Environment temperature affects cell proliferation in the spinal cord and brain of juvenile turtles
1 Departamento de Histología y Embriología, Facultad de
Medicina, Montevideo, Uruguay ZC 11800
2 Laboratorio de Neuroanatomía Comparada, Instituto de
Investigaciones Biológicas Clemente Estable, Avda. Italia 3318,
Montevideo, Uruguay ZC 11600
3 Unidad Asociada a la Facultad de Ciencias, Montevideo, Uruguay ZC
11600
* Author for correspondence (e-mail: omar{at}iibce.edu.uy)
Accepted 27 May 2003
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Summary |
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Key words: neurogenesis, cell proliferation, temperature acclimation, brain, spinal cord, BrdU, turtle, Chrysemys d'orbigny
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Introduction |
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Despite important advances made in the field, information concerned with
environmental factors influencing cell proliferation in the CNS of ectotherm
vertebrates is still scarce. Since metabolic activity in these animals is
largely dependent on heat transfer from the environment
(Prosser, 1952), temperature
appeared to be a plausible external factor that could affect post-natal cell
proliferation in the CNS. Confirming this hypothesis, Ramírez et al.
(1997
) and Peñafiel et
al. (2001
) have reported that
temperature increases neurogenesis and neuroblast migration in the brain of
adult lizards.
The purpose of the present paper is to demonstrate that environmental
temperature modulates cell proliferation in the CNS, including the spinal
cord, of juvenile turtles. Here, we employed bromodeoxyuridine (BrdU) to label
proliferating cells and other immunostaining procedures to identify the
temperature-affected cell population. Our studies have revealed that
warm-acclimated turtles (WATs) showed a statistically significant increase in
proliferating cells when compared with cold-acclimated turtles (CATs).
Multiple-labeling experiments showed that an important percentage of the
proliferating cells exhibited the morphological and immunostaining
characteristics of glial cells, including typical radial glia (RG). Since
unanimously accepted criteria for identifying stem/progenitor cells are still
lacking (Scheffler et al.,
1999; Seaberg and van der
Kooy, 2003
), we have preferred to use a purely descriptive term
such as `proliferating cells' to name the cell population that incorporated
BrdU.
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Materials and methods |
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BrdU-labeling of proliferating cells
Twenty turtles were divided into two groups of 10 animals, and each group
was maintained in separate aquaria at different temperatures. WATs were
maintained in a warm, controlled environment (27-30°C), while CATs were
maintained in an outdoor aquarium under the influence of the seasonal
fluctuating temperature (5-14°C). Since recent investigations performed in
mammals indicate the adverse influence of stress on post-natal neurogenesis
(Gould et al., 1998), a
fixed-temperature cold environment was avoided. On the other hand, the
selected warm temperature range was revealed to be stimulating for turtles,
increasing their motor activity, food intake and body mass. Both groups were
under seasonal daily light-dark rhythms and were provided with abundant food
(living Tubifex and small earthworms). In experiments performed with
CATs, the mean temperature during measurements made six days before the
injection time point was considered to be the environmental temperature for
each of the experiments. Turtles from both groups received a single
intraperitoneal dose of BrdU (100 mg kg-1) and were perfused with
the fixative solution 24 h later. It should be noted that, according to our
test experiments (N=4), a dose as high as 800 mg kg-1
seems to be innocuous and does not induce labeling of non-mitotic cells.
We also performed `paired experiments' (N=4) in which one WAT and
one CAT received the BrdU pulse on the same day at the same time, and both
animals were sacrificed 24 h later. These experiments were designed to rule
out unexplored biological rhythms
(Cermakian and Sassone-Corsi,
2001) that might be affecting cell proliferation in the CNS. To
minimize potential variations during immunostaining, tissues from both turtles
were processed together.
Fixation procedures were always performed in anesthetized animals unresponsive to nociceptive stimuli. To achieve complete anesthesia, 5 mg kg-1 of sodium methohexitone (Brietal, Lilly, Basingstoke, UK) were injected intraperitoneally. Saline used to wash the blood vessels as well as the fixative fluids were propelled into the vascular bed using a peristaltic pump. Brains and spinal cords were albumin-gelatin embedded and cross-sectioned using a vibrating microtome (each section was 60 µm thick). Sections were hydrolyzed (2 mol l-1 HCl for 1 h), passed through three washing buffered solutions and incubated overnight in a buffered solution containing 0.3% Triton X-100 and the anti-BrdU antibody (1:500 monoclonal; Dako A/S, Glostrup, Denmark). Detection of nuclei that had incorporated BrdU was achieved using horseradish peroxidase (HRP)-conjugated or fluorophore-conjugated secondary antibodies (anti-mouse made in goat; 1:500; Chemicon International, Inc., Temecula, CA, USA). The HRP was revealed using diaminobenzidine or the peroxidase substrate kit from Vector Labs (Burlingame, CA, USA).
Densities of BrdU-labeled nuclei (BrdU-LN) were calculated in the following
spinal cord regions: lateral funiculus (LF), dorsal funiculus (DF), ventral
funiculus (VF), dorsal horn (DH), ventral horn (VH), intermediate region (IR)
and central region (CR). Densities of BrdU-labeled nuclei were also calculated
in the dorsal cortex (DC) and medial cortex (MC) of the brain and in
paraventricular zones of the diencephalon (Dien). In the case of the spinal
cord, BrdU-LCs were counted within the limits of circles (radii, 50 µm)
distributed to explore the main gray matter and white matter regions. In these
counts, marked endothelial or blood cells were discarded. A similar procedure,
but adapted to the geometry of the organ (using squares of the same area
instead of circles), was employed to study different regions of the brain
parenchyma. The circle or square, reproduced at the appropriate magnification
on a transparent sheet, was overlaid onto the screen of a high-resolution
monitor displaying images of the spinal cord or brain sections. Density
(D) of labeled cells was calculated in each spinal cord region from
counts made in samples of eight sections obtained at each segment from
cervical to lumbar levels. Density of labeled cells in the brain was
calculated from three samples (R1-R3) taken from the ependymal epithelium
(EpE) towards the nervous parenchyma in each of the eight explored sections;
homologous regions of WATs and CATs were sampled. For the spinal cord, the
algorithm was:
D=NL/(NSAx32),
in which NL is the number of marked nuclei in each sample,
NSA is the number of sampled areas, and 32 is the number
of sections (eight) multiplied by the four spinal cord segments explored (C1,
C2, T and L). An analogous algorithm was used when dealing with the brain but
the multiplier was 24 instead of 32, since eight sections from three zones
(DC, MC and Dien) were explored (Fig.
1).
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Multiple immunostaining
Using appropriated fluorophore/cromophore combinations, we obtained
differential staining between nuclei that incorporated BrdU and the nuclear or
cytoplasmic proteins characterizing neurons or glial cells. For these
purposes, we employed eight turtles. Two basic criteria were established for
proper identification of double-labeled (BrdU-glial/neuronal marker) and
single-labeled (BrdU) cells in WATs and CATs: (1) close focus coincidence of
BrdU-stained nuclei and the glial/neuronal-specific staining (cytoplasmic or
nuclear) and (2) visualization of unstained cell compartments, alternating
between epi-fluorescence and Nomarski illumination
(Horner et al., 2000). For
quantification studies, sections were processed for revealing BrdU-marked
nuclei [we have selected sections from spinal cords and brains of WATs
(N=4) and CATs (N=4)]. Sections processed for revealing BrdU
(spinal cords and brains) were incubated in the following primary antibodies:
rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Chemicon
International, Inc.), rabbit anti-S100 proteins (S100; 1:200; Sigma-Aldrich,
Inc., St Louis, MO, USA), mouse anti-oligodendrocyte (1:200; Chemicon
International, Inc.), mouse anti-neuronal nuclei proteins (NeuN; 1:500;
Chemicon International, Inc.). GFAP stains cytoskeleton proteins of supporting
cells in the brain and spinal cord; S100 reacts with the S100 family of
proteins present in glial and ependymal cells; and NeuN reacts with most
neuronal cell types in the CNS of vertebrates (staining is primarily localized
in the nucleus, extending in some cases into the cytoplasm). It should be
noted that reliable results were not obtained with the anti-oligodendrocyte
antibody. 50-100 nuclei were examined in the sections incubated in each
antibody; single- and double-labeled cells were counted separately. The
percentage of BrdU-LCs immunolabeled with a second antibody was defined as the
double-labeling index (DLI) for that cell marker. When dealing with the spinal
cord, we also made triple-labeling experiments in WATs (N=2)
involving BrdU, GFAP and S100. After processing for BrdU detection, the
sections were incubated in a solution containing GFAP and S100 primary
antibodies. The sections were sequentially processed with two
fluorophore-conjugated secondary antibodies emitting light at different
wavelengths. In these preparations, we looked for single BrdU-LCs,
alternating, as described, between epi-fluorescence and Nomarski illumination.
In this particular case, quantification was expressed as the single-labeling
index (SLI), representing the percentage of BrdU-LCs that do not express
either S100 or GFAP. Control experiments were performed by omitting or
replacing primary antibodies with normal serum. In these experiments, no
detectable staining of cell structures was observed. Bright-field images were
captured indistinctly with a photographic camera (using fine grain film) or
with a CCD camera. In the latter, the images were processed with commercially
available software.
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Results |
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Data resulting from paired experiments (Fig. 2F) were of particular interest since, in these cases, the influence of unexplored biological rhythms should be ruled out. As already stated, in these circumstances tissues from both turtles were processed together to neutralize inherent variability of the immunostaining procedure. The obtained results also revealed significant differences (Fisher exact test, P<0.01) between density values of BrdU-LCs in members of each pair (Fig. 2E).
Multiple-labeling experiments
To determine the identity of the cell populations affected by temperature,
we performed double-labeling experiments involving BrdU and specific cell
markers. BrdU/S100-colabeled cells were found in different spinal cord regions
including the EpE (Fig. 3A-D). For WATs, the DLI was 49% (N=100 nuclei), while for CATs the DLI was
11% (N=100). Close inspection revealed that colabeled cells found in
the EpE were radial glia (RG) lining the central canal. These experiments also
revealed BrdU-LCs that did not express S100
(Fig. 3E-H). Double-labeling
experiments involving BrdU and GFAP also revealed BrdU/GFAP-colabeled cells
coexisting with single BrdU-LCs (Fig.
3I-K). For WATs, the DLI was 22.5% (N=100 nuclei) and for
CATs the DLI was 52% (N=100 nuclei). Triple-labeling experiments
involving BrdU, S100 and GFAP also revealed cells showing single BrdU labeling
(not shown; SLI=29%, N=50 nuclei). As expected, for short surviving
time points after BrdU administration, BrdU/NeuN-colabeled cells were not
found (Fernández et al.,
2002; Cooper-Kuhn and Kuhn,
2002
).
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Forebrain
BrdU-labeled cells - density distribution
Similar to results obtained from studies in the spinal cord, the density of
BrdU-LCs was significantly greater in animals maintained in a warmer than in a
cooler environment. The difference was noticed in the histological
preparations (Fig. 4A,B) and
validated by the quantitative topological studies. For R1 (the sample area
closest to the EpE), the mean density value of BrdU-LCs in WATs was
significantly different (independent t-test, P<0.01) from
the corresponding mean value of CATs. Most BrdU-LCs occurred within the limits
of R1, but a few marked nuclei were found in the other sampled areas of the
nervous parenchyma (R2-R3). However, in the latter regions, the differences
between means were not statistically significant
(Fig. 4C). Differences between
WATs and CATs were also evident when comparing mean density values from
particular brain zones such as the DC (P<0.05), MC
(P<0.01) and Dien (P<0.01)
(Fig. 5A). As occurred when
studying the spinal cord, the paired experiments
(Fig. 5B)confirmed that a warm
environment increases cell proliferation in the brain of juvenile turtles
(Fisher exact test, P<0.01).
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Double-labeling experiments
In the EpE lining the brain cavities, immunostaining experiments involving
both BrdU and S100 antibodies revealed cells colabeling for BrdU and S100 and
also cells only stained for BrdU (Fig.
6A-C). For WATs, the DLI was 42% (N=100 nuclei) and for
CATs the DLI was 12% (N=100 nuclei). The morphological
characteristics of the double-labeled cells were coincident with those of
typical RG (nuclei close to ventricle lumen, apical surface of the cells
contacting the ventricle lumen and a fine radial process extending to the
brain parenchyma). Double-labeling experiments also showed the occurrence of
RG cells with BrdU-stained nuclei expressing GFAP
(Fig. 6D-F). As in the
double-labeling experiments involving S100, RG with BrdU-stained nuclei but
not expressing GFAP were also found in the EpE (not shown). For WATs, the DLI
was 74% (N=100 nuclei), while for CATs the DLI was 12% (N=50
nuclei).
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Discussion |
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Our studies on the CNS of turtles of the genus Chrysemys revealed
a significant increase in the density of BrdU-LCs in turtles acclimated to
27-30°C when compared with turtles exposed to temperature fluctuating
within the 5-14°C range. In the spinal cord, differences between WATs and
CATs were particularly evident when exploring the central gray matter. These
results suggest that the CR-IR contains the major cell population sensitive to
the direct or indirect temperature effects. It has to be emphasized that
within the limits of the CR-IR lies the central canal lined by the EpE. If the
data obtained from the spinal cord are compared with those of the forebrain,
we find that differences between WATs and CATs are significant at the level of
R1 (the zone containing the EpE) and not significant in other regions of the
nervous parenchyma. This also points to the cell-proliferative capacity of the
EpE but does not exclude the presence of cells retaining mitotic activity in
other regions of the CNS. These results were confirmed by paired experiments
in which more elusive factors that might be affecting cell proliferation
(Cermakian and Sassone Corsi,
2001) were equalized.
To identify the cell population affected by temperature, we combined
BrdU-labeling with the labeling of glial and neuronal markers. Our results
indicate that temperature mainly affected a population of GFAP-positive and
S100-positive cells with the characteristics of pleomorphic neuroglia and
typical RG [it should be noted that mammalian-like astrocytes are uncommon in
reptiles (De Castro, 1920)].
The RGs reside in the EpE lining the brain cavities, including the central
canal of the spinal cord. Recent studies indicate that GFAP-positive cells
with the characteristics of astrocytes behave as neuronal precursors in the
subventricular zone of rodents (Doetsch et
al., 1999
; Seri et al.,
2001
; Alvarez-Buylla and
García-Verdugo, 2002
). In addition, in the case of
embryonic development, 'distinction between radial glial cells and
neuronal progenitors has recently collapsed'
(Fishell and Kriegstein,
2003
). The same line of thought is maintained by Noctor et al.
(2001
), suggesting that
neurogenic potentialities of RG may be extended into post-natal periods. We
have also found BrdU-LCs that do not express either GFAP or S100. Since our
double-labeling experiments with NeuN have excluded the neuronal nature of
these cells, it seems reasonable to consider them to be either
oligodendrocytes or perhaps cells close to a more primitive undifferentiated
lineage. (To test this hypothesis we tried oligodendrocyte markers, but
available antibodies do not work properly when assayed in turtles.) Therefore,
differences in the DLIs observed between WATs and CATs have to be considered
as suggestive clues to be explored in detail in future work. Moreover, the
current absence of modern glia cell descriptions in turtles contributes to the
difficulties in assessing the identity of the BrdU-LCs not expressing glial
markers. As mentioned above, we have not found BrdU-LCs expressing NeuN. This
is consistent with our previous results in turtles
(Fernández et al.,
2002
) and with the systematic studies performed by Cooper-Kuhn and
Kuhn (2002
) in rats. In both
species, BrdU/NeuN-colabeled cells appear several days after BrdU
administration.
Our results are in agreement with current views that emphasize the role
played by glial cells in the process of cell proliferation and neural
differentiation after birth. It can be concluded that temperature mainly
affects cells that have to be considered as `contained within the
neuroepithelial-radialglia-astrocyte lineage'
(Alvarez-Buylla et al., 2001).
This is particularly evident when dealing with the RG lining the brain
cavities and the central canal of the spinal cord.
There is little doubt that, within physiological limits, temperature could
increase per se the cell metabolism and the mitotic rate ['...the
duration of the metaphase pause becomes shorter as the temperature is
increased.' (DuPraw,
1970)]. There are, however, indirect factors that may be affecting
cell division. For example, WATs displayed an increased motor activity and
were more voracious than CATs. Consequently, both behavioral (van Praag et
al., 1999
,
2000
) and nutritional factors
should be taken into account when dealing with possible mechanisms that could
be operating in the temperature-induced increase of cell proliferation. With
respect to the biological significance of this phenomenon, it is reasonable to
relate it to the changing activity of turtles throughout the year. During
winter, turtles of the genus Chrysemys have a reduced motor activity
and a reduced food intake. These behavioral patterns are dramatically
increased in summertime. If, as suggested, cell proliferation may be reflected
in an increased number of nerve cells
(Fernández et al.,
2002
), the new neurons may facilitate the operation of circuits
vinculated with more-demanding behavioral tasks such as prey capture and
reproductive maneuvers. The differentiation fate of the increased number of
BrdU-LCs remains a subject for further studies.
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Acknowledgments |
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