text:
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NMDA receptor-mediated excitotoxic neuronal apoptosis in vitro
and in vivo occurs in an ER stress and PUMA independent manner
Caoimhı ´n G. Concannon,1Manus W. Ward,1Helena P. Bonner, Katsura Kuroki, Liam P. Tuffy,
Caroline T. Bonner, Ina Woods, Tobias Engel, David C. Henshall and Jochen H. M. Prehn
Department of Physiology and Medical Physics and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland,
Dublin, Ireland
Glutamate receptor excitation is known to play an important
role during neuronal injury associated with ischaemic stroke,
head trauma and prolonged seizures (Schanne et al. 1979;
Choi 1994). The activation of NMDA receptors, and the
subsequent Ca2+influx, have been identified as critical in
mediating this form of injury (Choi et al. 1987; Tymianski
et al. 1993a) with much research focused on the sequestra-
tion of Ca2+within the mitochondrial matrix following
excitation. The endoplasmic reticulum (ER) is a subcellular
compartment which also exhibits a high Ca2+sequestering
activity and plays a functional role in cellular Ca2+regula-
tion, however, the role of the ER during excitotoxic neuronal
injury is currently poorly understood.
Within cells the ER is the major site for protein folding and
maturation. Disruption of homeostasis within the ER such as
depletion of ER Ca2+can interfere with protein folding and
transport, and can lead to the activation of a highly conserved
response termed ER stress. This disturbance in ER homeo-
stasis is sensed by three ER-localized proteins: activating
transcription factor 6 (ATF6), RNA-dependent protein kinase-
like ER kinase (PERK) and inositol-requiring ER-to-nucleus
signal kinase 1 (IRE1) which under normal physiological
condition are kept in check bound to the molecular chaperone,
glucose regulated protein 78 (Grp78) (Zhang and Kaufman
2004). Upon accumulation of misfolded proteins within the
Received October 4, 2007; revised manuscript received December 5,
2007; accepted December 7, 2007.
Address correspondence and reprint requests to Jochen H. M. Prehn,
PhD, Department of Physiology and Medical Physics, Royal College of
Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland.
E-mail: firstname.lastname@example.com
1Both these authors contributed equally to this work.
Abbreviations used: ATF, activating transcription factor; BH, Bcl-2
homology; CHOP, CIEBP homology protein; EBP, enhancerbinding
protein; DG, dentate gyrus; ER, endoplasmic reticulum; Grp, glucose
regulated protein; IRE1, inositol-requiring ER-to-nucleus signal kinase
1; LDH, lactate dehydrogenase; MEM, minimal essential medium;
OGD, oxygen and glucose deprivation; PBS, phosphate-buffered saline;
PERK, protein kinase-like ER kinase; PI, propidium iodide; PUMA, p53
up-regulatedmediator ofapoptosis;
reticulum Ca2+-ATPase; TUNEL, terminal deoxynucleotidyl transferase-
mediated dUTP nick end labelling; UPR, unfolded protein response;
XBP1, X-box binding protein 1.
Disruption of endoplasmic reticulum (ER) Ca2+homeostasis
and ER dysfunction have been suggested to contribute to
excitotoxic and ischaemic neuronal injury. Previously, we
have characterized the neural transcriptome following ER
stress and identified the BH3-only protein, p53 up-regulated
mediator of apoptosis (PUMA), as a central mediator of ER
stress toxicity. In this study, we investigated the effects of
excitotoxic injury on ER Ca2+levels and induction of ER stress
responses in models of glutamate- and NMDA-induced
excitotoxic apoptosis. While exposure to the ER stressor
tunicamycin induced an ER stress response in cerebellar
granule neurons, transcriptional activation of targets of the ER
stress response, including PUMA, were absent following glu-
tamate-induced apoptosis. Confocal imaging revealed no
long-term changes in the ER Ca2+level in response to glu-
tamate. Murine cortical neurons and organotypic hippocampal
slice cultures from PUMA+/+ and PUMA)/) animals provided
no evidence of ER stress and did not differ in their sensitivity
to NMDA. Finally, NMDA-induced excitotoxic apoptosis in vivo
was not associated with ER stress, nor did deficiency in
PUMA alleviate the injury induced. Our data suggest that
NMDA receptor-mediated excitotoxic apoptosis occurs in vitro
and in vivo in an ER stress and PUMA independent manner.
Keywords: BH3-only protein, endoplasmic reticulum stress,
excitotoxicity, glutamate, NMDA.
J. Neurochem. (2008) 105, 891–903.
d JOURNAL OF NEUROCHEMISTRY
|2008| 105| 891–903 doi: 10.1111/j.1471-4159.2007.05187.x
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ER, Grp78 dissociates from each of the sensors leading to
activation of their associated signalling pathways, culminat-
ing in the attenuation of general protein synthesis and
increased transcription of ER resident chaperones and protein
foldases. However, under conditions of prolonged or severe
stress, apoptosis ensues.
We have previously characterized the gene expression
profile during ER stress-induced apoptosis of neural cells
treated with the inhibitor of N-glycosylation, tunicamycin
(Reimertz et al. 2003). p53 up-regulated mediator of apop-
tosis (PUMA), a member of the Bcl-2 homology 3 (BH3)-
only family of proteins was the most prominent pro-apoptotic
gene induced. BH3-only proteins comprise a pro-apoptotic
subset of the Bcl-2 family which function to couple cellular
stress events to the release of pro-apoptotic proteins from
mitochondrial in a Bax-dependent manner (Labi et al. 2006).
Expression of PUMA has been demonstrated to be both
necessary and sufficient for inducing ER stress-mediated
apoptosis in a number of cell models (Reimertz et al. 2003;
Luo et al. 2005; Li et al. 2006; Nickson et al. 2007).
Recently, increased X-box binding protein 1 (XBP1) splicing
and CIEBP homology protein (CHOP) mRNA levels have
been observed during nitric oxide- and excitotoxicity-induced
inactivation of protein-disulphide isomerases (Uehara et al.
2006), suggesting that excitotoxic injury may activate an ER
stress response. Moreover, NMDA-mediated excitotoxic
apoptosis has been previously demonstrated to occur in a
Bax-dependent manner (Xiang et al. 1998). In the present
study, we therefore sought to clarify whether an ER stress
response is induced following NMDA-receptor mediated
excitotoxic apoptosis in vitro or in vivo, and explored the
potential role of PUMA in mediating such injury.
Materials and methods
Foetal calf serum, horse serum, B27 supplement, neurobasal medium
and minimal essential medium (MEM) were from Invitrogen
(Paisley, Strathclyde, UK). Tunicamycin was purchased from Alexis
(Axxora, Nottingham, UK). All other chemicals came in analytical
grade purity from Sigma-Aldrich (Tallaght, Dublin, Ireland).
Preparation of primary cerebellar granule neurons
Cerebellar granule neurons were prepared as described previously
(Ward et al. 2000) with minor modifications. Cerebella from 7-day-
old Wistar rats of both sexes were dissected and pooled. The tissue
was placed in 20 mL of filter sterilized phosphate-buffered saline
(PBS) supplemented with 0.25 mg/mL trypsin and incubated at 37?C
for 20 min. Trypsinization was terminated by addition of an equal
volume of PBS supplemented with 0.05 mg/mL soyabean trypsin
inhibitor, 3 mmol/L MgSO4and 30 U/mL Dnase I. The neurons
were then triturated and the resulting neurons were resuspended in
supplemented culture medium. Cells were then plated on poly-
L-lysine coated glass Willco dishes (Willco BV, Amsterdam, The
Netherlands) or six-well plates at 1 · 106cells per mL and
maintained at 37?C in a humidified atmosphere of 5% CO2: 95%
air. All experiments were performed on days 7–9 in vitro.
Preparation of primary cortical neurons
Cortical neurons were prepared as described previously (Kushnar-
eva et al. 2005). Briefly days 17–19 (E17–19) were isolated from
inbred PUMA+/+ and PUMA)/) C57BL6/J mouse embryos by
hysterectomy of the uterus, using an abdominal injection of 0.2 lL
of pentobarbital as lethal anaesthesia. The embryos were transferred
to a dissection medium on ice (PBS with 0.25% glucose and 0.3%
bovine serum albumin). The cerebral cortices from each of the
embryos were isolated, the surrounding meninges were removed and
the tissue was pooled in dissection media on ice. The tissue was
incubated with trypsin (0.125 mg/mL) at 37?C for 15 min.
Soyabean trypsin inhibitor was used to stop the reaction (0.5 mg/
mL). The neurons were dissociated from tissue in 5 mL of the
trypsin inhibitor by gentle pipetting. The neurons were resuspended
in plating media MEM containing 5% foetal calf serum, 5% horse
serum, 100 U/mL penicillin/streptomycin, 0.5 mmol/L L-glutamine
and 6% glucose) and plated at 7 · 105cells/mL on poly-lysine
coated plates. Cells were incubated at 37?C, 5% CO2in media (new
basal medium (NBM)-embryonic containing 180 lmol/L glutamate,
100 U/mL of penicillin/streptomycin, 1% B27 and 0.5 mmol/L
L-glutamine) was exchanged every 3–4 days. Experiments were
carried out on day 11. The generation of PUMA knockout mice was
described previously (Villunger et al. 2003).
Lactate dehydrogenase release and Hoechst staining
Cerebellar granule neurons (1 · 106cells per well) were plated on
poly-L-lysine coated 24-well plates. In each case neuronal injury
was assessed at each of the time points by quantitative measurement
of lactate dehydrogenase (LDH) release using a cytotoxicity
Detection Kit (Roche Diagnostics Ltd, Bell lane, Lewes, UK).
Total cell death was obtained by incubating cultures in 0.8% Triton-
X 100 for 45 min. Levels of cell death in cultures were normalized
as a percentage of total cell death as per manufacturers instructions.
All conditions were carried out in triplicate in each experiment and
each experiment was carried out three times. For analysis of the
nuclear structure of cortical neurons, cell were stained live with
Hoechst 33258 (Sigma, Dublin, Ireland) at a final concentration of
1 lg/mL for 10 min, fixed and nuclear morphology was observed
using an Eclipse TE 300 inverted microscope (Nikon, Du ¨sseldorf,
Germany) and a 63· oil objective.
Organotypic hippocampal slice cultures
Organotypic hippocampal slice cultures were prepared and grown
according to the modified procedure described previously (Kristen-
sen et al. 2001). Briefly 10-day-old mice were decapitated; brain was
removed and gently submerged in dissection medium containing
Hank's balanced salt solution (Gibco, Biosciences, Dublin, Ireland),
20 mmol/L HEPES, 100 U/mL penicillin and 100 lg/mL strepto-
mycin and 0.65% glucose. After separation of both hemispheres,
each hippocampus was carefully removed. Both hippocampi were
placed on the Teflon plate of a Mc Illwain tissue Chopper (Mickle
Laboratory, Guldfort, UK), and cut into 450-lm thick sections. The
slices were transferred into fresh dissection medium, and selected for
clear hippocampal morphology [intact CA regions and dentate gyrus
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892| C. G. Concannon et al.
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(DG)]. The slices were then transferred onto the porous (0.4 lm)
membrane of the millicell inserts (Millipore, Cork, Ireland). Per
animal up to six sections per hemisphere could be obtained and three
were placed on each membrane. The inserts were placed in 24-well
tissue culture plate with 250 lLof culture medium consisting of 50%
MEM (Sigma, Dublin, Ireland), 25% horse serum (Biosciences),
4 mmol/L L-glutamine, 6 mg/mL D-glucose, 2% B27, 50 U/mL
Penicillin G and 50 lg/mL streptomycin/mL (Sigma, Dublin,
Ireland) The slices were placed in a humidified chamber with 5%
CO2at 35?C. Cultures were maintained for 10 days in vitro with
media changes every other day.
Stimulus of organotypic hippocampal slices, quantification of
injury and OGD experiments
Slices were pre-stained with propidium iodide (PI) at 5 lg/mL prior
to initiation of experiments. Healthy slices from PUMA+/+ and
PUMA)/) animals were treated with 0.05 mmol/L NMDA for
30 min in experimental buffer (120 mmol/L NaCl, 3.5 mmol/L
KCl, 0.4 mmol/L KH2PO4, 5 mmol/L NaHCO3, 20 mmol/L
HEPES and 1.2 mmol/L Na2SO4) supplemented with glucose
(15 mmol/L) and CaCl2(1.2 mmol/L) for 30 min at 35?C. Control
slices contained were treated with experimental buffer without the
NMDA. In alternative experiments healthy slices from PUMA+/+
and PUMA)/) were exposed to tunicamycin (30 lmol/L) for 24 h
(vehicle addition was used as control). Observation of cell death was
performed by PI staining (5 lg/mL) for 15 min prior to image
acquisition. Images were acquired with an Eclipse TE 300 inverted
microscope (Nikon) and a 4· dry objective with WASABI Software
(Hamamatsu Photonics, Germany). The CA1 and DG region were
selected from PUMA+/+ and PUMA)/) hippocampal slices
following NMDA receptor over-activation and the addition of
tunicamycin respectively. The regional grey-level intensity was
measured using the WASABI interactive drawing tool, background
grey-level was corrected for and the average intensity plotted for
nine slices (triplicates from three separate cultures) for each of the
conditions. Cell death induced via each condition was expressed as a
percentage of total cell death within the slice region induced via
treatment with 1 mmol/L NMDA for 24 h which induced total cell
death in the CA1 or via treatment with 30 lmol/L tunicamycin for
72 h which induced total cell death in the DG. For the oxygen and
glucose deprivation (OGD) experiments OGD solution containing
124 mmol/L NaCl, 2.5 mmol/L KCl, 1.25 mmol/L KH2PO4,
26 mmol/L NaHCO3, 1.5 mmol/L MgSO4and 2.5 mmol/L CaCl2
(pH 7.4) was bubbled with oxygen-free nitrogen for 1 h prior to
being placed in a COY hypoxic chamber (COY Lab Products Inc.,
Grass Lake, MI, USA) saturated with 94% N2, 1% O2and 5% CO2
at 35?C for a further hour. The hippocampal slices were taken from
the incubator and placed in the hypoxic chamber, the media was
rapidly replaced with O2depleted Roswell Park Memorial Institute
(RPMI) containing zero glucose and slices were maintained in the
hypoxic chamber for 30 min. The slices were removed from the
chamber and the experimental media replaced with normal culture
media and returned to the incubator under normoxic conditions until
neuronal injury was evaluated 24 h later with PI.
Confocal microscopy
Cerebellar granule neurons on Willco dishes were loaded with Fluo-
4AM (3 lmol/L) for 30 min at 37?C (in the dark), in experimental
buffer (120 mmol/L NaCl, 3.5 mmol/L KCl, 0.4 mmol/L KH2PO4,
20 mmol/L HEPES, 5 mmol/L NaHCO3, 1.2 mmol/L Na2SO4,
1.2 mmol/L CaCl2, 1.2 mmol/L MgCl2and 15 mmol/L glucose, pH
7.4). The cells were washed in fresh medium after loading before
being mounted in a thermostatically regulated chamber (37?C) and
placed on the stage of a LSM 510 Meta Zeiss (Jena, Germany)
confocal microscope. An objective heater was also utilized enabling
themaintenance of asteady temperature of 37?C ± 0.26?C within the
extracellular media. The neurons (1 · 106) were bathed in 2 mL of
experimental buffer and a thin layer of mineral oil was added to
that involve the addition of glutamate. For glutamate-induced
apoptosis, neurons were exposed to glutamate and glycine (100 and
10 lmol/L) for 5 min with MK801 (10 lmol/L) added to block
glutamate receptor activation. Fluo-4AM was excited at 488 nm with
anArgonlaser(1%)andtheemissioncollectedthrougha505–550 nm
barrier filter. Images were collected at 15 s intervals during glutamate
excitation and every 5 min during the rest of the experiment and the
resulting fluorescent images were processed using METAMORPH
Software (Universal Imaging Co., West Chester, PA, USA). To
with Fluo-4AM as above and placed on a LSM 510 Meta Zeiss
confocal microscope and images were taken every 2 min. Thapsigar-
gin (10 lmol/L) was added to control neurons (non-stimulated) after
0.5 h and peak Ca2+responses measured. In addition thapsigargin
(10 lmol/L) was added to glutamate-stimulated neurons at 0.5 and
5 h post-excitation and the peak Ca2+responses measured. Images
were again processed using METAMORPH Software and the data
presented as a percentage of the baseline response.
Real-time qPCR
TotalRNAwas extracted using theRNeasy mini Kit (Qiagen, Hilden,
Germany). First strand cDNA synthesis was performed using 2 lg of
total RNA as template and reverse transcribed using Superscript II
(Invitrogen) primed with 50 pmol of random hexamers. Quantitative
real-time PCR was performed using the LIGHTCYCLER 2.0 (Roche
Diagnostics, Basel, Switzerland) and the QuantiTech SYBR Green
PCRkit (Qiagen) as per manufacturer'sprotocol. Specific primers for
each gene analysed were designed using PRIMER3 Software (http://
sense primers were: TCTCAGTGCAATGGCTTCC and CAATG-
CATTCTCCACACCAG for Grp78; TCACAAGCACCTCCCAA-
AGC and AGCAAGCTGTGCCACTTTCC for CHOP; GGTCT-
GAA for XBP1s; ATGGACTCAGCATCGGAAGG and TGGCTC-
ATTTGCTCTTCACG for PUMA; TCGTTCCTCCAGTCCGA-
GAG and TGAGTGGGGGTCGGTGTAGT for c-Jun; and AGC-
CATCCAGGCTGTGTTGT and CAGCTGTGGTGGTGAAGCTG
for b-Actin.Each primer pairwas tested with alogarithmic dilution of
a cDNA mix to generate a linear standard curve, which was used to
calculate the primer pair efficiency. The PCR reactions were
performed in 20 lL volumes with following parameters: 95?C for
15 min followed by 40 cycles of 94?C for 20 s, 59?C for 20 s and
72?Cfor20 s.ThegenerationofspecificPCRproductswasconfirmed
by melting curve analysis and gel electrophoresis. The data was
analysed using the LIGHTCYCLER Software 4.0 with all samples
normalized to b-actin.
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ER stress and excitotoxicity |
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Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and
western blotting
Preparation of cell lysates and western blotting was carried out as
described (Reimertz et al. 2003). The resulting blots were probed
with a mouse monoclonal anti-KDEL antibody (StressGen,
Victoria, BC, Canada) diluted 1 : 1000, a rabbit polyclonal anti-
CHOP antibody (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) diluted 1 : 250, a rabbit polyclonal anti-XBP1 antibody
(Santa Cruz Biotechnology), a mouse monoclonal anti-Ubiquitin
antibody (Affiniti, Victoria, BC, Canada) diluted 1 : 1000, a
rabbit polyclonalanti-protein
(StressGen) diluted 1 : 1000, or a mouse monoclonal anti-b-Actin
antibody (clone DM 1A; Sigma), diluted 1 : 5000. Horseradish
peroxidase-conjugated secondary antibodies diluted 1 : 10 000
(Pierce,Northumberland, UK)
Signal West Pico Chemiluminescent Substrate (Pierce) and
imaged using a FujiFilm LAS-3000 imaging system (Fuji,
Sheffield, UK).
disulphideisomerase antibody
weredetectedusing Super-
NMDA-induced hippocampal lesions
In vivo excitoxicity was modelled using bilateral intra-hippocampal
NMDA injections to trigger death of CA1 pyramidal neurons.
Briefly, adult (20–25 g) male C57BL/6 mice were anaesthetized with
isoflurane and placed in a stereotaxic frame (Stoelting, Wood Dale,
IL, USA) equipped with a neonatal rat/mouse adaptor. Mice were
maintained normothermic by means of a feedback controlled
homeothermic blanket. Following a midline incision, two craniec-
tomies were performed. Coordinates from Bregma: AP = )1.9 mm
posterior,±1.5 mm lateral. NMDA (1 lg) or vehicle was injected at a
depth of 1.3 mm below the dura in 0.1 lL volume. Mice were killed
2, 8, 24 or 48 h later, perfused with saline to remove intravascular
blood components and brains fresh-frozen and processed for
immunohistochemical analysis. Ethical approval was provided by
the Royal College of Surgeons in Ireland for all animal work carried
out within this study.
Immunohistochemistry and TUNEL staining
Immunohistochemistry was performed as previously described
(Yamamoto et al. 2006). Mouse brain sections (12 lm) were air
dried (15 min), post-fixed in 4% formalin (30 min), washed in PBS
and then processed for immunohistochemistry and detection of
DNA fragmentation. Following blocking in 5% goat serum, sections
were incubated overnight at 4?C with an anti-KDEL antibody
(mouse monoclonal; Stressgen Biotechnologies, Victoria, Canada).
Sections were washed in PBS and then incubated for 2 h at 20–25?C
in a 1 : 1000 dilution of Rhodamine goat anti-mouse secondary
antibody (Jackson Immunoresearch, Plymouth, PA, USA). Detec-
tion of DNA fragmentation was performed using a fluorescein-based
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labelling (TUNEL) technique (Roche Molecular Biochemicals,
Indianapolis, IN, USA) as per manufacturer's instructions. After
additional washes, sections were counterstained with 4¢, 6 diami-
dino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA)
for assessment of nuclear morphology. Images were visualized using
a Hamamatsu Orca 285 camera attached to a Nikon 2000s
epifluorescence microscope (Micro-optica, Dublin, Ireland) under
Ex/Em wavelengths of 330–380/420 nm (blue), 472/520 nm (green)
and 540–580/600–660 nm (red).
Data are given as mean ± SEM. For statistical comparison, one-way
ANOVA followed by Tukey's test were employed. Values of p < 0.05
were considered to be statistically significant.
Transient activation of glutamate receptors induces a
time-dependent increase in excitotoxic apoptosis of
cultured rat cerebellar granule cells in the absence
of long-term ER Ca2+level changes
Excitotoxic neuronal injury has been shown to be highly
dependent on excessive Ca2+uptake (Tymianski et al. 1993b;
Sattler et al. 1998; Ward et al. 2005). We have previously
established a model in which a 5 min transient activation of
glutamate receptors results in a delayed, apoptotic neuronal
injury in cultured rat cerebellar granule neurons (Ward et al.
2006). In agreement with previous studies (Ward et al. 2006),
cerebellar granule neurons loaded with the Ca2+sensitive
probe Fluo-4AM and exposed to glutamate went through a
delayed collapse of Ca2+homeostasis 8–16 h downstream of
glutamate excitation (Fig. 1a). In order to quantify the onset
and development of injury within our model we measured
LDH release over a 24 h period post-glutamate excitation
(Fig. 1b). The LDH release showed a similar trend to the Ca2+
data in that there was no significant increase in cell death prior
to the 4 h time point with most of the injury occurring
between 8 and 24 h. In order to investigate whether transient
glutamate receptor activation affected Ca2+levels within the
ER, we treated cells with thapsigargin, an inhibitor of Sarco/
Endoplasmic Reticulum Ca2+-ATPase pumps, which mobi-
lizes Ca2+from ER stores to the cytosol. Control cerebellar
granule neurons and glutamate excited (5 min) neurons were
loaded with Fluo-4AM at 0.5 and 5 h post-glutamate
excitation, time points which preceded the onset of injury
(Fig. 1a and b). The neurons were subsequently exposed to
10 lmol/L thapsigargin and changes in cytosolic Ca2+levels
were monitored. Addition of thapsigargin to the neurons
resulted in a rapid increase in cytosolic Ca2+levels. Quan-
tification of this thapsigargin sensitive Ca2+response between
glutamate excited and control neurons revealed no significant
difference in the level of peak Ca2+responses between the
two groups (Fig. 1c). Taken together, this data suggests that
although transient glutamate receptor activation acutely
increases cytosolic Ca2+levels uptake of Ca2+into ER is
not suppressed post-insult.
Glutamate receptor activation does not induce changes in
the gene expression of UPR associated genes
To determine if activation of glutamate receptors was
associated with activation of the three main signalling
pathways associated with accumulation of unfolded proteins
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within the ER, we exposed cerebellar granular cells to
glutamate for 5 min and extracted RNA at various time
periods. In parallel, neurons were treated with 3 lmol/L
tunicamycin, an inhibitor of N-glycosylation, and a classical
mediator of the ER stress response. Activation of ATF6
results in increased transcription of the ER resident chaper-
ones Grp78 (Yoshida et al. 1998). As illustrated in Fig. 2a,
glutamate treatment was not associated with a significant
modulation of Grp78 mRNA expression. In contrast,
tunicamycin treatment resulted in a robust and prolonged
increase in Grp78 expression (Fig. 2a). Signalling through
IRE1a is associated with increased splicing of the transcrip-
tion factor, (XBP1 (Yoshida et al. 2001). In order to detect
XBP1 splicing we selected primers which detect only the
spliced form of XBP1 as described by Hirota et al. (2006).
Real-time qPCR analysis revealed a highly potent and rapid
increase in splicing of XBP1 following tunicamycin treat-
ment which was sustained over the time period investigated,
an event which was only modestly affected in glutamate-
treated samples (Fig. 2b). Similarly, PERK-dependent sig-
nalling is associated with increases in CHOP mRNA
expression via increased translation of ATF4 (Harding et al.
2000). In line with the previous observations, increased
CHOP mRNA expression was only evident in tunicamycin-
treated samples and not in the glutamate model (Fig. 2c). ER
stress is also associated with the transcriptional activation of
ATF4 (Reimertz et al. 2003) and ATF6 (Namba et al. 2007),
however, despite being induced by tunicamycin treatment,
glutamate treatment failed to result in their increased
expression (data not shown). To confirm the results of the
gene expression studies, we performed western blot analysis
of lysates from glutamate- and tunicamycin-treated samples.
As demonstrated in Fig. 2d, western blot analysis using an
anti-KDEL antibody, which detects Grp94 and Grp78
expression, revealed only the tunicamycin-treated samples
exhibited increased levels of the ER chaperones, Grp94 and
Grp78. Similar results were found by western blotting for
CHOPand protein disulphide
(Fig. 2d). Furthermore, western blot analysis revealed the
presence of the spliced form of XBP1 following tunicamycin
treatment, an observation which was absent in the glutamate-
treated samples.
isomerase expression
Fig. 1 Activation of glutamate receptors is not associated with the
modulation of Ca2+levels within the ER. Cerebellar granule neurons
were exposed to glutamate/glycine (100/10 lmol/L) for 5 min, following
which receptor activation was inhibited by the addition of MK801 and
neuronal injury monitored over a 24 h period. (a) Representative Ca2+
traces [Fluo-4 (3 lmol/L) for 30 min at 37?C] for neurons exposed to
glutamate (traces selected from five separate experiments from dif-
ferent cultures). (b) LDH release was measured at indicated time points
(4, 8, 16 and 24 h) following the initial insult (n = 3 experiments in
triplicate) (c) Quantification of the thapsigargin sensitive response in
glutamate excited (0.5 and 5 h after stimulus) and control neurons as
described in the Material and methods (0.5 h). Experiments were
carried out in three separate cultures with similar results. No significant
(ns) difference in Ca2+transients were observed between glutamate
treated (n = 13, 0.25 h and n = 16, 5 h) and control neurons (n = 32)
following the addition of thapsigargin.
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ER stress and excitotoxicity|
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Proteasome function is not affected by glutamate receptor
Given that disturbances in protein folding may also modulate
the rate of protein degradation and the activity of the
proteasome, the major site of protein degradation within a
cell, we investigated whether glutamate-induced excitotoxic
apoptosis was associated with the modulation of proteasome
function. Proteins designated for proteasomal degradation are
tagged with poly-ubiquitin chains resulting in their targeting
to the proteasome complex where they are degraded. As
demonstrated in Fig. 3a, treatment with the proteasomal
inhibitor, epoxomicin (Epoxo), resulted in the accumulation
of mono- and poly-ubiquitinylated proteins, an observation
which was absence in glutamate-treated samples. We have
previously profiled the transcriptional changes which occur
following inhibition of the proteasome in neural cells
(Concannon et al. 2007). Amongst the most prominently
induced genes is the transcription factor c-Jun. Real-time
qPCR analysis revealed a potent time-dependent increase in
c-Jun transcription following inhibition of the proteasome
with epoxomicin (Fig. 3c). This potent increase in c-Jun
transcription was absent following glutamate treatment
(Fig. 3b). Taken together, these data suggest that gluta-
mate-induced excitotoxic apoptosis does not result in mod-
ulation of proteasome function and associated stress events.
NMDA receptor-mediated excitotoxic apoptosis of
cultured mouse cortical neurons does not require PUMA
Endoplasmic reticulum stress-induced apoptosis is mediated,
at least in part, by induction of the BH3-only protein PUMA
(Reimertz et al. 2003), with increased transcription of PUMA
mRNA preceding the onset of apoptosis. Real-time qPCR
Fig. 2 Tunicamycin but not transient glu-
tamate excitation induces transcriptional
activation of ER stress response genes.
Cerebellar granule neurons were exposed
to glutamate/glycine (100/10 lmol/L) for
5 min and allowed to recover for the indi-
cated time periods (left panels) or were
treated with tunicamycin (3 lmol/L) for
indicated time periods (right panels). Fol-
lowing treatments mRNA expression of (a)
Grp78, (b) XBP1 and (c) CHOP were
analysed by real-time qPCR. Expression
levels were normalized to control treated
cells anddata are
mean ± SE from n = 3 separate experi-
ments. (d) Cerebellar granule neurons were
treated as described above. Left panel; the
expression levels of Grp94, Grp78, CHOP
and protein disulphide isomerase (PDI)
were examined by western blotting (left
panel) with actin as loading control. As a
positive control, cells were treated with
3 lmol/L tunicamycin for 24 h (Tuni). Right
panel; the expression levels of both the
unspliced (XBP1u) and spliced variants of
XBP1 (XBP1s) were examined by western
blotting. As a positive control, cells were
treated with 3 lmol/L tunicamycin for 16 h
(Tuni). Actin served as a loading control.
Similar results were obtained in two sepa-
rate experiments.
Journal Compilation ? 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 891–903
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896| C. G. Concannon et al.
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analysis revealed that the expression of levels of PUMA
mRNA was not significantly altered in glutamate-treated
cerebellar granule neurons (Fig. 4a). In contrast, tunicamycin
treatment resulted in time-dependent increase in the expres-
sion of PUMA mRNA (Fig. 4b). Furthermore, this increase
in PUMA mRNA within tunicamycin-treated samples pre-
ceded injury onset (Fig. 4c), supporting a requirement for
PUMA in ER stress-mediated injury.
We next investigated the requirement for PUMA expres-
sion in an NMDA-mediated model of excitotoxic apoptosis
in mouse cortical neurons. Primary cortical neurons were
derived from both PUMA+/+ and PUMA)/) mice on the
same day and maintained in culture for 11 days prior to
NMDA treatment. Neurons were stimulated with 300 lmol/
L NMDA for 15 min and allowed to recover for 24 h and
Fig. 3 Transient glutamate excitation does not correlate with inhibition
of proteasome function. (a) Cerebellar granule neurons were treated
as described above. Whole cell extracts were analysed by western
blotting using an antibody that recognizes mono- and poly-ubiquitiny-
lated proteins. As a positive control, neurons were treated with
50 nmol/L epoxomicin for 16 h (Epoxo). Actin served as a loading
control. Similar results were obtained in two separate experiments.
Cerebellar granule neurons were exposed to glutamate/glycine (100/
10 lmol/L) for 5 min and allowed to recover for the indicated time
periods (b) or were treated with the proteasomal inhibitor, epoxomicin
(50 nmol/L), for indicated time periods (c). Following treatments
expression of c-Jun was analysed by real-time qPCR. Expression
levels were normalized to control treated cells and data is represented
as mean ± SE from n = 3 separate experiments. *, p < 0.05.
Fig. 4 PUMA expression is unaffected by activation of glutamate
receptors. Cerebellar granule neurons were exposed to glutamate/
glycine (100/10 lmol/L) for 5 min and allowed to recover for the indi-
cated time periods (a) or were treated with tunicamycin (3 lmol/L) for
indicated time periods (b). Following treatments expression of PUMA
was analysed by real-time qPCR. Expression levels were normalized to
control treated cells and data is represented as mean ± SE from n = 3
separate experiments. (c) Cerebellar granule neurons were treated
with 3 lmol/L tunicamycin for the indicated time periods and LDH
release measured as described in Material and methods. *, p < 0.05.
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ER stress and excitotoxicity|
Page 8
cell viability in the cultures assessed by the ability to exclude
the vital dye, PI. This treatment regime induced morpholog-
ical changes indicative of apoptotic cell death including
condensation and fragmentation of nuclei as assessed by
Hoechst staining (Fig. 5a). Subsequent quantification of the
number of PI-positive cells in PUMA+/+ and PUMA)/)
cultures revealed that the loss of PUMA expression had no
effect on the levels of cell death resulting from excitation
with NMDA (Fig. 5b). In contrast, loss of PUMA expression
significantly attenuated the levels of cell death following
tunicamycin treatment (Fig. 5c). In agreement with these
results, western blot analysis revealed an absence of
increased expression of the ER stress markers, Grp78 or
CHOP, in NMDA-treated cortical cultures (Fig. 5d), further
demonstrating the absence of an ER stress response in
NMDA receptor dependent glutamate toxicity.
Loss of PUMA expression does not protect against
NMDA-induced excitotoxic apoptosis in an organotypic
hippocampal slice culture model
In order to explore whether ER stress may be involved in
mediating excitotoxic cell death in selectively vulnerable
brain regions, we employed a mouse model of organotypic
hippocampal slice culture. A transient activation of glutamate
receptors with 0.05 mmol/L NMDA for 30 min resulted in
cell death and a progression of apoptosis over time within the
CA1 region of the organotypic hippocampal slice cultures.
4¢, 6 Diamidino-2-phenylindole staining of these neurons
within this CA1 region revealed the presence of condensed
nuclei indicative of apoptotic cell death (Fig. 6a). Subse-
quent quantitative analysis of the levels of injury induced in
slices derived from either PUMA+/+ and PUMA)/) slices
mice revealed similar levels of cell death in PUMA+/+ and
PUMA)/) slices suggesting that loss of PUMA expression
did not protect from NMDA-mediated excitotoxic apoptosis
(Fig. 6a and b). In contrast, loss of PUMA expression offered
significant protection from ER stress mediated via tunica-
mycin treatment. As demonstrated in Fig. 6d, addition of
30 lmol/L tunicamycin for 24 h resulted in increased PI
staining and damage with the DG region of the hippocampus.
However, in mice deficient for PUMA expression, the levels
of injury were dramatically impaired supporting a central role
of PUMA in mediating ER stress-dependent injury in this
model (Fig. 6c and d). It has been previously demonstrated
(a) (b)
Fig. 5 (a) Cortical neurons were exposed to Sham conditions or
NMDA (300 lmol/L) for 15 min and nuclear morphology assessed by
Hoechst 33258. Scale bar: 10 lm (b) Cortical neurons derived from
PUMA+/+ and PUMA)/) mice were treated as described above and
the extent of injury was assessed after 24 h with propidium iodide (PI).
Experiments were carried out in triplicate in cortical neurons derived
from three separate cultures for each strain of mice. No significant
difference in the extent of neuronal injury was identified between
PUMA+/+ and PUMA)/) mice. (c) Cortical neurons derived from
PUMA+/+ and PUMA)/) mice were treated with 1 lmol/L tunicamycin
for 2 h. Following treatment, medium was replaced with conditioned
medium and cell death assessed after 24 h with PI. Experiments were
carried out in triplicate in cortical neurons derived from three separate
cultures for each strain of mice. *p < 0.05 compared with PUMA+/+
treated mice. (d) Whole-cell extracts were prepared from mouse cor-
tical cultures slices treated with 300 lmol/L NMDA for 15 min and
allowed to recover for the indicated time points. Grp78 and CHOP
expression levels were assessed by western blotting. Tunicamycin
(Tuni) treated cells were used as a positive control. Actin served as a
loading control. Similar results were obtained in two separate experi-
Journal Compilation ? 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 891–903
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898| C. G. Concannon et al.
Page 9
that mice injected intraperitoneally with tunicamycin have
marked neuronal damage within the DG of the hippocampus
(Chae et al. 2004). Consistent with the absence of a role for
PUMA in mediating NMDA-induced excitotoxic apoptosis,
western blot analysis revealed increased levels of Grp78 only
in tunicamycin-treated slices and not following NMDA
treatment, demonstrating an absence of an ER stress response
(Fig. 6e). Furthermore, in order to investigate whether
conditions of OGD, which mimic the effects of ischaemia,
could potentially induce an ER stress response we incubated
hippocampal slice cultures in OGD conditions for 30 min
and allowed them to subsequently recover under normal
culture conditions. As demonstrated in Fig. 6f, 30 min OGD
was sufficient to induce CA1 damage (Fig. 6f). Interestingly,
a time-dependent increase in the expression of both Grp78
and CHOP was evident in this model (Fig. 6g).
(a) (b)
Fig. 6 Organotypic hippocampal slices isolated from PUMA+/+ and
PUMA)/) mice show no difference in sensitivity to NMDA excitation.
(a) Top panels: 4¢, 6 diamidino-2-phenylindole stained neurons from
the CA1 region of wild-type mice exposed to Sham conditions or
NMDA (0.05 mmol/L) for 30 min at 24 h post-insult. Scale bar:
100 lm. Lower panel: Organotypic hippocampal slices derived from
PUMA+/+ and PUMA)/) mice were exposed to Sham conditions or
NMDA (0.05 mmol/L) for 30 min and the extent of injury assessed
24 h post-treatments via propidium iodide (PI) and quantification as
described in the Material and methods. Experiments were carried out
in triplicate in three separate cultures for each strain of mice. No sig-
nificant difference in the extent of neuronal injury was identified be-
tween PUMA+/+ and PUMA)/) mice. (b) Representative images of
Sham and NMDA treated hippocampal slices derived from PUMA+/+
and PUMA)/) mice. Scale bar: 100 lm. (c) Hippocampal slices from
derived from PUMA+/+ and PUMA)/) mice were treated with di-
methylsulphoxide or 30 lmol/L tunicamycin for 24 h. Injury induced
was assessed by PI staining as previously described. Different from
tunicamycin treated PUMA+/+; *p < 0.05. (d) Representative images
of dimethylsulphoxide and Tuni treated slices from derived from
PUMA+/+ and PUMA)/) mice. Scale bar: 500 lm. (e) Whole-cell
extracts were prepared from wild-type hippocampal slices treated with
0.05 mmol/L NMDA for 30 min and allowed to recover for the indicated
time points. Grp78 expression levels were assessed by western
blotting. Tunicamycin (Tuni) treated cells were used as a positive
control. Actin served as a loading control. Similar results were ob-
tained in two separate experiments. (f) Representative images of
Sham and OGD (30 min) treated hippocampal slices organotypic
hippocampal slices derived from C57BL6 mice. Injury induced was
assessed by PI staining as previously described. (g) Whole-cell ex-
tracts were prepared from hippocampal slices treated to 30 min OGD
and allowed to recover under normoxic conditions for the indicated
time points. Grp78 and CHOP expression levels were assessed by
western blotting. Actin served loading control.
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ER stress and excitotoxicity|
Page 10
NMDA-induced lesions in the mouse hippocampus occur
independent of ER stress and PUMA expression
In order to examine whether excitotoxicity-induced apoptosis
in vivo may be mediated in a PUMA-dependent manner,
excitotoxic hippocampal lesions were induced in wild-type
mice by bilateral intra-hippocampal NMDA injections.
Injury in this model is characterized by widespread degen-
eration of CA1 neurons, which became TUNEL-positive at
8 h post-injection (Fig. 7a). Immunohistochemical analysis
of levels of KDEL revealed modest immunoreactivity in
CA1 neurons in control mice. Following NMDA injection
KDEL immunostaining rapidly declined as early as 2 h post-
treatment, supporting an absence of ER stress in this model.
A similar immunostaining pattern was observed for calnexin,
an additional ER stress marker (Supplemental Fig. S1). We
next compared neuronal death induced by intra-hippocampal
NMDA between PUMA+/+, PUMA+/) and PUMA)/) mice.
Importantly, counting of neuronal within the hippocampus
revealed similar counts in PUMA+/+, PUMA+/) and
PUMA)/) mice (Supplemental Fig. S2). PUMA+/+ mice
exhibited near-complete death of CA1 neurons when exam-
ined 48 h following NMDA injection. Quantification of the
levels of apoptosis induced in this model revealed no
significant differences between PUMA+/+, PUMA+/) and
PUMA)/) mice (Fig. 7b), confirming that NMDA-induced
excitotoxic apoptosis is mediated in an ER stress and PUMA
independent manner.
Activation of glutamate receptors has been inextricably
linked with the neuronal injury associated with acute
neurological insults such as cerebral ischaemia, trauma,
and seizures. Several studies have suggested the hypothesis
that ER dysfunction and ER stress are responsible for
transducing excitotoxic and ischaemic injury (Paschen
2003; Paschen et al. 2003; Tarabal et al. 2005; Uehara
et al. 2006). In this study, we have demonstrated that the
activation of NMDA receptors per se is not associated with
significant modulation of ER stress signalling pathways.
Fig. 7 In vivo excitotoxic injury is mediated
in an ER stress and PUMA independent
manner. (a)Mice
NMDA injections and killed after 2–24 h
post-NMDA injection. Images show repre-
sentative CA1 fields from 4¢, 6 diamidino-
TUNEL stained sections for each of the
treatment groups. Images shown are rep-
resentative of n = 3 mice per treatment
group. Scale bar: 100 lm. (b) PUMA+/+,
PUMA+/) and PUMA)/) mice were treated
were injected with NMDA as described in
(a). Forty-eight hours post-NMDA injection
hippocampal slices were the number of
TUNEL-positive cells were quantified as
described in the Material and methods. No
significant difference in the extent of neu-
ronal injury was identified between groups.
Data are mean ± SE from n = 4–8 animals
per genotype.
Journal Compilation ? 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 891–903
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900| C. G. Concannon et al.
Page 11
Moreover, loss of expression of the BH3-only protein,
PUMA, a previously implicated mediator of ER stress-
induced cell death had no effect in modulation the injury
associated with excitotoxicity.
The classical ER stress response involves modulation of
three distinct signalling pathways: ATF6, PERK and IRE1a
pathways. Analysis of the expression levels of target genes of
each of these pathways failed to detect significant changes in
their expression levels in several models of delayed excito-
toxic injury. The activation of a typical ER stress response is
associated with a rapid and sustained increase in splicing of
XBP1 mRNA, resulting in an alteration in the open reading
frame of the XBP1 protein leading to the translation of a
highly active transcription factor. In our studies glutamate-
induced injury resulted in a very moderate increase in the
splicing of XBP1 mRNA as assessed by qPCR. However,
subsequent western blot analysis failed to demonstrate any
increase in the expression of spliced XBP1 on a protein level.
In contrast, tunicamycin-induced injury was associated with
a rapid and robust increase in XBP1 splicing at both mRNA
and protein level. Furthermore, induction of the classical
XBP1 target gene, grp78, was unaffected, both at the mRNA
and protein level. A recent study by Uehara et al. (2006)
demonstrated increased splicing of XBP1 mRNA following
NMDA-mediated excitotoxic injury, however, no increases
in the translation of this potent transcription factor or
induction of its target genes were demonstrated suggesting
that although XBP1 splicing may be moderately affected it is
not sufficient to allow for increases on a protein level.
Finally, we demonstrate that the transcription rates of the
BH3-only protein, PUMA, which has previously been
demonstrated to be central to mediating ER stress-mediated
cell death (Reimertz et al. 2003; Luo et al. 2005; Li et al.
2006; Nickson et al. 2007), remained unaltered during
NMDA receptor-mediated excitotoxic apoptosis. Indeed,
utilizing primary cortical neurons, organotypic hippocampal
slices and in vivo hippocampal NMDA injection, we were
able to demonstrate that excitotoxic injury does not require
PUMA expression.
A previous study has demonstrated acute increases in ER
Ca2+levels following NMDA receptor activation (Wang
et al. 2006). Our Fluo-4 experiments revealed that the influx
of Ca2+associated with transient NMDA receptor activation
does not significantly alter the overall rate of Ca2+uptake
into the ER in the long-term. In line with this observation,
previous studies have illustrated that mitochondria appear to
be the major subcellular site for accumulation of Ca2+
following its influx into neurons after excitation of ionotropic
receptors (Budd and Nicholls 1996; Stout et al. 1998; Ward
et al. 2005). Of note, several studies have demonstrated a
decrease in ER Ca2+stores post-ischaemia (Kohno et al.
1997; Xing et al. 2004) which may also potentially account
for the increased expression of ER stress markers following
Several studies have demonstrated that ischaemic injury in
neurons is associated with activation of several pathways of
the unfolded protein response (UPR) including phosphoryla-
tion of PERK (Kumar et al. 2001) and activation of IRE1a-
dependent signalling (Paschen et al. 2003). The accumulation
of protein aggregates containing ubiquitinylated proteins in
the cytosol of neurons is also an early evident following
cerebral ischaemia (Hu et al. 2000, 2001; Asai et al. 2002).
Defects in proteasome-mediated protein degradation trigger
certain similar cellular stress responses as observed during ER
stress, such as attenuation of protein synthesis and induction
of transcription factors of the ATF and C/enhancer binding
protein (EBP) families (Jiang and Wek 2005), although the
global pattern of gene expression pattern is quite distinct from
that induced during ER stress (Reimertz et al. 2003; Concan-
non et al. 2007). In our in vitro and in vivo models we were
also unable to detect increased levels of ubiquitinylated
proteins or gene expression changes indicative of proteasomal
dysfunction in response to excitotoxic injury. However, OGD
in organotypic hippocampal slices was sufficient to induce
increased expression of both Grp78 and CHOP. Our results
suggest that application of exogenous glutamate/NMDA both
in vitro and in vivo does not appear to contribute to ER stress
responses or defects in protein degradation. Interestingly,
hypoxia alone has been shown to increase the expression of
ER stress response genes (Blais et al. 2004; Bi et al. 2005),
suggesting that the activation of ER stress during ischaemia
may be the result of the events linked to the absence of
sufficient levels of oxygen and/or glucose. Indeed, hypoxia
within tumour cells has recently been described to activate
both the IRE1a and PERK signalling pathways of the UPR
(Feldman et al. 2005). We and others have previously
demonstrated a transcriptional activation of CHOP expression
in neural cells exposed to hypoglycaemic conditions (Carlson
et al. 1993; Ikesugi et al. 2006; Kogel et al. 2006). Further-
more, Hori et al. (1996) have demonstrated that hypoxia and
subsequent reoxygenation results in a depletion of intracellu-
lar glucose levels and the subsequent induction of Grp78.
Indeed, Grp78 and Grp94 were first identified on the basis of
their induction in cells following glucose starvation (Pouysse-
gur et al. 1977). Similarly, the generation of reactive oxygen
intermediates during reperfusion following ischaemia may
also be detrimental to ER homeostasis and may contribute to
ischaemia-induced ER stress or defect in protein degradation.
In conclusion, our study demonstrates that NMDA-med-
iated excitotoxic apoptosis does not appear to significantly
alter ER stress responses and does not require PUMA
expression. They also suggest that the contribution of
NMDA-mediated pathways to the activation of the ER stress
response seen during OGD or ischaemia may not be as
significant as previously thought. This study has important
implications for the reassessment of the design of therapies
aimed at the ER stress response for the treatment of injuries
associated with excitotoxic processes.
? 2008 The Authors
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ER stress and excitotoxicity|
Page 12
The authors wish to thank Drs A. Strasser and A. Villunger for gifts
of knockout mice. This study was supported by Grants from Science
Foundation Ireland (03/RP/B344) and the Health Research Board
(RP/2005/206) to JHMP. DCH is supported by the Wellcome Trust,
and LPT is a recipient of an IRCSET Post-graduate Studentship
Supplementary material
The following supplementary material is available for this article
Fig. S1 C57BL/6 mice.
Fig. S2 NeuN-positive cells.
This material is available as part of the online article from http://
Please note: Blackwell Publishing are not responsible for the
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directed to the corresponding author for the article.
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and Prehn J. H. (2006) Real time single cell analysis of Bid
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ER stress and excitotoxicity|


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Save the date for Connecticut's only Spring Apple Hunt! Find apples hidden in a Hay Maze; Enjoy Magic Shows, Pony Rides and Horse-drawn Wagon Rides, too! Children ages 1 to 10 years may participate. (Event rain date: Sunday, April 19th)
If you're getting married, this event is just for you! Each bride will enjoy a glass of complimentary champagne upon arrival, and will get to speak to the many talented vendors the show has to offer. There will be raffles every hour on the hour and a GRAND PRIZE GIVEAWAY!
(Children will have a great time dressing up like a real baker to decorate an Easter Cupcake Basket and Cookie. Pre-paid reservations are required. Please call 860.349.6043 to reserve your space.
Your first opportunity to try the latest and greatest golf technology. All the top brands in golf will be at the Golf Club with their newest equipment and best deals!
Lyman Orchards weekly Junior Golf School provides a safe and fun instructional environment where your young golfer will learn, refine, and improve their skills. Fun and half day sessions available. Call 860.398.6480 for details.
Spend the day with us and see what we have to offer for women and golf in 2015. Free clinics, giveaways, meet the staff, have fun and more!
Kids will love decorating a delicious fresh-baked cake, just for Mom! This hands-on workshop takes place in the Apple Barrel. All decorating materials are provided, including tons of frosting, special candies, and a delicious pre-made cake. Bring your camera! Reservations required. More info to come.
Treat Mom to a memorable experience she will treasure forever with a delicious holiday brunch in the historic Lyman Homestead. Catered by our Apple Barrel chefs. Reservations required. More information to come.
It's golf with a twist! Nine holes played at night with glow-in-the-dark balls and glow sticks!
Focus on lifestyle of golf and learn to play together. Free clinics, giveaways, discounts and more. Where else can you spend uninterrupted quality family time?
Our annual Strawberry Fest kicks off our summer event season, celebrating family fun, Lyman's fresh grown strawberries, and sweet strawberry treats from the Apple Barrel. Fun activities include Breakfast on the Deck, Strawberry picking in the orchard, live music, complementary food tastings and demos, pie eating contests, pony rides, and interactive activities for children throughout the day.
Celebrate Dad on his day and bring him to Lyman Orchards for a mouth-watering barbecue picnic. Prepared by our Apple Barrel chefs, this BBQ includes traditional BBQ fare, including chicken, fall-off-the-bone ribs, burgers and hotdogs. Make this year's Father's Day one he'll always remember! More information to come.
Lyman Orchards' weekly Junior Golf School provides a safe and fun instructional environment where young golfers will learn, refine, and improve their skills. Full day and half day sessions available. Call 860.398.6480 for more details.
It's golf with a twist! Nine holes played at night with glow-in-the-dark balls and glow sticks! Enjoy a delicious BBQ dinner, too!
Join us as we celebrate our homegrown Blueberries, Jostaberries, and Raspberries with a day of sweet family fun! Come berry picking, kick-back and relax to some live music on the Apple Barrel deck, and enjoy hours of family-oriented activities, food tastings, treats from the Outdoor Grill, and much more!
Lose yourself amid 350,000 blooming red and yellow sunflowers; search for the exit along miles of pathways and enjoy fun trivia along the way! Experience Connecticut's original Sunflower Maze and help a great cause. $1 of each admission is donated to Connecticut Children's Medical Center.
Last year, over 12,000 golfers at 118 Billy Casper Golf Courses nationwide raised more then $887,000 to benefit Wounded Warrior Project®. Come out to play and raise money to help those who served our country!
Bring family and friends! Enjoy a fun day in the country with live music, great food and bushels of outdoor activities for all ages. Go peach picking in the orchards; visit the Apple Barrel for cider donuts and fresh-baked Peach Pies! Activities of the day include pony rides, horse-drawn wagon rides, face-painting for children, and more!
PBS Kids' Mr. Steve blends smart lyrics, catchy melodies and thoughtful messages into fun, participatory songs that kids love and adults find irresistible. Bring a blanket or chairs and relax in the field outside the Apple Barrel!
It's golf with a twist! Nine holes played at night with glow-in-the-dark balls and glow sticks!
"Air Cooled in the Orchard! Enjoy viewing a selection of these great classic automobiles on the Apple Barrel grounds. Presented by Connecticut CORSA and the Connecticut VW Club.
An old-fashioned New England tradition continues at the Apple Barrel tent where we're serving up fresh steamers, mussels, lobsters, and all the fixings! Pre-paid reservations are required. More information to come.
With more than two miles of twisting and winding pathways carved within four acres of towering cornstalks, a hundred decision points, and just one way out, getting lost in our Corn Maze will be a lot of fun, and it's all for a great cause! $1 of every admission is donated to the American Cancer Society.
Come join the fun as Nicole Frechette and her favorite regional and local bands perform live at the Apple Barrel tent to raise money for St. Jude Children's Research Hospital!
October is Breast Cancer Awareness Month! Have a great time painting a pumpkin while supporting a worthy cause! You pick out the pumpkin, we provide all the paints, glitter, and special decorating materials you can use! Located on the Apple Barrel grounds; proceeds will benefit the Middlesex Comprehensive Breast Center.
This event is the most fun you'll have on the golf course as our Superintendents line the course with challenging obstacles and more!
From May 23rd through October 12th, come enjoy a hearty outdoor country breakfast prepared by our chefs on the Apple Barrel deck! Available Saturday and Sunday mornings, 8:00am - 11:00am.
Enjoy cooking demos and free tastings of holiday dishes, appetizers, and desserts throughout the day. Take home some delicious holiday recipes created by our Chefs!
Join us for a 3.7 mile run and walk through the picturesque hills of our orchard during fruit blossom time to benefit children with cancer. Also, kids can participate in a quarter-mile fun run!

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I'm going to brew a sweet stout this weekend and am considering switching out half, or maybe all of the Pale malt and using Munich instead. Has anybody tried this and what did you think? I'm thinking the Munich would really up the maltiness in the taste to go with the roasted malts. I will be adding either 1/2 or 1 lb of lactose also.
listen to the jamil show on the brewing network about sweet stouts, a lot of good info there.
For any beer, I would not recommend Munich at those levels unless you are very experienced at mashing with enzymes at 1/3 their normal levels. Munich malt and I assume you are talking Munich 10, has an alpha amylase level of around 30 (base malt is about 45 -60) and a DP of about 50 (base malt about 130 - 160). The addition of Munich at the 10 - 20% level will enhance the malt flavor sufficiently without the potential of a long and difficult mash. If you do try a 50% addition of Munich with base malt, extend your mash time as needed doing periodic iodine testing for conversion of the starch.
Dr Malt
When I get around to brewing it, I've got an oatmeal stout on the docket that I'm planning on a Munich addition to. I can't remember how much I have - I think around 2.5#s. Same thought process as you're having; more malt profile to underlie to roastiness.
marris otter would add a more "malty" profile which might go well with a sweet stout.
if you use an english pale malt (like marris otter) instead of an american 2-row you should get more maltiness too.
I really like Munich! I've mashed 50% Munich grain bills before. I'm even contemplating an all Munich lager this winter. I've just never gone this high on an ale and was curious if others had. The Pale I'm using is British (Optic).
I'm probably going to do 40% - 50% for the dopplebock I've got planned; I'm pretty sure that's typical for that style. Don't know why it wouldn't work here, too.
My rye ale is 60% Munich (60/40 Munich/rye). LOTS of malt character. I tried a bit of Munich (about 10% I think) in an oatmeal stout once and I didn't much care for it. Just personal preference in that case, I wouldn't presume to say it doesn't work. For me I just preferred what crystal gave me.
I would second the use of a 3-5 L british pale instead of all munich. I've found out the hard way (first debacle of a AG batch) that too much munich can become extremely cloying if not used properly. Sure, if you have the perfect water, mash temps, additionla base and specialty grains, etc. then it may be OK, but I'd stay away from an all munich-as-base-grain stout unless experimentation is your primary motivating factor - in that case, good luck and post the results!


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In 2014 Walt Disney World donated more to Central Florida Community than ever before
With nearly 75,000 Cast Members who call Central Florida home, the Walt Disney World Resort has a significant stake in the success of Central Florida as a community. As such they're committed to making a difference in the community. In … Continue reading
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February 17 · 6:30 PMSouth Florida Android Developers User GroupJoin us on Tuesday, February 17th, at 6:30pm, at MEC 261, for a talk on 8 elements to make a game successful, by Carlos Icaza. About the topic: In order for a game to be... Learn more
January 22 · 6:30 PMSouth Florida Android Developers User GroupWe are excited to announce our next South Florida Android Developers UG meeting. Please join us on Thursday, January 22nd, at 6:30pm, at MEC, for a chat on Dissecting... Learn more
October 25, 2014 · 5:00 PMAndroid Meetup at Tech Weekend @ The DolphinsCome join us for a special meetup during Tech Weekend @ The Dolphins. We have a guest speaker, Chuck Hirstius, who is going to show us how to use GSON and web APIs. ... Learn more
January 28, 2014 · 6:30 PMXamarin PresentationJim Blizzard of Microsoft will be presenting the Xamarin development environment, allowing C# Programming for iOS, Android and Windows devices.
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Glenwood won the battle but lost the war.
The Rams got five total touchdowns from Corey Bertini and racked up 422 yards rushing in a 48-20 victory over Atlantic to close on the regular season at Ram Memorial Field on Friday. But with Council Bluffs Lewis Central's 38-10 victory over Creston, Glenwood falls just short of a trip to the state playoffs.
Spencer Phillips rushed for a career-high 287 yards and four touchdowns as Fremont-Mills cruised to a 61-12 victory over Lenox on Friday.
The Knights played the game without head coach Jeremy Christiansen who missed the game due to a death in the family. Assistant coaches Greg Ernster and Kyle Fichter served as co-head coaches in Christiansen's absence.
"The kids came out fast and wanted to get this game done early and get ready for Wednesday," Ernster said.
Fremont-Mills vs. Coon Rapids-Bayard
Wednesday, 7 p.m. at Tabor
The East Mills football team posted it's third shutout of the season and rushed for 367 yards in a 66-0 victory over West Monona in the regular-season finale for both teams in Class A, District 8 on Friday.
"This was one of those games we knew if we took care of business we could get the win," said East Mills coach Andy Irwin.
With the victory the Wolverines (7-2) wrap up the No. 2 spot in District 8. East Mills will host Woodward-Granger in the Wednesday's opening round of the playoffs. Kick off is set for 7 p.m. in Malvern.
TABOR - Someone said long ago it's not easy to beat a team three times in the same season.
Sure didn't look that way last Tuesday as Fremont-Mills dominated Heartland Christian from the first serve en route to a 25-15, 25-12, 25-10 Class 1A, Region 2 opening round victory in Tabor.
The Glenwood girls cross country team placed eighth and boys ninth at the Class 3A State Qualifying Meet in Winterset on Thursday.
Perry's Atem Akok was the boys individual champion with a time of 16:24. Ballard's Abby Kohut-Jackson was the girls champion with a top time of 14:32.
The top 10 runners in each regional/district site qualify for this week's state cross country meet.
MALVERN - If "three yards and a cloud of dust" is as old of a football adage as it is passé in today's era of the spread offense then Friday night in Malvern must have looked like throwback night.
A-H-S-T and East Mills engaged in a good, old fashioned slug fest that featured a combined 93 rushing attempts as the Vikings rode the game's lone touchdown to a 10-0 victory over the Wolverines to stay perfect on the season and seal up the top spot in Class 1A, District 8.
For one game, the Fremont-Mills volleyball team showed defending nine-time champion Stanton it came to play in the Corner Conference championship match Thursday.
"The girls came out like gangbusters," said Fremont-Mills coach Tracy Malcom. "We went into this game driven. We say that before every game, and we were on as far as setting and hitting. We were sharp and polished. We were on target exactly where we wanted to be."
Fremont-Mills scored touchdowns on plays of 30, 33, 39, 43 and 68 yards as the Knights cruised to a 52-12 victory over Nishnabotna in Eight-Man, District 8 football Friday in Hamburg.
"This was a very up and down game for us," said Fremont-Mills coach Jeremy Christiansen. "We overwhelmed them at times but we also had a lot of breakdowns here and there."
Trevor Frain raced 55 yards for a touchdown on Creston's opening play from scrimmage and Glenwood never seemed to recover in a 49-21 Class 3A, District 1 loss on Friday.
Creston (5-2) sprinted out to a 28-0 lead before Glenwood got on the board just before halftime to make it 28-7 at the break.
Copyright © 2015
The Opinion Tribune is your source for local news, sports, events and information in the city of Glenwood and Mills County, Iowa.


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Style Watch PollFashion Faceoff
Sometimes, stylish star minds think alike. You decide: Who wears it better?
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The Mysterious Cities of Gold: Secret Paths
Published by Ynnis, Developed by -
Genres: Adventure (1 players)
US release date: Mar 6th, 2014 | EU release date: Feb 27th, 2014
Prices: $14.99 (US) | £13.49 (UK) | €14.99 (EUR)
A mystery worth puzzling over?
Review written by
April 16th, 2014
The Mysterious Cities of Gold: Secret Paths is a charming adventure game based off the French-Japanese animated series from 1982. Recently, the show received a second season, and this game follows the adventures of its three protagonists.
The opening animation shows the cast in their giant mechanical Condor flying above a vibrant and misty view of China, searching for the lost city of gold. Fortunately, the game does exactly that. With its colorful cel-shaded style, the art mimics the cartoon surprisingly well.
As you explore the maps, you are presented with obstacles that can be overcome by a specific skill that one of the protagonists possess. Esteban can call on the power of the sun to remotely access switches, Zia can fit through small areas the others cannot, Tao can decipher ancient Mu text for clues to puzzles, and use his pet bird Kokapetl to snag keys from guards unnoticed.
The great level design and interesting switch mechanism also introduces enemies and stealth to the gameplay. When the player enters the view of an enemy, a dotted line appears between them and it becomes a solid line the closer the two are. If the player breaks the line of sight by running away or jumping into a barrel, the guard will lose track and return to their patrol. While the penalty for being captured isn't that hindering, the player will lose time for being captured. Also, your score on each level is dependent on how few times you are captured, how many parchments you've collected, and how quickly you complete the stage within the time limit.
The stages themselves are well designed puzzles that lay out a series of even more puzzles for each hero, so it's a good idea to use the camera function to plan out your route with each character. However, you will sometimes need to plan quickly as some levels will leave you with fewer seconds to spare, leaving the player with the task of racing the clock while solving various puzzles.
Although the levels are well thought out, the same cannot be said for the puzzles they contain. Typically you will find the same puzzles throughout the game in different combinations, making them a little repetitive and simple. Most of the challenges come from completing these puzzles in the time limit while also acquiring hidden artwork and collecting every parchment.
The overall gameplay balances risk and reward, making for a challenging and fun experience for any age.
No posts yet for this game. The posts are in another castle.
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Michael Jordan said:
No they're making it from the movie ... Speed Racer: The Video Game
daveoneal said:
Thanks Rob. I had no idea this one was published. I'm very happy about it! Yes, Jonathan, it is hard as all get-out, ... Street Fighter 2010: The Final Fight review
daveoneal said:
This was a very well-written review. Apparently Super Hexagon was developed by Terry Cavanagh (who made vvvvvv), so ... Cosmophony review
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:p p ... TMNT: Smash Up
dirk said:
cool :p ... TMNT: Smash Up
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CORVALLIS, Ore. — Five million dollars is how much is at stake. Corvallis, one of 50 communities in the nation, is competing for a Georgetown University Energy Prize to continue promoting energy conservation. And if Corvallis were to win, residents in the community could also win some of the big prize money. Energize Corvallis, a … Continue reading »
OSU Named a Green University
CORVALLIS, Ore. – Oregon State University was named a green university for the fourth year in a row, Tuesday. OSU received 95 points out of a possible 99 as a Green School in the latest edition of The Princeton Review's Guide to 332 Green Colleges: 2014 Edition. The Princeton Review tallies its Green Rating scores … Continue reading »
Non-Profits Receive Greenpower Grants
EUGENE, Ore. — Saving energy is generating a lot of green for two local non-profits. The Eugene Hearing and Speech Center and the Relief Nursery, each won a 2014 Greenpower Grant from EWEB. The organizations were presented $50,000 this weekend. Both plan to use the money to install solar panel systems at their Eugene centers. … Continue reading »
Motorcyclist Dies in HWY 97 Crash
MADRAS, Ore. — Oregon State Police says the motorcyclist involved in a Highway 97 crash on August 8 has died. Troopers say Donald W. Heckathorn, 64, of Madras, died Wednesday from the injuries he received in a crash at the intersection of Highway 97 and Dover Lane south of Madras. OSP says around 3 p.m. … Continue reading »
Civil War Sitdown With Keanon Lowe
Duck wide receiver Keanon Lowe is one of a few players on the roster who hail from the state of Oregon. He's made an impact this year with 22 catches for 244 yards and three touchdowns. The Jesuit High School product sat down with our own Erik Elken to talk about what the Civil War … Continue reading »