett., 13 (1979) 225-230. Tarrade, T. and Crain, S.M., Regional localization of patterned spontaneous discharges during maturation in culture of fetal mouse medulla and spinal cord explants, Develop. Neurosci., I (1978) 119-132. Winer, B.J., Statistical Principles in Experimental Design, McGraw-Hill, Kogakuska Ltd., Tokyo, 1971.