Mechanisms of Ageing and Development, 9 (1979) 553-566 ©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
553
GLIAL CELLS: MODULATORS OF NEURONAL ENVIRONMENT*
ANTONIA VERNADAKIS, RAEL NIDESS, BRUCE CULVER and ELLEN BRAGG ARNOLD Departments of Psychiatry and Pharmacology, Developmental Psychobiology Research Group, University of Colorado, School of Medicine, Denver, Colorado 80262 (U.S.A.) (Received December 5, 1977; in revised form April 20, 1978) SUMMARY Studies of glial cells in neural tissue culture systems suggest that glial cells subserve different functions during development and aging of the central nervous system and that they may help modulate the neuronal environment by virtue of their responsiveness to hormones and other intrinsic factors. There is a marked proliferation of glial cells during early stages of brain development, probably reflecting the involvement of glial cells in myelination and other growth processes. Studies in culture suggest that proliferation of glial cells can be induced by steroid hormones. The migration rate of glial cells from cerebellar explants of embryonic chick brain grown in organotypic culture was measured in control and hormone-treated explants. Treatment with cortisol, corticosterone, estradiol, and progesterone significantly elevated glial cell migration from the tissue explants. The influence of steroid hormones on glial cells may be mediated via a steroid intracellular mechanism. In C-6 glioma cells and in chick embryo dissociated brain cell cultures consisting predominantly of glial cells, 3H-corticosterone was shown to accumulate by a saturable but non-specific retention mechanism. In contrast, the accumulation of aH-corticosterone by predominantly neuronal cultures was both saturable and specific. Glial cells in culture exhibit certain age-related changes, including changes in resting membrane potentials and in cellular responses to hormone treatment, as measured by changes in incorporation of 3H-leucine into protein and incorporation of 3H-uridine into RNA. The possibility that glial cells in rive may likewise exhibit differential responses to hormones throughout the lifespan suggests that hormones may markedly influence cellular aging.
INTRODUCTION In view of the fact that glial cells in the central nervous system outnumber neurons by a factor of ten, their role in CNS function continues to be the subject of much investigation. Glial cells have been proposed to be involved in myelination [ 1], to act as spatial ionic buffers [2] to be electrica/ly coupled to neurons [3] and to proliferate with in*Based on a paper presented at a specialgroup of Symposiaentitled, "Frontiers in AgingResearch", arranged by the program committee of the Biological SciencesSection of the Gerontological Society, San Francisco Meeting, November 18-22, 1977.
554 creased neuronal activity [4, 5]. More recently, glial ceils have been shown to accumulate neurotransmitters [6] and to be involved in the regulation of cyclic nucleotides [7]. The role ofglial cells in the aging of the central nervous system is only beginning to be explored. Neural tissue culture systems, derived either from normal tissue or neoplastic cell lines, provide models with which to investigate physiological, morphological, and biochemical properties of glial cells as well as their coupling to neuronal function. This paper will discuss some of our findings in glial cells using neural culture techniques. These findings lead us to suggest that glial cells subserve different functions during development and aging of the CNS and that they modulate the neuronal microenvironment by virtue of their responsiveness to intrinsic factors, such as hormones.
PROLIFERATION OF GLIAL CELLS Using the chicken as the experimental animal, we have studied changes in the concentration of nucleic acids and the activity of butyrylcholinesterase (BuChE) in various brain areas throughout the lifespan of this animal [8]. Based on the premise that DNA is located almost exclusively within the nucleus and is constant in amount within the diploid nucleus of a resting cell of any species [9], DNA content has been interpreted as an index of cell density. Thus, during early embryonic development the decrease in DNA content observed in both the cerebral hemispheres and cerebellum (Fig. 1) reflects a decrease in cell density due to an increase in cell size, i.e., cell body growth and dendritic branching which are known to occur with maturation [ 10]. This active cellular growth also is reflected in the high RNA content observed in the cerebral hemispheres and cerebellum during early embryonic maturation (Fig. 2). o
o Cerebrol hemispheres
o------e O p t i c
x- ..........x C e r e b e l l u m
~---a
lobes
Oiencepholon- midbroin
7
OI EMBRYONE". !
AGE
POS T-H,4TCHING AGE
EMBRYONIC AGE
POST-HATCHING AGE
]c 4.~
k.
"t..
/
::, l " ' ~ J ~ /
'.,-
"')-- ~
Z C:3
~ ac I-.....i../6
/--~\
,.
ON( DAY ~4 . Ooys
Months
.
~6 .
.
m
Ooys
~o
)
~53 . .
.
. ,z.
2o
~o;,
Mo~ths
Fig. 1. Changes in DNA content in various CNS areas of chicks during embryonic age (days) and posthatching age (months). Points with bracketed vertical lines represent means ±S.E. (from Vernadakis
(81).
555
o
o Cerebral hemispheres
x ..............~
•
Cerebellum
6.C EMBRYONIC AGE
..... 4 Optic lobes
~---~
POST-HATCHINGAGE
Diencephalon-midbroin
EMBRYONIC AGE
POST-HATCHING AGE
5.(3 .~ 4.0
-$ ~n
3.C
E 2.C
~-~.~.:~
r~n
""
I.C ON~ DAY
,;~ ;6 ~ ~t . Days
ONE DAY
.
.
.
Months
t,, [~3ys
Months
Fig. 2. As in Fig. 1 except changes are in RNA content.
The pronounced increase in DNA content (unit weight) of the cerebellum between 20 days of embryonic age and one day after hatching is interpreted to reflect the marked increase in cell density, predominantly of granular cells and glial cells, during this period [11]. If one uses the activity of BuChE as an index of glial cell numbers [12] one can deduce that there is a marked proliferation of glial cells between 16 days of embryonic age and 3 months after hatching, a decrease between 3 and 12 months after hatching, and a second period of proliferation between 12 and 36 months (Fig. 3). The proliferation of glial cells during early growth has been associated with the involvement of glial cells in myelination and other neural growth processes [1]. It has been hypothesized [13] that this early proliferation of glial cells is induced by hormones, especially steroid hormones. This hypothesis is consistent with findings in neural tissue culture as discussed below. The later proliferation ofglial cells with aging has been postulated to represent a compensatory process of the brain to overcome morphologic and functional neuronal loss or neuronal changes occurring with age [ 14]. However, studies by Murray [ 15] have demonstrated an increase in aH-thymidine labelling of glial cells in the rat hypothalamus during dehydration which is induced by administering to the animals as 1% saline solution instead of water for a two-week period. In view of the fact that there is a decrease in water content of the brain with aging [13, 14], the later evident proliferation of glial cells could reflect a response to dehydration. Several neural culture systems have been used to elucidate the function of glial cells in the CNS and have been recently reviewed by us [16]. In early studies we employed the neural organotypic culture to study morphological and electrophysiological aspects of neural growth and differentiation and the role of hormones thereon [17]. A schematic representation of the Maximow double coverslip assembly for organotypic culture is illustrated in Fig. 4. The cerebellum is removed from a 15-day-old chick
556 o---o
EMBRYONIC AGE
.~_
E
e ----e Optic lobes
x.............~ C e r e b e l l u m
4.0
.~-
Cerebral hemispheres
t~---,, Diencephalon - midbrain
POST-HATCHINGAGE
EMBRYONIC AGE
,,,
POST-HATCHING AGE
:tO
i/ i
o ~:
2.C
¢.~ ¢-
I.~
/tJ
il
!J il
......
~,/
II'L
\\ \ /
!I
~r
~. Lc
,) /
g~ ~ 0~
o I ~ new ",
0
~W
Months
Days
Months
Fig. 3. As in Fig. 1 except changes are in the activity of butyrylcholinesterase.
- -
/
TOP ViEW
MAXIMOW DOUBUE COVERSLIP ASSEMBLY
Fig. 4. Diagram of a Maximow double coverslip assembly: organotypic culture set-up(from Vernadakis
[17]), embryo and placed into a sterile dish containing balanced salt solution. The tissue is stripped of its meningeal coverings, cut into 0.5 rnm 3 fragments, and two tissue fragments are explanted on Gold Seal rectangular coverslips, previously coated with a thin film of reconstituted rat tail collagen [ 18]. A single drop of nutrient medium is added to cover the two tissue fragments, and each coverslip is then incorporated into a Maximow double coverslip assembly and sealed with paraffin. The nutrient medium consists of 50% human ascitic fluid, 45% Gey's balanced salt solution, 5% embryo extract prepared from 9-dayold chick embryo, and 600 rag% glucose. When hormones were added to the medium, the final concentrations to which the explants were exposed were as follows: cortisol, 2.76 X 10- s M; corticosterone, 2.89 X 10- s M; estradiol dipropionate, 2.65 X l0 - s M; proge-
557 sterone, 3.8 × 10 - s M; and testosterone, 3.47 × 1 0 - a M . Cultures were incubated in the lying drop position at approximately 36 °C. As has been observed by Murray [19] microglial cells are the first to emigrate from the explant. These cells are followed by neuroglial elements, most notably oligodendroglia. As the culture advances in age and complexity of organization, oligodendroglia are overshadowed by a variety of astrocytes. The migration rates o f cells from the explant were compared with control and hormone-treated explants (Table I). This rate was calculated as follows: outlines of the explants were traced at 1 and 5 days by focusing the image of the explant on translucent paper placed on the translucent back o f a camera attached to the microscope. The images were cut from the paper and the papers were weighed to determine relative surface areas. The ratio between the surfaces of 1-day- and 5-day-old cultures was taken to represent the migration rate. The migration rates in the cortisol-, corticosterone-, estradiol-, and progesterone-treated explants were markedly higher than in the corresponding control explants (Table I). In addition, DNA content was significantly higher in explants cultured in the presence of cortisol or estradiol (evidence not shown here). If one assumes that the higher DNA content in the explants cultured in the presence of hormones reflects a greater number of cells, the increase in migration rate induced by these hormone treatments is probably also related to an increased number of cells, predominantly glial cells.
TABLE I EFFECTS OF HORMONES ON THE MIGRATION RATE OF CELLS IN CULTURED CEREBELLAR EXPLANTS* Culture medium
Number o f explants
Migration rate (mean ratio +-SE}
Control for cortisol Cortisol
67 50
Control for eortieosterone Corticosterone
16 21
4.07 -+0.12"* 5.06 -+0.23 (
553
GLIAL CELLS: MODULATORS OF NEURONAL ENVIRONMENT*
ANTONIA VERNADAKIS, RAEL NIDESS, BRUCE CULVER and ELLEN BRAGG ARNOLD Departments of Psychiatry and Pharmacology, Developmental Psychobiology Research Group, University of Colorado, School of Medicine, Denver, Colorado 80262 (U.S.A.) (Received December 5, 1977; in revised form April 20, 1978) SUMMARY Studies of glial cells in neural tissue culture systems suggest that glial cells subserve different functions during development and aging of the central nervous system and that they may help modulate the neuronal environment by virtue of their responsiveness to hormones and other intrinsic factors. There is a marked proliferation of glial cells during early stages of brain development, probably reflecting the involvement of glial cells in myelination and other growth processes. Studies in culture suggest that proliferation of glial cells can be induced by steroid hormones. The migration rate of glial cells from cerebellar explants of embryonic chick brain grown in organotypic culture was measured in control and hormone-treated explants. Treatment with cortisol, corticosterone, estradiol, and progesterone significantly elevated glial cell migration from the tissue explants. The influence of steroid hormones on glial cells may be mediated via a steroid intracellular mechanism. In C-6 glioma cells and in chick embryo dissociated brain cell cultures consisting predominantly of glial cells, 3H-corticosterone was shown to accumulate by a saturable but non-specific retention mechanism. In contrast, the accumulation of aH-corticosterone by predominantly neuronal cultures was both saturable and specific. Glial cells in culture exhibit certain age-related changes, including changes in resting membrane potentials and in cellular responses to hormone treatment, as measured by changes in incorporation of 3H-leucine into protein and incorporation of 3H-uridine into RNA. The possibility that glial cells in rive may likewise exhibit differential responses to hormones throughout the lifespan suggests that hormones may markedly influence cellular aging.
INTRODUCTION In view of the fact that glial cells in the central nervous system outnumber neurons by a factor of ten, their role in CNS function continues to be the subject of much investigation. Glial cells have been proposed to be involved in myelination [ 1], to act as spatial ionic buffers [2] to be electrica/ly coupled to neurons [3] and to proliferate with in*Based on a paper presented at a specialgroup of Symposiaentitled, "Frontiers in AgingResearch", arranged by the program committee of the Biological SciencesSection of the Gerontological Society, San Francisco Meeting, November 18-22, 1977.
554 creased neuronal activity [4, 5]. More recently, glial ceils have been shown to accumulate neurotransmitters [6] and to be involved in the regulation of cyclic nucleotides [7]. The role ofglial cells in the aging of the central nervous system is only beginning to be explored. Neural tissue culture systems, derived either from normal tissue or neoplastic cell lines, provide models with which to investigate physiological, morphological, and biochemical properties of glial cells as well as their coupling to neuronal function. This paper will discuss some of our findings in glial cells using neural culture techniques. These findings lead us to suggest that glial cells subserve different functions during development and aging of the CNS and that they modulate the neuronal microenvironment by virtue of their responsiveness to intrinsic factors, such as hormones.
PROLIFERATION OF GLIAL CELLS Using the chicken as the experimental animal, we have studied changes in the concentration of nucleic acids and the activity of butyrylcholinesterase (BuChE) in various brain areas throughout the lifespan of this animal [8]. Based on the premise that DNA is located almost exclusively within the nucleus and is constant in amount within the diploid nucleus of a resting cell of any species [9], DNA content has been interpreted as an index of cell density. Thus, during early embryonic development the decrease in DNA content observed in both the cerebral hemispheres and cerebellum (Fig. 1) reflects a decrease in cell density due to an increase in cell size, i.e., cell body growth and dendritic branching which are known to occur with maturation [ 10]. This active cellular growth also is reflected in the high RNA content observed in the cerebral hemispheres and cerebellum during early embryonic maturation (Fig. 2). o
o Cerebrol hemispheres
o------e O p t i c
x- ..........x C e r e b e l l u m
~---a
lobes
Oiencepholon- midbroin
7
OI EMBRYONE". !
AGE
POS T-H,4TCHING AGE
EMBRYONIC AGE
POST-HATCHING AGE
]c 4.~
k.
"t..
/
::, l " ' ~ J ~ /
'.,-
"')-- ~
Z C:3
~ ac I-.....i../6
/--~\
,.
ON( DAY ~4 . Ooys
Months
.
~6 .
.
m
Ooys
~o
)
~53 . .
.
. ,z.
2o
~o;,
Mo~ths
Fig. 1. Changes in DNA content in various CNS areas of chicks during embryonic age (days) and posthatching age (months). Points with bracketed vertical lines represent means ±S.E. (from Vernadakis
(81).
555
o
o Cerebral hemispheres
x ..............~
•
Cerebellum
6.C EMBRYONIC AGE
..... 4 Optic lobes
~---~
POST-HATCHINGAGE
Diencephalon-midbroin
EMBRYONIC AGE
POST-HATCHING AGE
5.(3 .~ 4.0
-$ ~n
3.C
E 2.C
~-~.~.:~
r~n
""
I.C ON~ DAY
,;~ ;6 ~ ~t . Days
ONE DAY
.
.
.
Months
t,, [~3ys
Months
Fig. 2. As in Fig. 1 except changes are in RNA content.
The pronounced increase in DNA content (unit weight) of the cerebellum between 20 days of embryonic age and one day after hatching is interpreted to reflect the marked increase in cell density, predominantly of granular cells and glial cells, during this period [11]. If one uses the activity of BuChE as an index of glial cell numbers [12] one can deduce that there is a marked proliferation of glial cells between 16 days of embryonic age and 3 months after hatching, a decrease between 3 and 12 months after hatching, and a second period of proliferation between 12 and 36 months (Fig. 3). The proliferation of glial cells during early growth has been associated with the involvement of glial cells in myelination and other neural growth processes [1]. It has been hypothesized [13] that this early proliferation of glial cells is induced by hormones, especially steroid hormones. This hypothesis is consistent with findings in neural tissue culture as discussed below. The later proliferation ofglial cells with aging has been postulated to represent a compensatory process of the brain to overcome morphologic and functional neuronal loss or neuronal changes occurring with age [ 14]. However, studies by Murray [ 15] have demonstrated an increase in aH-thymidine labelling of glial cells in the rat hypothalamus during dehydration which is induced by administering to the animals as 1% saline solution instead of water for a two-week period. In view of the fact that there is a decrease in water content of the brain with aging [13, 14], the later evident proliferation of glial cells could reflect a response to dehydration. Several neural culture systems have been used to elucidate the function of glial cells in the CNS and have been recently reviewed by us [16]. In early studies we employed the neural organotypic culture to study morphological and electrophysiological aspects of neural growth and differentiation and the role of hormones thereon [17]. A schematic representation of the Maximow double coverslip assembly for organotypic culture is illustrated in Fig. 4. The cerebellum is removed from a 15-day-old chick
556 o---o
EMBRYONIC AGE
.~_
E
e ----e Optic lobes
x.............~ C e r e b e l l u m
4.0
.~-
Cerebral hemispheres
t~---,, Diencephalon - midbrain
POST-HATCHINGAGE
EMBRYONIC AGE
,,,
POST-HATCHING AGE
:tO
i/ i
o ~:
2.C
¢.~ ¢-
I.~
/tJ
il
!J il
......
~,/
II'L
\\ \ /
!I
~r
~. Lc
,) /
g~ ~ 0~
o I ~ new ",
0
~W
Months
Days
Months
Fig. 3. As in Fig. 1 except changes are in the activity of butyrylcholinesterase.
- -
/
TOP ViEW
MAXIMOW DOUBUE COVERSLIP ASSEMBLY
Fig. 4. Diagram of a Maximow double coverslip assembly: organotypic culture set-up(from Vernadakis
[17]), embryo and placed into a sterile dish containing balanced salt solution. The tissue is stripped of its meningeal coverings, cut into 0.5 rnm 3 fragments, and two tissue fragments are explanted on Gold Seal rectangular coverslips, previously coated with a thin film of reconstituted rat tail collagen [ 18]. A single drop of nutrient medium is added to cover the two tissue fragments, and each coverslip is then incorporated into a Maximow double coverslip assembly and sealed with paraffin. The nutrient medium consists of 50% human ascitic fluid, 45% Gey's balanced salt solution, 5% embryo extract prepared from 9-dayold chick embryo, and 600 rag% glucose. When hormones were added to the medium, the final concentrations to which the explants were exposed were as follows: cortisol, 2.76 X 10- s M; corticosterone, 2.89 X 10- s M; estradiol dipropionate, 2.65 X l0 - s M; proge-
557 sterone, 3.8 × 10 - s M; and testosterone, 3.47 × 1 0 - a M . Cultures were incubated in the lying drop position at approximately 36 °C. As has been observed by Murray [19] microglial cells are the first to emigrate from the explant. These cells are followed by neuroglial elements, most notably oligodendroglia. As the culture advances in age and complexity of organization, oligodendroglia are overshadowed by a variety of astrocytes. The migration rates o f cells from the explant were compared with control and hormone-treated explants (Table I). This rate was calculated as follows: outlines of the explants were traced at 1 and 5 days by focusing the image of the explant on translucent paper placed on the translucent back o f a camera attached to the microscope. The images were cut from the paper and the papers were weighed to determine relative surface areas. The ratio between the surfaces of 1-day- and 5-day-old cultures was taken to represent the migration rate. The migration rates in the cortisol-, corticosterone-, estradiol-, and progesterone-treated explants were markedly higher than in the corresponding control explants (Table I). In addition, DNA content was significantly higher in explants cultured in the presence of cortisol or estradiol (evidence not shown here). If one assumes that the higher DNA content in the explants cultured in the presence of hormones reflects a greater number of cells, the increase in migration rate induced by these hormone treatments is probably also related to an increased number of cells, predominantly glial cells.
TABLE I EFFECTS OF HORMONES ON THE MIGRATION RATE OF CELLS IN CULTURED CEREBELLAR EXPLANTS* Culture medium
Number o f explants
Migration rate (mean ratio +-SE}
Control for cortisol Cortisol
67 50
Control for eortieosterone Corticosterone
16 21
4.07 -+0.12"* 5.06 -+0.23 (
