Endogenous amyloidogenesis in long-term rat hippocampal cell cultures.

Abstract

Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal development and senescence.
In the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the cellular conditions in the process of hippocampal culture development and aging. It was found that there was an increase in endogenous production of Aβ(1-42) and an increase in the accumulation of Congo Red-binding amyloidal aggregates associated with the aging of neurons in primary culture. In vitro changes in the morphology of co-existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of in vivo brain aging at the cellular level.
In conclusion, this study suggests that long-term primary CNS culture is a viable model for the study of basic mechanisms and effective methods to decelerate the process of neuronal senescence.

Full text

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RESEARCH ARTICLEOpen Access
Endogenous amyloidogenesis in long-term rat
hippocampal cell cultures
Sarah J Bertrand*, Marina V Aksenova, Micheal Y Aksenov, Charles F Mactutus and Rosemarie M Booze
Abstract
Background: Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes
associated with neuronal senescence. Improvements in available technology make it possible to observe
maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro.
Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell
cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of
long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal
development and senescence.
Results: In the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation
and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal
and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term
cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from
cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace
nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the
cellular conditions in the process of hippocampal culture development and aging. It was found that there was an
increase in endogenous production of Ab1-42and an increase in the accumulation of Congo Red-binding amyloidal
aggregates associated with the aging of neurons in primary culture. In vitro changes in the morphology of co-
existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of in
vivo brain aging at the cellular level.
Conclusion: In conclusion, this study suggests that long-term primary CNS culture is a viable model for the study
of basic mechanisms and effective methods to decelerate the process of neuronal senescence.
Keywords: Cell Culture Amyloid Peptide, Neurodegeneration/Aging
Background
As a result of the advances in isolation and culturing of
fetal and adult central nervous system (CNS) cells
achieved during the last two decades [1,2], primary neu-
ronal cell culture has become a powerful tool for isolat-
ing cellular and molecular mechanisms of neuronal
development and death [1,3,4]. Despite the routine suc-
cess at long-term culturing of primary rodent neurons
[5-9], the application of this potentially very useful
approach for modeling of neuronal cell aging remains
limited. The standard protocols that allow prolonged
CNS cell culturing (including culturing of hippocampal
neurons) are now widely available [10,2], however, sys-
tematic studies of the differentiation state, cytochemical,
and morphological characteristics of brain cells remain-
ing viable over long term in vitro are needed.
Although in vivo animal models can be used to reveal
many important aspects of neuronal development, dys-
function, and degeneration, the inherent complexity of
nervous tissue often obscures the overall understanding
of molecular, biochemical, and structural observations.
The in vitro system lacks an intact tissue environment;
nevertheless, the presence of a homogenous cell popula-
tion allows us to identify specific mechanisms underly-
ing the chemical and morphological changes seen in
vivo [6]. Using long term primary cell culture to
* Correspondence: bertrans@email.sc.edu
University of South Carolina, Program in Behavioral Neuroscience,
Department of Psychology, Columbia, SC 29208, USA
Bertrand et al. BMC Neuroscience 2011, 12:38
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© 2011 Bertrand et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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decipher the underlying development of the cells of the
hippocampus allows examination of where the initial
dysfunction takes place that leads to some of the symp-
toms of neurodegeneration. Understanding what a
mature neuron looks like, and when it matures in cul-
ture, will help facilitate the development of more effi-
cient in vitro testing.
In the current study we used the lack of nestin immu-
noreactivity, as well as various morphological categoriza-
tions, to classify our cultures as mature. Nestin is a type
VI neurofilament expressed in the developing CNS, spe-
cifically in progenitor cells undergoing differentiation
[11-13]. Specific mature neuronal and astrocytic mar-
kers, MAP-2 and GFAP, have been found to be co-
expressed with nestin in early, primary, neuronal cell
cultures [14,11]. The co-localization of these markers
and nestin indicate the event of differentiation. Conver-
sely, there should be little to no expression of the inter-
mediate fiber marker nestin in mature primary neuronal
cultures [14].
Increased amyloid-beta peptide (Ab) formation is a
common accompaniment of brain aging [15-17]. Accu-
mulation of abnormal protein aggregates, including the
misfolded Ab protein, is the manifestation of progres-
sively deteriorating capacity of aging biological systems
to withstand extrinsic and intrinsic hazards [18-20]. Our
current study was designed to investigate the possibility
of endogenous amyloid beta peptide formation and
accumulation of misfolded protein aggregates in hippo-
campal neurons during their development and matura-
tion in vitro.
Ab peptides arise from the cleavage of amyloid pre-
cursor protein (APP) [21]. b-and g-secretases cleave the
unprocessed APP in succession, resulting in the forma-
tion of Ab peptide [15,22,16,23]. Numerous studies have
used rodent primary hippocampal cell cultures as an
experimental model of normal biogenesis of Ab [24,13].
Rat hippocampal cells in primary cultures express
APP770, APP751, and APP695[24,25] and the amyloido-
genic route of APP processing resulting in the endogen-
ous Ab1-40or Ab1-42generation may naturally occur in
primary neuronal cultures [26].
The Ab monomers released from cells transform into
b-pleated sheet aggregates to initiate the process of neu-
ronal degeneration associated with the decline in meta-
bolic activity and oxidative damage of cultured
hippocampal neurons [27,28]. It was reported that the
rodent Ab is less likely to form large fibrillary structures
due to three amino acid substitutions compared to the
human sequence. Nevertheless, neurotoxic and pro-oxi-
dant properties of the rat variety of Ab 1-42 have pro-
ven to be the same as human [29]. Most amyloid
peptides interact strongly with cell membranes and this
interaction is enhanced by conditions which favor b-
sheet formation. The excessive release of Ab from cul-
tured rat hippocampal neurons can result in the increase
of amyloid-binding dye fluorescence in the culture med-
ium and Ab-mediated toxicity [30].
Although increased Ab formation and the production
of aggregates have been observed accompanying the
process of neuronal aging [17,18], low levels of Ab
derived from the natural processing of APP isoforms
have been found both in vivo and in vitro [17,22]. Nor-
mal physiologic levels of Ab are thought to be important
in regulating neurotransmission [15,16,23,24], conse-
quently many researchers suggest that Ab should only
be regarded as toxic when the production and degrada-
tion are imbalanced [15,16].
Overall, the purpose of this study is to investigate the
usefulness of long term primary hippocampal cell culture
for understanding the link between endogenous Ab pro-
duction, differentiation and maturation in vitro. In order
to discern this link we aim to identify criteria by which a
culture can be classified as mature. These classifications
can help us properly assess the endogenous levels of Ab
and the accumulation of misfolded Ab proteins into
aggregates as physiological, and not pathological, pro-
cesses of aging. The ability to classify the maturity of
neurons will also strengthen the cell culture model as a
whole, giving researchers the ability to more definitively
speak about influences on the aging nervous system.
Results
The long-term hippocampal cell culture maturation and
aging
Primary embryonic hippocampal culture preparations
used in this study are classified as moderate-high density
(160-180 cells/mm2). Soon after the adherence to the
surface of the cell culture plate, viable hippocampal cells
began expressing marker proteins that indicate their
commitment to either the neuronal or astrocytic differ-
entiation. The representative DIC microscopy images in
figure 1A illustrate that the cells were initially small
with no, or a very small number of, neuritic outgrowths
at 4 DIV, and later form a visible network by 14 DIV.
Extensive networking of neurites and the defined
appearance of pyramidal cells at DIV 21-35, indicated
this was a stage of maturity for the cell culture. Aging
hippocampal cells (DIV 40-65) appeared to have pro-
gressively less defined connections with excessive bund-
ling of neurites and larger, more defined cell bodies.
Consistent with the DIC imaging of developing neural
networks were images of Nestin/MAP-2 immunofluores-
cence from the long-term hippocampal cultures of dif-
ferent ages (Figure 1B). In 7-14 DIV hippocampal cells
nestin immunoreactivity was generally localized within
the axonal growth cones. Neuronal maturation asso-
ciated with extensive dendritic arborization was
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characterized by a gradual decline of the expression of
this immature neurofilament protein and the increase of
MAP-2 expression. As shown in figure 1B, after 14 DIV
the majority of the neuronal population in the long-
term hippocampal cell culture became predominantly
MAP-2 expressing principle pyramidal neurons.
Nevertheless, nestin immunoreactivity could be detected
in some cells until DIV 21. Hippocampal neuronal cells
expressing nestin along with MAP-2 were typically
found in cell clusters at this age (21 DIV).
Observations of the mature hippocampal primary cul-
tures during their long-term maintenance clearly

B
A
Figure 1 Rat hippocampal neurons show distinct morphology (MAP-2; green) at different time points (A) and stop expressing the
intermediate filament VI, nestin (red), over time (B). Cell bodies are stained with Hoerscht. A. Young (4 DIV) cells display small soma and
short, thin neurites. As time passes the somas become larger and more defined, while neurites elongate (7 - 40 DIV). Once mature (21-40 DIV),
the network takes on a progressively more intricate and bundled appearance. At 65 DIV, excessive bundling is present. B. The expression of
nestin degrades over time (4 DIV- 21 DIV). MAP-2 expression appears to gain intensity up to 40 DIV. At 60 DIV, the MAP-2 expression becomes
faded, and takes on a “beaded” appearance on some neurites.
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showed that the stationary phase, during which cultured
cells showed no noticeable changes in morphology, may
continue until DIV 40. Signs of neuritic network dete-
rioration began to occur after 40 DIV. Immunoreactivity
of MAP-2 in these aged hippocampal cell cultures also
revealed varicosities along the processes, giving them a
‘beaded’ appearance, as shown in figure 1B at 60 DIV.
Primary rat fetal hippocampal cultures were neuron-
enriched throughout long-term maintenance. The fully
mature hippocampal cell culture (21-35DIV) contained
15 ± 5% GFAP positive cells. The number of astrocytes
remained stable throughout the cell culture aging period
and did not exceed 20% even in very old (65 DIV) cul-
tures (data not shown). However, the astrocytic cell
morphology and GFAP expression changed with the
aging of the cell culture age (Figure 2). In long-term
hippocampal cultures of 45-60 DIV, astrocytes exhibited
very intensive GFAP immunostaining and displayed an
extensively branched network of processes (Figure 2B).
GFAP expressing cells had thicker processes relative to
MAP-2 expressing cells, and their nuclei appeared to be
flat and more oblong than MAP-2 expressing cells.
Results of immunoblotting analyses of neuronal
(MAP-2, NSE) and astrocytic (GFAP, GS) marker pro-
teins in hippocampal cell lysates (Figure 3A, B) were
consistent with the results of the immunofluorescent
microscopy and neuronal/astrocytic cell counting. The
MAP-2/GFAP immunoreactivity ratio significantly (P =
0.002 < 0.05) decreased from 2.70 ± 0.22 to 1.50 ± 0.08
between 14 and 25 DIV when hippocampal cell cultures
reached the peak of their development. No significant
changes in the MAP-2/GFAP ratio have been found in
fully mature hippocampal cultures between 25 DIV and
35 DIV (1.50 ± 0.08 and 1.34 ± 0.07; P = 0.191 > 0.05).
The end of the stationary phase (35 DIV) was marked
by the significant decrease of the MAP-2/GFAP ratio at
45 DIV (0.64 ± 0.03; P = 0.0001 < 0.05), which was
attributed to the sharp drop in MAP-2 immunoreactiv-
ity but not to the increase in GFAP immunoreactivity.
The observed changes in levels of immunoreactive
MAP-2 corresponded well with the results of anti-MAP-
2 immunofluorescent microscopy, revealing signs of
neuronal dendritic network degeneration at this time
point (40-50 DIV). Consistently, as shown by the images
of the Western blots in Figure 3, the intensity of protein
bands corresponding to synaptodendritic markers MAP-
2 and PSD 95 exhibited pronounced decreases between
35 DIV and 45 DIV compared to NSE, GS and b-actin.
Amyloid beta -peptide release and deposition of b-
pleated sheet protein aggregates
The presence of specific Ab 1-42 immunofluorescence
in cultured hippocampal neurons indicates the possibi-
lity of endogenous Ab production. Only negligible levels
of extracellular Ab 1-42 immunoreactivity were detected
by direct ELISA in the cell culture medium at DIV 7
and DIV 14 (Figure 4A). The progressive accumulation
of Ab 1-42 in the growth medium was starting in the
stationary phase of cell culture development (21-35
DIV) and peaked at 120-150 nM during the period of in
vitro aging between 40 DIV and 60 DIV (Figure 4A).
In this study, the long-term maintenance of hippo-
campal cell cultures continued until 65 DIV. Live-dead
staining was routinely performed to determine cell viabi-
lity. Double labeling with calcein AM and Congo Red
revealed the presence of cells with membrane integrity
and the capability to retain the fluorescent product of
the calcein AM hydrolysis fairly long into culturing (65
DIV; Figure 4D).
Results of the Congo Red staining of long-term hippo-
campal cultures at different stages of their development
are shown in Figure 4B. Congo Red staining of cell
bodies or processes was rarely observed in hippocampal
cell cultures until 21 DIV. However, after 35 DIV the
Congo Red stained amyloid-like depositions were fre-
quently found in association with the dendritic network
and in the cell body (Figure 4C). In cultures older than
40 days, increasing numbers of cells with Congo Red-
positive somata were observed (Figure 4E). Total num-
ber of cells containing Congo-red positive amyloidal
aggregates was much higher in older cells (60 DIV; Fig-
ure 4E). Co-labeling with calcein AM and Congo red
detected many calcein AM-positive cells some with the
presence of Congo Red, as shown by the yellow arrows
in Figure 4D.
Discussion
The technology of CNS cell culturing continues to
evolve, bringing forward novel perspectives to advance
the understanding of basic principles of neural function-
ing by using isolated neural cells in a controlled envir-
onment. In this study we used the techniques for
culturing rat fetal hippocampal neurons developed by
Brewer and co-authors [1-3,7,31]. When this method is
used, isolated neurons remain viable for long periods of
time without the need for astroglial feeder co-cultures
[10]. Our own experience [5] and reports from other
investigators [7,8,6,32], have documented the successful
maintenance of viable cultures containing primary
rodent fetal neurons for 60-70 days or longer. The abil-
ity to maintain cell cultures for extended periods of
time allows further understanding of the fundamental
processes of development and aging within the neuronal
populations.
Despite the abundance of published studies carried
out in primary CNS cultures, important aspects of neu-
ronal differentiation in vitro remain ambiguous. The
data reported here describe the long-term time course
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of maturation of isolated primary rat hippocampal cells
in culture. We report that at 21 DIV, isolated hippocam-
pal neurons completely switch from the expression of
the intermediate type VI neurofilaments (nestin) to the
synthesis of more advanced neurofilaments containing
MAP-2. These data suggest that hippocampal cultures
younger than 21 DIV do not resemble fully mature neu-
rons as seen in vivo [11,6].
This culturing protocol uses a medium specifically for-
mulated to promote neuronal survival and does not sup-
port glial proliferation [1]. However, it does not prevent
the development of viable progenitor cells from
differentiating into astrocytes during the development of
the cell culture. Astrocytic development is sometimes
viewed as a necessary event for the long-term survival of
neuronal cells in culture [6,14]. We found that the pre-
valence of the neuronal phenotype (MAP-2 expressing
cells) is maintained through all stages of the cell culture
development. Cell populations in fully mature, long-
term hippocampal cell cultures could include up to 20%
GFAP-positive cells and relative numbers of astrocytes
did not show further increase during the cell culture
aging period. The gradual increase in GFAP expression
and enrichment of the network of GFAP-positive cell











A
B
Figure 2 Astrocytic growth consists of arborization rather than division. A. A representative image of MAP (red) and GFAP (green)
expression. Cell nuclei are stained with Hoerscht (blue). Although there are multiple cell bodies present, only one is shown to be expressing
GFAP. B. At earlier time points, GFAP expression is limited due to the culture medium which does not support astrocytic growth. Although there
appears to be an increase in GFAP expression as cultures age, cell bodies associated with GFAP expression remain at similar levels, but there is
an increase in arborization.
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processes, may be explained by slow (in comparison to
neurons) maturation of existing astrocytes in B27-sup-
plemented Neurobasal medium, which is not optimized
to support astrocytic growth [6,1]. Alternatively, the
enhanced arborization and GFAP expression may be
attributable to astrocytic activation adjunct to the cell
culture aging [33,5,13]. As in vivo, primary long-term
cell culture aging is associated with the progressive oxi-
dative damage of hippocampal neurons [5] and astro-
cytes are known to respond to various physiological or
noxious stimuli including oxidative stress with increased
expression of GFAP [14]. However, the results of our
current study show signs of progressive degeneration of
both MAP-2 and GFAP-containing networks during the
later stages in the long-term hippocampal cell cultures.
Our study adds to the literature that investigates the
endogenous release of Ab [34,17,16,15,22,26] and poten-
tial deposition of amyloid-like aggregates in the process
of development and aging of isolated hippocampal cells
[17]. Consistently, we have detected the expression of
APP and the presence of intracellular Ab 1-42 immu-
noreactivity in long-term hippocampal cultures. The
longitudinal monitoring of extracellular Ab 1-42 immu-
noreactivity in primary hippocampal cell culture from
DIV 4 until DIV 60 has shown that young, actively dif-
ferentiating hippocampal cells have very low levels of
physiological secretion of amyloidogenic Ab. Direct Ab
ELISA measurements for 7-15DIV cell cultures that we
report are consistent with similar data, which can be
found in recent publications [26]. Notably, our experi-
ments show that the increase of extracellular Ab coin-
cided with the complete maturation of synaptodendritic
networks in hippocampal cultures, which indicates a
potential relationship between increasing neuronal activ-
ity and the secretion of Ab. Indeed, several recent stu-
dies [15,22,23,34] suggested that the formation of Ab
monomers play a role in the regulation of synaptic
transmission.
Amyloid-like protein aggregates are thought to disrupt
normal neuronal functioning by irreversible spontaneous
insertion into cell membranes. The existing evidence
suggests that the misfolded and aggregated proteins are
essential elements in the vast majority of age-related
neurodegenerative diseases [19]. Therefore, we used the
Cell culture age, DIV
0 10 20 3040 5060
MAP-2/GFAP ratio, arbitrary units
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*
**
A B
Figure 3 MAP-2 and GFAP expression changes over time. A. A comparison of Western blots of specific cell proteins and ubiquitous proteins
in both astrocytes and neurons. There is a steady expression of both GS and GFAP throughout the life of the culture, indicating that astrocytes
do not over take the dish. MAP-2 and PSD 95 expression increases until DIV 35, at DIV 45 there is a marked drop in the expression of these
proteins. This may be due to the fragmentation of neurites. The steady expression of NSE indicates a steady cell population. The steady
expression of b-actin indicates that the cell population remains unchanged throughout the life cycle of the cell. B. The ratio between MAP-2 and
GFAP decreases over time, supporting the western blots for MAP-2 and GFAP. At later time points, although there is an unchanged presence of
GFAP, there is a decreased expression of MAP-2.
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Cell culture age, DIV
0 1020 3040 5060 70
A? ? 1-42, nM
0
20
40
60
80
100
120
140
160
180
A
B
D
14 DIV 60 DIV
Congo red + cells, N per 100
0
5
10
15
20
25
*
E
C
Figure 4 Long term primary hippocampal cell cultures naturally release Ab1-42 in increasing amounts over time. Ab1-42 is also seen to
form an increasing amount of aggregates over time, and is present in live cells. A. Ab1-42 immunoreactivity in the CM samples collected from
individual cultures (n = 4 per time point) was measured by a direct ELISA. There was a marked increase in Ab release beginning at DIV 21. Data
are presented as mean values ± SEM. Cell viability in hippocampal cell cultures was determined using Live/Dead assay. B. Live cells stained with
Congo red dye. Staining with Congo Red is a classical method of the detection of amyloids. Congo red exhibits very low non-specific binding.
Congo Red positive staining increases as cells age, beginning at DIV 21 and increasing until DIV 40. C. Congo red staining can also be observed
in what appears to be the inside of cells, as well as the network. D. Congo red (red) can also be observed in live cells at DIV 60. Live cells are
marked with calcein (green), and an overlap of orange indicates an overlap of markers. Other Congo red marked cells may be dead at this time
point. E. Counts of Congo red positive cells significantly (P < 0.05) increase over time.
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Congo Red staining to determine whether the changes
in extracellular levels of Ab 1-42 were associated with
the deposition of b-amyloids in long-term hippocampal
cell cultures. The presence of Congo Red-positive aggre-
gates associated with the network of processes and cell
bodies of hippocampal cells appeared to mark the begin-
ning of the period of decay in the long-term cell culture.
Congo Red stained aggregates were consistently
observed in long-term cultures after 30 DIV when the
levels of extracellular Ab 1-42 grew higher than 100
nM. The appearance of cell-associated amyloid-like
aggregates became more frequent as the cell cultures
continued to age and correlated well with morphological
signs of deterioration of dendritic networks. Intriguingly,
the cell culture ages when the presence of b-amyloidal
aggregates were observed in the current study corre-
sponded well with previously reported time course of
protein oxidative damage in aging long-term hippocam-
pal cell cultures [5,35,36].
The Ab release and the following changes of the spe-
cific b-amyloid labeling preceded the decline of cell den-
sities in aging hippocampal cultures. The pronounced
increase in the number of cells exhibiting positive
Congo Red staining of the somata in 60 DIV long-term
cultures could be explained by the increased membrane
permeability of decaying hippocampal cells which
allowed Congo Red access to the intracellular compart-
ment. Nevertheless, the overlap between the calcein AM
and the Congo Red positive labeling indicated that, even
in very old cultures, amyloid like aggregates could be
found in the remaining alive hippocampal cells, which
continue to maintain intact membrane integrity. On the
other hand, the appearance of Congo Red fluorescence
in dead (calcein AM-negative) cells is consistent with
the suggestion that the presence of b-sheet structured
protein aggregates may be a marker of neural cell injury
and decay [37,38].
Studies of primary neuronal cultures beyond DIV 30
are infrequent [6,5,1]. Moreover, studies characterizing
the development and senescence of neuronal cells in
vitro without any treatment are difficult to identify in
the literature. The current study not only joins the lim-
ited literature of long term cultures past DIV 30, we
also characterized some important developmental mile-
stones of note when using primary cell cultures. We
found that neurons in primary culture undergo distinct
developmental phases: differentiation, maturation, steady
state, and senescence. Viability of these cell cultures was
verified using immunoblotting of important structural
proteins, immunoreactivity of important neuronal and
glial markers, and differential interference contrast
images to track visible damage on the cells.
Differentiation (DIV4-14) was characterized by the
presence of nestin immunoreactivty and the prevalence
of small cells with short processes and little to no
branching in DIC images. Maturation (DIV 14-21) was
classified as a time when the majority of nestin expres-
sing cells began expressing MAP-2 (or GFAP), and a
more defined network of neurites and larger, more
defined cell bodies were present in DIC images. The
steady state (DIV 21-35) was defined as the point in
time when the neuronal cells in culture no longer dis-
played any noticeable changes. Senescence (DIV40-60)
was defined as a decrease in total cell number, the pre-
sence of “beading” of the processes, a steady rise in Ab
production and an increase in aggregate formation.
Conclusions
The phenomenon of cellular aging takes place in long-
term primary neuronal cell cultures [13,4,5]. The pre-
sent study found that endogenous cell senescence-asso-
ciated amyloidogenesis in vitro occur in long-term
hippocampal cell cultures. This outcome presents simi-
lar observations seen in the patterns of neuronal aging
seen in animals, therefore, suggesting the possibility that
long-term primary cell culture could serve as a model
for particular aspects of aging, such as the study of neu-
rodegenerative diseases. In sum, our results indicate that
long-term primary cell cultures may be a useful instru-
ment for designing and testing effective ways to delay
neuronal senescence and other age related phenomenon.
Methods
Primary hippocampal cell cultures were prepared from
18-day-old Sprague-Dawley rat fetuses. Procedures were
carried out in accordance with the University of South
Carolinas Institutional Animal Care and Use Committee.
Rat hippocampi were dissected and incubated for 10
min in a solution of 2 mg/ml trypsin in Hank’s balanced
salt solution (HBSS) buffered with 10 mM HEPES
(GIBCO Life Technologies, Paisley, Scotland). The tissue
was then exposed for 2 min to soybean trypsin inhibitor
(1 mg/ml in HBSS) and rinsed 3 times in HBSS. Cells
were dissociated by trituration and distributed to glass
bottom 35 mm and 96-well poly-L-lysine-coated plastic
culture dishes (Costar, Cambridge, MA). Dishes were
coated with a poly-L-lysine 48 hours prior to culturing.
Initial plating densities were about 160-180 cells/mm2.
At the time of plating, plates contained DMEM/F12
(GIBCO) supplemented with 100 mL/L fetal bovine
serum (Sigma Chemicals, St. Louis, MO). After a 24-hr
period, DMEM/F12 was replaced with an equal amount
of Neurobasal medium supplemented with 2% v/v B-27,
2 mM GlutaMAX supplement and 0.5% w/v D-(1) glu-
cose (all ingredients are from GIBCO). Neurobasal, B27-
supplemented medium was used because it favors neu-
ronal cell development and is not supportive for astro-
cytic proliferation. Cultures were maintained at 37°C in
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a 5% CO2/95% room air-humidified incubator at all
times [5]. The long term maintenance of hippocampal
cell cultures was carried out by replacing two thirds of
the neurobasal medium with fresh medium after two
weeks.
The morphology of hippocampal cells was examined
at different time points (4, 7, 14, 21, 40, and 60 days in
vitro (DIV) using differential interference contrast
(DIC) microscopy as shown in Figure 1A. The fluores-
cent Hoescht staining of cell nuclei was used to moni-
tor age-related changes in the cell density. The
viability of hippocampal cells in long-term cultures
was monitored by the fluorescent calcein AM/ethidium
bromide cell labeling (Live/Dead cell viability kit, Invi-
trogen, Carlsbad, CA).
Immunofluorescent labeling with primary antibodies
against nestin, MAP-2, and GFAP were used to assess
the maturity of hippocampal neurons and detect cells
with the neuronal astrocytic phenotype in long-term
cultures of different ages (See Table 1 for antibodies
used in this study). Nestin is a type VI intermediate fila-
ment protein transiently expressed by progenitor cells at
the early stages of neurogenesis. Upon differentiation,
nestin becomes down-regulated and replaced by neuron
or astrocytic specific filaments, in this case MAP-2 and
GFAP were used, respectively. Cells were fixed with an
acetic acid/methanol solution for 5 minutes prior to
blocking with a 10% horse serum solution. Secondary
antibodies were Alexa Dye-conjugated host animal-spe-
cific IgG (Invitrogen). In all immunolabeling analyses
the specificity of staining was confirmed by the exclu-
sion of the subsequent primary antibody. The deposition
of amyloid-like aggregates in the long-term hippocampal
cell cultures was studied using the b-amyloid-binding
Congo Red (Sigma Aldrich). Compared to other amy-
loid-binding dyes, such as thioflavin H or S, Congo Red
exhibits very low non-specific binding and cannot be
internalized by the cells, due to the presence of two
hydrophilic sulfonic groups. Staining of non-fixed hippo-
campal cells with Congo Red and microscopic imaging
of the results was carried out as previously described
[39].
Total cell lysates prepared from hippocampal cells that
had reached the ages of 14, 25, 35, and 45 DIV as pre-
viously described [40] were analyzed by Western blot-
ting and using primary antibodies against neuron- or
astrocyte-specific cytoskeletal (MAP-2 and GFAP) and
cytosolic (neuron- specific enolase, NSE and glutamine
synthetase, GS) marker proteins. In parallel Western
blots of the hippocampal cell lysates were immunos-
tained with antibodies against synaptic (PSD 95) and
ubiquitous cytoskeletal proteins (b actin).
Direct ELISA measurements of the Ab 1-42 immunor-
eactivity in cell-conditioned medium (CM) samples were
performed using the rabbit polyclonal anti-Ab 1-42 anti-
body. Serial dilutions of freshly prepared stocks of the
synthetic rat Ab 1-42 (0-2500 nM) (Anaspec, CA) with
cell culture medium were used for calibration.
Immunofluorescent imaging was carried out under
20× magnification using the inverted fluorescent micro-
scope (Nikon Eclipse TE2000-E) as previously described
[4,40]. Immunofluorescent images, images of specific
Congo Red binding, and DIC images were captured
using a CCD camera. Merged images were produced
and analyzed by the NIS Elements imaging software
(Nikon). Hippocampal neurons (MAP-2 labeled cells)
and astrocytes (GFAP labeled cells) were counted using
the Object counting option of the NIS Elements imaging
software package in 4 random fields of vision in cell cul-
tures of different ages. For each field of vision total
numbers of hippocampal cells were determined using
the counting of Hoescht fluorescent stain of cell nuclei.
Statistical comparisons were made using ANOVA
and planned comparisons were used to determine
Table 1 Antibodies Used
Name Method Dilution Animal ConjugateManufacturer
MAP-2 ICC/IF; WB 1:500; 1:1000 Rabbit MonoclonalSanta Cruz
GFAP ICC/IF;WB1:5000 Chicken MonoclonalAbcam
Glutamine SynthaseWB 1:10000 Mouse Monoclonal Chemicon
Neuron Specific EnolaseWB 1:5000 Chicken PolyclonalAves
PSD-95
b Actin
Ab 1-42
WB 1:1000 Goat PolyclonalAbcam
WB
WB; ICC/IF1:500 Abcam
NestinICC/IF1:1000Rabbit Polyclonal Abcam
Anti-Rabbit IgGWB 1:1500Sigma
Anti-Mouse IgGWB 1:1500Sigma
Anti-Goat IgG WB 1:1500Abcam
Anti-Chicken IgG WB1:1500Abcam
Bertrand et al. BMC Neuroscience 2011, 12:38
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specific treatment effects. Significant differences were
set at P < 0.05.
Abbreviations
Aβ: Amyloid Beta; GFAP: Glial fibrillary acidic protein; APP: Amyloid precursor
protein; GS: Glutamine synthase; CNS: Central Nervous System; MAP-2:
Microtubule associated protein 2; DIC: Days in vitro; NSE: Neuron specific
enolase; DIV: Differntial interference contrast; PSD 95: Post-synaptic density
95
Acknowledgements
This work is supported by University of South Carolina Research Foundation
grant, NIH Grant # DA013137, and DA031604.
Authors’ contributions
SJB assisted in carrying out immunohistochemistry, morphologically
characterized cells, and drafted the manuscript. MVA carried out ELISA, and
assisted in immunohistochemistry. MYA carried out Western Blot analysis,
aided in manuscript preparation, and performed the statistical analysis. MVA
and MYA conceived of the study, and participated in its design. CFM and
RMB participated in the design and coordination of the experiments, as well
as helping to draft the manuscript. All authors read and approved the final
manuscript.
Received: 20 December 2010 Accepted: 10 May 2011
Published: 10 May 2011
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doi:10.1186/1471-2202-12-38
Cite this article as: Bertrand et al.: Endogenous amyloidogenesis in
long-term rat hippocampal cell cultures. BMC Neuroscience 2011 12:38.
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