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RESULTS
to established protocols (Bozon et al., 2003) (Figure 1A). because group-housed males that were intermittently
The same animals were tested sequentially at different separated for testing fought over time and had to be singly
time points throughout the course of infection, using dif- housed, excluding them from further testing. The differ-
ferent sets of objects at each time point. The percentage ence in exploratory preference ratios, or memory, for all
of time spent exploring the novel compared to the familiar time points after PrP depletion compared to performance
object was expressed as a ratio of exploratory preference at 8 wpi prior to depletion is highly significant: p < 0.00001.
and used as a measure of memory. Random exploration of ANOVA analysis of all data sets for group effects also re-
the two objects results in a ratio of 1; preferential explora- sulted in p < 0.0001. Uninfected mice examined at repre-
tion of the novel object compared to the other gives a ratio sentative time points retained exploratory capacity
greater than 1. Statistical significance of data was calcu- (Figure S2), ruling out the possibility that PrPC depletion it-
lated using parametric tests (Student’s t test; two-tailed) self might alter object-recognition memory.
after confirmation that data were normally distributed. The loss of preferential exploration of a novel object in
All mice tested showed random exploration of the tg37 mice was not due to prion-related changes in motor
equally unfamiliar objects during the learning phase of function. Indeed, we found that activity in the open field in-
the test, with a baseline exploratory ratio of 1 (see Fig- creased, rather than decreased, with disease progression
ure S1 in the Supplemental Data available online). At 7 up to 12 wpi in tg37 animals (Figure 1D) (p = 0.013 for
wpi, all prion-infected animals displayed normal novel- activity of tg37 mice at 12 wpi compared to 8 wpi, and
object discrimination during the test phase (Figure 1B), p = 0.015 compared to 10 wpi), but not in NFH-Cre/tg37
with mean exploratory preference ratios of 1.70 ± 0.19 mice or in uninfected animals (Figure S3). This is consis-
SEM (tg37 controls) and 1.81 ± 0.07 SEM (NFH-Cre/tg37 tent with previous observations in prion-infected mice of
mice). But at 8 wpi, both groups of infected mice showed several strains (Cunningham et al., 2005; Deacon et al.,
significant inability to discriminate a novel object during 2001; Dell’Omo et al., 2002; Guenther et al., 2001). Limb
the test phase, with mean exploratory preference ratios power, assessed by testing grip strength, was unaffected
of 1.05 6 0.05 SEM and 0.90 6 0.03 SEM, respectively, in all mice, and coordinated movement, assessed here by
for the novel object: i.e., no preference. These differences the ability of the mice to reorientate themselves on a verti-
in exploration at 7 wpi versus 8 wpi were significant for cal wire grid after inversion, was also maintained (data not
mice of both genotypes: p = 0.0001 for NFH-Cre/tg37 shown).
mice and p = 0.0498 for tg37 mice, and p = 0.0001 when Loss of PrP expression due to Cre-mediated recombi-
mice of both groups are pooled at 7 and 8 wpi and com- nation in the hippocampi of NFH-Cre/tg37 mice after
pared. The loss in novel-object discriminatory capacity 8 wpi (or 9 weeks of age for uninfected mice) was con-
coincided with the appearance of early spongiform firmed by immunohistochemistry and RT-PCR for PrP
change in all prion-infected mice (Figure 1C, center mRNA expression at representative time points (Figure 2).
panels) and with reduction of synaptic responses (see be-
low and Figures 5A–5C). In prion-diseased tg37 mice, this Burrowing Declines in Early Prion Infection
impairment of memory persisted throughout the course of We also tested the mice for changes in burrowing and
infection (Figure 1B). By 12 wpi, when they were beginning nesting previously described in early prion infection. In
to show early signs of prion disease (such as reduced the burrowing task, mice actively burrow in a container
grooming, but not overt motor signs), the exploratory pref- that is filled with objects, pushing or carrying them out
erence ratio was inverted at 0.64 ± 0.05 SEM (p = 0.0012 into the cage until the container is almost emptied
for exploration compared to 7 wpi), possibly reflecting (Figure 3A). We found that all mice burrowed actively, dis-
preference for the familiar object and neophobia (anxiety placing 70%–80% of the pellets in a 24 hr period at 6
induced by novel objects) at this stage in tg37 mice. (mean 78% ± 5 SEM) and 7 wpi (73% ± 4 SEM), respec-
tively (Figure 3B). Mice burrowed less from 8 wpi (to
PrP Depletion Reverses Early Cognitive Deficits a mean of 66% ± 6 for tg37 and 54% ± 7 for NFH-Cre/
In contrast to tg37 mice, NFH-Cre/tg37 mice recovered tg37 mice, falling to 42% ± 6 and 45% ± 6, respectively,
the ability to discriminate novel objects after neuronal at 9 wpi). The timing of onset of the impairment is consis-
PrPC depletion. The exploratory preference for the novel tent with observations in other prion-infected mouse
object returned at 9 wpi (mean ratio of 1.40 ± 0.05 SEM; strains (Cunningham et al., 2003, 2005; Deacon et al.,
Figure 1B), very soon after PrP depletion, (p = 0.0004 for 2001; Guenther et al., 2001). The decline was significant
NFH-Cre/tg37 mice at 9 wpi compared to 8 wpi). The dif- for burrowing at 8 and 9 wpi compared to activity at 6
ference between mice with and without PrP depletion at and 7 wpi for both tg37 mice and NFH-Cre/tg37 mice
9 wpi is also significant (p = 0.0451). This functional recov- (p = 0.001 in each case). In tg37 mice, burrowing con-
ery occurred in parallel with reversal of spongiform pathol- tinued to decline until the mice reached onset of clinical
ogy seen in NFH-Cre/tg37 mice examined histologically disease, when they displaced just 21%–28% of pellets.
over this period (Figure 1C, panels E and F). Further, the
recovery in memory was sustained up to 20+ wpi, with PrP Depletion Reverses Burrowing Deficits
a mean exploratory ratio of 1.67 ± 0.25 SEM. Numbers In contrast, in mice with Cre-mediated PrP knockout,
available for testing after 20 weeks were diminished there was recovery of burrowing behavior by 10 wpi,
which was significant when compared to control tg37 relatively later than burrowing behavior in the disease
mice at this time point (p = 0.004) and highly significant process, when Cre-mediated recombination and reversal
at 12 wpi (p = 0.0001). The increase in burrowing activity of early pathology has already occurred.
in mice 10–14 wpi (post-PrP depletion) compared with As motor impairment is not a factor here, these data
the reduction seen in mice at the earlier time points of support a motivational—rather than a motor—impairment
8 and 9 wpi (prior to/around time of PrP depletion) was as a basis for loss of burrowing activity, as proposed by
also significant (p = 0.001). Uninfected mice of both geno- Deacon et al. (Deacon et al., 2001).
types showed sustained burrowing activity when tested at
representative time points pre- and post-PrP depletion Behavioral Recovery Parallels Reversal of Pathology
(Figure S4). ANOVA analysis of all data to account for Histological examination of age-matched RML prion-
group effects confirmed significance of data; p < 0.0001. infected mice confirmed absence of detectable prion
The later recovery of burrowing (at 10 wpi) compared pathological change at 6 and 7 wpi, when all mice showed
to that of novel-object recognition (at 9 wpi) likely reflects normal learning, memory, and burrowing behavior (Fig-
the fact the animals were tested at the beginning of ures 1C, panels A and D and Figures 4A–4C and 4J–4L).
the week for burrowing and at the end for novel-object Loss of memory and burrowing correlated with the devel-
recognition, so that the actual difference in age at testing opment of spongiform change in neurons at 8 wpi (Fig-
is 2–3 days. ure 1C, panels B and E), and recovery of these behaviors
All mice constructed nests from nestlets up to 10 wpi, correlated with the reversal of spongiosis by 10 wpi in
when tg37 mice then stopped shredding their nestlets. mice with PrP depletion (Figure 1C, panel F). Reduced
Mice with Cre-mediated PrP depletion did not show any levels of synaptophysin 1 in the dorsal hippocampus
change in this behavior at any stage. This is probably have been correlated with reduced burrowing behavior
due to the fact that in this model nesting is affected in prion-infected mice (Cunningham et al., 2003). We
found marked loss of synaptophysin immunoreactivity infected tg37 and NFH-Cre/tg37 mice showed similar
throughout the hippocampus in tg37 mice from 10 wpi, significant reductions in evoked excitatory postsynaptic
most notable in regions CA1-3 (Figure 4, panel I). We field potentials (EPSPs) at 50% of values seen in both
also found reduction in total synaptophysin levels in scra- uninfected mice and in recovered NFH-Cre/tg37 animals
pie sick tg37 animals on Western blotting of hippocampal (Figures 5A–5C). Cre-mediated neuronal PrP depletion in
homogenates (Figures S5A), but we did not detect NFH-Cre/tg37 mice resulted in rapid recovery of EPSPs
reduced levels at earlier time points in correlation with to control levels, a recovery sustained in mice examined
behavioral change here (Figure S5B), suggesting that up to 18 wpi. In contrast, synaptic responses in the
these behavioral deficits occur before there is significant prion-infected tg37 mice continued to decline, and at
loss of synapses structurally. Importantly, normal levels 9 wpi were 22% of those of uninfected control mice. The
of synaptophysin immunoreactivity were maintained in stimulus-response curves differed significantly between
infected NFH-Cre/tg37 mice up to 40 wpi (Figure 4, panel groups at different time points (p < 0.001, general linear
U), suggesting that synaptic integrity and density remains model [GLM]; pair-wise differences described above as
intact long term despite the extensive accumulation of significant had p < 0.001 with Bonferroni correction for
non-neuronal PrPSc (Figure 4, panel T). multiple comparisons). Action potentials in the presynap-
tic axons give rise to ‘‘fiber volleys’’ (Winegar and MacIver,
CA1 Synaptic Responses Are Impaired but Rapidly 2006) that can be seen immediately after the stimulus
Recover after PrP Depletion artifact. We found that the fiber volleys’ amplitudes were
We found that behavioral changes were paralleled by neu- linearly related to the EPSPs (correlation coefficient 0.97;
rophysiological changes (Figure 5). We tested synaptic Figure 6A), suggesting that the change in EPSP was
responses and plasticity in the stratum radiatum of the directly due to a change in the presynaptic action poten-
CA1 region of hippocampal slices. At 8 wpi, both prion- tials, rather than at a synaptic level.
Further, both their occurrence and recovery are inde- in effective synaptic integration itself, implicating mecha-
pendent of PrPSc accumulation, with novel implications nisms other than CA1 LTP in deficient memory. Dissocia-
for both mechanisms of neurotoxicity and therapeutic tion of mechanisms underlying the acquisition and storage
possibilities. of information and the endpoint of such changes in
We found that relatively early in prion infection, by 8 wpi, expressed behavior have been reported elsewhere (Frag-
mice lost the capacity for novel-object recognition, a test kouli et al., 2005), and learning and memory have been
of nonspatial memory, and showed significantly reduced found associated with impaired synaptic responses and
burrowing behavior (Figures 1 and 3). These were not intact LTP in other rodent models (Brace et al., 1985).
due to impaired motor function (Figure 1D) and thus reflect However, LTP alterations have been described in prion-
impaired cognitive and motivational function. Further, PrP infected mice, of different mouse strains than those
depletion itself had no effect on behavior and cognition in used here, at a relatively later stage in the disease process
uninfected animals (Figures S1–S4). Neurophysiologically, and infected with different prion strains and using
we observed reduced synaptic responses in the CA1 more intense conditioning stimulation to induce LTP
region at the same time points (Figure 5). Remarkably, (Chiti et al., 2006).
by 9 wpi, soon after neuronal PrP depletion in NFH-Cre/ Both cognitive and neurophysiological impairment and
tg37 mice both synaptic and cognitive/behavioral deficits recovery in our model appeared to be independent of
recovered, and this recovery was sustained up to 30 wpi. PrPSc accumulation. Impaired object-recognition mem-
The development of phenotypic deficits thus paralleled ory, burrowing behavior, and reduced postsynaptic
the development of early spongiform change, and recov- potentials in CA1 occur at 8 wpi in both mouse lines,
ery of these deficits in mice with PrP depletion paralleled before extensive deposits of PrPSc are apparent. Further,
its reversal. We found no objective evidence of synapse both the loss and recovery of memory and species-typical
loss in the early stages of infection associated with early behaviors appear to be independent of aggregated PrPSc
cognitive failure (Figure 4 and Figure S5), in contrast to deposition, up to 20+ wpi, as is synaptic function up to
observations made by others in association with burrow- 18 wpi.
ing decline in prion infection (Cunningham et al., 2003), Indeed, early synaptic pathology and dendritic dysfunc-
which may reflect differences in the strains of mice and tion precede PrPSc accumulation (Jamieson et al., 2001),
of infectious prions used. The relationship between spon- consistent with our observations on behavioral and neuro-
giform change and the functional deficits we observed is physiological changes in the absence of extensive aggre-
not clear. Spongiform degeneration in prion disorders in- gated PrPSc deposits here.
volves the formation of membrane-bound vacuoles within Our data are also consistent with findings in other
neurons and neuropil—predominantly within dendrites— models of neurodegenerative disorders: that neuronal
and intradendritic distensions and dendritic spine loss dysfunction and cognitive defects occur before cellular
colocalize with vacuolar pathology in the hippocampus degeneration and independently of pathological aggrega-
(Jeffrey et al., 1997). The deficits we observe may thus tion of disease-associated proteins (Lambert et al., 1998;
reflect the earliest neuronal and dendritic dysfunction, Santacruz et al., 2005; Yamamoto et al., 2000), and they
although more detailed neurophysiological analysis impli- can result from synaptic dysfunction rather than neuronal
cates impaired function at the level of the presynaptic loss (Buttini et al., 2002; Mucke et al., 2000; Walsh et al.,
axon also. 2002; Wang et al., 2002). In several models of Alzheimer’s
Thus, we found that synaptic responses were de- disease there is increasing evidence that this may be due
pressed in our mouse model, which can be explained to soluble Ab oligomers rather than amyloid (Billings et al.,
by the proportional loss of the presynaptic fiber volley 2005; Dodart et al., 2002; Kotilinek et al., 2002), explaining
(Figure 6B): the correlation between reduced field synap- plaque-independent cognitive failure in these animals.
tic potentials and presynaptic fiber volleys is very strong at Consistent with these studies, our findings support the
0.97, strongly supporting a causal link (Figure 6A). Depres- concept that a transient—as yet unidentified—neurotoxic
sion of presynaptic fiber volleys has been reported late in species is generated within neurons when PrPC is con-
the progression of another scrapie model (Chiti et al., verted to PrPSc, which rapidly impairs neuronal function
2006), where it was tentatively attributed to loss of the and synaptic responses. Depleting neuronal PrPC would
presynaptic neurons (CA3 pyramidal cells). The present halt formation of such an intermediate, whether it be
loss of the fiber volley was rapidly reversible and therefore a new molecular species or an oligomeric form of PrPSc,
cannot be due to neuronal loss; more likely it was due to allowing rapid recovery of neuronal function.
axonal dysfunction. Targeting PrPC therapeutically remains somewhat con-
Synaptic plasticity appears to be maintained, however: troversial, however, as the physiological role of the native
the presence of LTP recordings that we found in all mice at protein is still unclear. While both embryonic (Bueler et al.,
all stages of disease implies that those synapses still 1992; Manson et al., 1994) and postnatal (Mallucci et al.,
remaining in prion-infected tg37 mice have the intact 2002) PrP knockout mice are viable and phenotypically
molecular apparatus both to release transmitter and to essentially normal, there is conflicting evidence for a role
sustain LTP, for review see Collingridge et al., 2004. It for PrPC in some learning tasks (Criado et al., 2005) but
appears that here memory is impaired due to reduction not others (Roesler et al., 1999). Our data suggest that at
least nonspatial memory tasks are independent of PrPC Inoculation with Prions
expression specifically, depending instead on neuronal in- One-week-old mice were anesthetized and inoculated intracerebrally
into the right parietal lobe with 20 ml of 1% brain homogenate of
tegrity and effective synaptic transmission. Also, we know
Chandler/RML mouse-passaged scrapie strain (RML, Rocky Mountain
that PrPC depletion does affect certain intrinsic properties Laboratories) and subsequently examined daily for signs of scrapie, as
of CA1 cells, causing reduction in the late afterhyperpola- described (Mallucci et al., 2003).
rization potential (AHP) (Colling et al., 1996; Mallucci et al.,
2002). However, the only known phenotypic correlate of Behavioral Testing
late AHP reduction in transgenic mice is loss of social All testing took place in a room with sombre lighting and constant
transference of food preference between mice, which background noise. Mice were handled for 10 min a day for several
days and habituated to the test environment prior to testing. Only
can be reversed by environmental enrichment (Giese
group-housed males were used. Groups of eight to ten mice were
et al., 1998). tested sequentially over time for each paradigm, unless otherwise
Recent work implicates PrP expression in neurogenesis stated. Mice were handled, habituated, trained, and tested at the
and in the the proliferation of multipotent neuronal precur- same time for each experiment.
sor cells in the dentate gyrus in vivo. Despite having
reduced numbers of proliferating precursor cells com- Novel Object Recognition Task
This was performed as described (Bozon et al., 2003). Briefly, mice
pared to wild-type or PrP-overexpressing animals, mice
were tested in a dark cylindrical arena (69 cm diameter) mounted
constitutively lacking in PrP nonetheless have equal final with a 96 LED cluster infra-red light source and Watec video camera
numbers of neurons in the dentate gyrus as PrP-express- (both Track-sys). Various plastic objects were constructed from inter-
ing mice (Steele et al., 2006). Consistent with these data, locking plastic building blocks and were used in all experiments. (Pilot
we also found that numbers of granule cell neurons in studies confirmed all objects were of equal inherent interest.) On day 1
the dentate gyrus were unaltered after PrP depletion in in- of each experiment (learning phase), two objects were placed in the
center of two 15 cm diameter circles inside the arena. Each mouse
fected and uninfected NFH-Cre/tg37 mice (Figure S7).
was placed in the arena for two blocks of 10 min for exploration of
Overall, we conclude that the dramatic benefits to neuro-
the objects with an intertrial interval of 5 min. Forty-eight hours later,
nal function and survival in prion-infected mice we have a novel object was randomly exchanged for one of the familiar ones,
shown here support targeting neuronal PrPC directly as and retention was tested by placing the mice back in the arena for
a therapeutic approach. a 5 min session (test phase). The amount of time spent exploring all
Our findings of early reversible neurophysiological and objects was measured for each animal with the examiner blind to
cognitive deficits occurring prior to neuronal loss open genotype and time point for the animals. All objects and arena were
cleansed thoroughly between trials to ensure the absence of olfactory
new avenues in the prion field. To date, prion infection in
cues. Criteria for exploration were based strictly on active exploration,
mice has conventionally been diagnosed when motor where mice had at least both forelimbs within a circle of 15 cm around
deficits reflect advanced neurodegeneration. Now the an object, head oriented toward it or touching it with their noses. Mice
identification of earlier dysfunction helps direct the study with overt motor symptoms were not used. New sets of objects were
of mechanisms of neurotoxicity and therapies to earlier used at each time point.
stages of disease, when rescue is still possible. Eventually
Burrowing and Nesting
it may also enable preclinical testing of therapeutic strate-
Two hours before the start of the dark period, mice (which had not
gies through cognitive endpoints. These data now lead to been food deprived) were placed in individual plastic cages containing
the hope that early intervention in human prion disease, a gray plastic tube 20 cm long 3 6.8 cm diameter, filled with 200 g of
will not only halt clinical progression but allow reversal of normal food pellets as described (Cunningham et al., 2003). The weight
early behavioral and cognitive abnormalities. of pellets remaining in the tube after 24 hr was measured, and the
percentage displaced (burrowed) was calculated. A pressed cotton
square (‘‘Nestlet’’) was also placed in each cage, and the occurrence
of nest building after 24 hr was observed.
EXPERIMENTAL PROCEDURES
Open-Field Activity
Animals Each mouse was placed in the corner of an empty, clear plastic arena
tg37 and NFH-Cre transgenic mice (Mallucci et al., 2002) were bred in- (50 3 32 3 20cm) divided into 10 3 10 cm squares and observed for
house and were housed in a temperature- and light-controlled mouse 3 min. The number of squares entered (all four paws inside) and num-
colony room (12 hr light/dark cycle) in groups of four to six. All mice had ber of rears was recorded.
free access to food and water. All cages contained Perspex boxes
(‘‘igloos’’), cylindrical cardboard tubes, and folded sheets of tissue Inverted Screen Test of Muscular Strength and Reorientation
paper in view of the positive effects of such enrichment on cognition Each mouse was placed onto a horizontal wire mesh of 12 mm squares
in rodents. All animal work conformed to the United Kingdom regula- made from 2 mm diameter wire. The mesh was slowly inverted, and the
tions and institutional guidelines and was performed under Home time for the mouse to fall off was measured up to 1 min. For orientation,
Office project license. Generation and characterization of these the mouse was first placed facing upward on the vertical mesh, which
mice, including onset and timing of recombination and genotyping was then rotated through 180 in the vertical plane so that the mouse
by PCR screening of DNA from tail biopsy samples are described faced the floor. The time taken for the mouse to reorientate itself and
(Mallucci et al., 2002). The mice were generated on a Prnp0/0 back- return to its original position was recorded.
ground so that all PrP expression is from the PrP (MloxP) transgene.
The genetic background is predominantly FVB after ten generations Electrophysiology
of backcrossing. All animals are hemizygous for one or both trans- Mice were anesthetized by intraperitoneal injection of a mixture of
genes (MloxP alone or also with NFH-Cre). medetomide (1 mg/kg) and ketamine (76 mg/kg) prior to being killed
by cervical dislocation, their brains removed for preparation of 400 mm Business-stat/otherapplets/Normality.htm). Student’s t tests were
horizontal slices of ventral hippocampus, using a remote control Vibro- applied to all data sets with two tails (two samples; unequal var-
slice (Campden Instruments, Loughborough, UK) housed in a HEPA- iance). ANOVA testing was performed using one-way analysis for
filtered thin-film isolator to contain contaminated aerosols. Slices group effects (http://www.physics.csbsju.edu/stats/anova.html). Mann
were kept in an interface holding chamber in the isolator until trans- Whitney U tests (Wilcoxon rank sum tests) were performed where
ferred to an interface chamber for recording. ACSF comprised (in data was not normally distributed; notably for motor testing. Analysis
mM) 135 NaCl, 16 NaHCO3, 1.25 NaH2PO4, 3 KCl, 2 CaCl2, 1 MgCl, of neurophysiological data were performed using general linear model
and 10 glucose, pH 7.4, gassed with 95% O2, 5% CO2, maintained (GLM) in SPSS version 13.
at 32 C, and directed to a container containing 2 M NaOH after single
passage through the chamber.
Supplemental Data
Stimuli were applied through a bipolar stimulating electrode placed
The Supplemental Data for this article, including seven figures and
in the stratum radiatum approximately 150 mm from the CA1 pyramidal
Supplemental Experimental Procedures, can be found online at
cell layer. Extracellular recordings of EPSPs were made from the same
http://www.neuron.org/cgi/content/full/53/3/325/DC1/.
layer of stratum radiatum, 500 mm away, using glass microelec-
trodes, an Axoclamp 2B amplifier, and a 1401plus signal acquisition
system running Signal version 2.15 (CED, Cambridge, UK). Stimulus ACKNOWLEDGMENTS
response curves used a fixed set of stimulus strengths, and the field
EPSPs were measured as the initial slope (between 20%–80% of We thank Julie Underwood, Michelle Hainsworth, and Jackie Linehan
maximum). LTP was induced by a high-intensity theta-burst protocol for technical assistance; Ray Young for help with graphics; Sam Cooke
(Morgan and Teyler, 2001) comprising five trains of four pulses at and Tim Bliss (National Institute of Medical Research, Mill Hill) and
100 Hz separated by 200 ms, repeated six times with an interburst in- Colm Cunningham (University of Southampton) for advice and guid-
terval of 10 s, delivered after at least 30 min of recording half-maximal ance on behavioral testing. This work was funded by the Medical Re-
stimuli delivered every 30 s. The same test stimuli were then delivered search Council of Great Britain. The neurophysiological experiments
for a further 45 min. were funded by a Physiological Society summer studentship (H.K.)
and the Biotechnology and Biological Sciences Research Council
Neuropathological Examination of Brain Sections (A.D.P.).
PrPSc in brain tissue was detected after denaturation of fixed sections
in formic acid (Hill et al., 1997) using monoclonal antibody to PrP, ICSM Received: September 28, 2006
35 (Asante et al., 2002), and an automated immunostaining system Revised: December 11, 2006
(www.ventanamed.com) as described (Joiner et al., 2002). Astrocyto- Accepted: January 5, 2007
sis was detected using anti-GFAP polyclonal antiserum (Dako; 1:500 Published: January 31, 2007
dilution), and synaptophysin was detected with polyclonal rabbit anti-
serum (Zymed; prediluted) using the same system. Neu N staining was REFERENCES
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