Targeting Cellular Prion Protein Reverses Early
Cognitive Deficits and Neurophysiological
Dysfunction in Prion-Infected Mice
Giovanna R. Mallucci,1,* Melanie D. White,1Michael Farmer,1Andrew Dickinson,1Husna Khatun,2
Andrew D. Powell,2Sebastian Brandner,1John G.R. Jefferys,2and John Collinge1
1MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG,
2Department of Neurophysiology, Division of Neuroscience, University of Birmingham, Birmingham, B15 2TT, United Kingdom
Currently, no treatment can prevent the cogni-
tive and motor decline associated with wide-
spread neurodegeneration in prion disease.
However, we previously showed that targeting
endogenous neuronal prion protein (PrPC) (the
precursor of its disease-associated isoform,
PrPSc) in mice with early prion infection re-
versed spongiform change and prevented clin-
that cognitive and behavioral deficits and im-
paired neurophysiological function accompany
early hippocampal spongiform pathology. Re-
markably, these behavioral and synaptic im-
pairments recover when neuronal PrPCis de-
pleted, in parallel with reversal of spongiosis.
Thus, early functional impairments precede
neuronal loss in prion disease and can be res-
cued. Further, they occur before extensive
PrPScdeposits accumulate and recover rapidly
after PrPCdepletion, supporting the concept
that they are caused by a transient neurotoxic
species, distinct from aggregated PrPSc. These
data suggest that early intervention in human
prion disease may lead to recovery of cognitive
and behavioral symptoms.
Changes in motivation, mood, and behavior are common
early symptoms in human prion disorders, especially var-
iant Creutzfeldt-Jakob disease (vCJD), often occurring
long before diagnosis is made. By the time characteristic
dementia and motor deficits are established, there is typ-
ically advanced neuronal loss, with no realistic potential
for curative treatment or recovery. In experimentally in-
fected mice, clinicaldisease isdiagnosed bythe presence
of locomotor changes, but again, these occur during
nal loss. However, there are earlier pathological and phe-
notypic changes. Spongiform change and synapse loss
precede neuronal loss (Jeffrey et al., 2000), and changes
in species-typical behaviors also occur long before typical
motor symptoms (Betmouni et al., 1999; Cunningham
et al., 2005; Deacon et al., 2001; Guenther et al., 2001)
and correlate with early loss of presynaptic terminals in
the dorsal hippocampus (Cunningham et al., 2003).
We previously showed that early spongiform degenera-
tion in the hippocampus of prion-infected mice reverses
when PrPCis depleted in neurons (Mallucci et al., 2003).
We now ask whether this early pathological change pro-
duces functional deficits and whether its reversal is
reflected in functional recovery. As the hippocampus is
particularly targeted by several strains of murine prions
and is easily accessible for neurophysiological measure-
ments, we focused on tests of hippocampal function.
We used prion-infected transgenic mice with and without
‘‘induced’’ PrP depletion for our experiments. Thus, tg37
mice express PrP from ‘‘floxed’’ PrP sequences (MloxP
transgenes) and succumb to Rocky Mountain Laboratory
(RML) prion infection ?13 weeks postinoculation (wpi)
(Mallucci et al., 2002). In these mice, earliest prion patho-
logical changes, including spongiosis, gliosis, and PrPSc
deposition, appear by 8 wpi (Mallucci et al., 2003). In
double-transgenic NFH-Cre/tg37 mice, PrP expression
is the same as in tg37 mice until floxed PrP sequences
are excised by the DNA recombinase, Cre (Sauer and
Henderson, 1989), at ?9–10 weeks of age, when neuronal
PrP is depleted (Mallucci et al., 2002). NFH-Cre/tg37 mice
infected with prions at 1 week of age develop early hippo-
campal pathology in parallel with control tg37 mice, but
spongiosis reverses soon after Cre-mediated PrP deple-
tion ?8–9 wpi, and the animals survive long term (Mallucci
et al., 2003).
We tested memory function and spontaneous etholog-
ical behaviors in vivo and measured synaptic responses
neurophysiologically in vitro. We used the novel object
recognition task, a nonspatial learning task based on the
spontaneous preference of both mice and rats for novelty
and their ability to remember previously encountered
Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc. 325
objects, that is rapidly learned (Clark et al., 2000; Dodart
et al., 1997; Ennaceur and Delacour, 1988; Messier,
1997). In rodents, the hippocampus is thought to be in-
volved in delay-dependent object-recognition memory
ing phase), whereas recall over shorter retention intervals
is thought to involve parahippocampal structures (Clark
et al., 2000; Hammond et al., 2004; Mumby et al., 2005).
Novel-object recognition is extensively used for testing
declarative memory in mice, and, critically, performance
is independent of mouse strain or genetic background,
unlike most other memory tasks for which performance
is highly strain dependent (Sik et al., 2003). We also tested
the spontaneous behaviors of burrowing and nesting,
(Betmouni et al., 1999; Cunningham et al., 2005; Deacon
et al., 2001; Guenther et al., 2001) and also localize to
the dorsal hippocampus. These behaviors have been pro-
posed as powerful tools for elucidating brain function
nization and executive function, and are thought to reflect
motivational aspects of spontaneous behavior in rodents.
at 1 week of age and tested groups of eight to ten mice of
each genotype at each time point behaviorally and neuro-
physiologically from 7 wpi. Tg37 mice were tested up to
?12 wpi, after which they developed clinical signs of prion
disease and were culled. NFH-Cre/tg37 mice were tested
up to ?30 wpi. Groups of six mice infected in parallel were
tion. Equal numbers of uninfected mice were tested at
three representative time points (pre-, during-, and post-
PrP depletion) to exclude unforeseen effects of loss of
PrP expression itself.
Object Memory Is Impaired Early in Prion Infection
We first assessed animals in the novel object recognition
task (Figure 1). We used male mice, as they are generally
considered superior to females at localizing and recogniz-
intensive training in an arena containing two objects that
they could explore freely (learning phase). Forty-eight
hours later, they were re-exposed to two objects, one of
which was familiar and one new (test phase) according
Figure 1. The Ability to Discriminate Novel Objects Is Lost in
Prion-Infected Mice but Recovers when Neuronal PrPCIs
Depleted, in Parallel with Reversal of Pathological Change
(A) Mice are allowed to explore two objects in an arena during the
learning phase of the test; then, after a retention interval, they are ex-
posed to a novel object (test phase). Time spent actively exploring the
novel object compared to the familiar one is a measure of object-
recognition memory and is expressed as the ratio of exploratory pref-
bars) showed normal object-recognition memory at 7 wpi, with prefer-
ential exploration of the novel object. This was significantly impaired in
all mice by 8 wpi, but recovered in mice with Cre-mediated PrP deple-
tion at 9 wpi. Recovery was sustained up to 30 wpi. (C) Hematoxylin
and eosin-stained sections of hippocampi from prion-infected tg37
and NFH-Cre/tg37 mice have normal appearance at 6 wpi (panels
[A] and [D]). Spongiosis develops in all animals (panels [B] and [E]) by
8 wpi when memory is impaired, but by 10 wpi this has reversed in
mice with Cre-mediated depletion (panel [F]), in parallel with recovery
of novel-object memory. (D) Reducedexploration of novel object is not
due to prion-induced reduction in motor activity. Mice were tested for
activity in the open field by measuring the number of squares crossed
in an arena in 3 min. There was no decline in activity, but at 12 wpi tg37
mice were significantly more active than NFH-Cre/tg37 mice, consis-
tent with previous findings in prion infection. n = 8–10 mice for all
groups except at 8, 9, and 10 wpi when two groups of 8–10 mice
each were tested. N = 7 at 12 wpi for tg37, and at 20–30 wpi for
NFH-Cre/tg37 mice. Error bars represent standard error of the mean
(SEM). Dashed line representsrandom exploration (exploratory prefer-
ence = 1). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test; two tails).
Open arrow indicates onset of earliest signs of scrapie in tg37 mice
(ss). Closed arrow indicates onset of Cre-mediated knockout. Scale
bar represents 160 mm.
326 Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc.
Reversal of Prion-Induced Cognitive Defects
to established protocols (Bozon et al., 2003) (Figure 1A).
The same animals were tested sequentially at different
time points throughout the course of infection, using dif-
ferent sets of objects at each time point. The percentage
of time spent exploring the novel compared to the familiar
object was expressed as a ratio of exploratory preference
the two objects results in a ratio of 1; preferential explora-
tion of the novel object compared to the other gives a ratio
greater than 1. Statistical significance of data was calcu-
lated using parametric tests (Student’s t test; two-tailed)
after confirmation that data were normally distributed.
All mice tested showed random exploration of the
equally unfamiliar objects during the learning phase of
the test, with a baseline exploratory ratio of ?1 (see Fig-
ure S1 in the Supplemental Data available online). At 7
wpi, all prion-infected animals displayed normal novel-
object discrimination during the test phase (Figure 1B),
with mean exploratory preference ratios of 1.70 ± 0.19
SEM (tg37 controls) and 1.81 ± 0.07 SEM (NFH-Cre/tg37
mice). But at 8 wpi, both groups of infected mice showed
significant inability to discriminate a novel object during
the test phase, with mean exploratory preference ratios
of 1.05 6 0.05 SEM and 0.90 6 0.03 SEM, respectively,
for the novel object: i.e., no preference. These differences
in exploration at 7 wpi versus 8 wpi were significant for
mice of both genotypes: p = 0.0001 for NFH-Cre/tg37
mice and p = 0.0498 for tg37 mice, and p = 0.0001 when
mice of both groups are pooled at 7 and 8 wpi and com-
pared. The loss in novel-object discriminatory capacity
coincided with the appearance of early spongiform
change in all prion-infected mice (Figure 1C, center
panels) and with reduction of synaptic responses (see be-
low and Figures 5A–5C). In prion-diseased tg37 mice, this
impairment of memory persisted throughout the course of
infection(Figure1B).By12wpi, whentheywere beginning
to show early signs of prion disease (such as reduced
grooming, but not overtmotor signs), the exploratory pref-
erence ratio was inverted at 0.64 ± 0.05 SEM (p = 0.0012
for exploration compared to 7 wpi), possibly reflecting
preference for the familiar object and neophobia (anxiety
induced by novel objects) at this stage in tg37 mice.
PrP Depletion Reverses Early Cognitive Deficits
In contrast to tg37 mice, NFH-Cre/tg37 mice recovered
the ability to discriminate novel objects after neuronal
PrPCdepletion. The exploratory preference for the novel
object returned at 9 wpi (mean ratio of 1.40 ± 0.05 SEM;
Figure 1B), very soon after PrP depletion, (p = 0.0004 for
NFH-Cre/tg37 mice at 9 wpi compared to 8 wpi). The dif-
ference between mice with and without PrP depletion at
9 wpi is also significant (p = 0.0451). This functional recov-
eryoccurredin parallel withreversal of spongiform pathol-
ogy seen in NFH-Cre/tg37 mice examined histologically
over this period (Figure 1C, panels E and F). Further, the
recovery in memory was sustained up to 20+ wpi, with
a mean exploratory ratio of 1.67 ± 0.25 SEM. Numbers
available for testing after 20 weeks were diminished
because group-housed males that were intermittently
separated for testingfought over time and had to be singly
housed, excluding them from further testing. The differ-
ence in exploratory preference ratios, or memory, for all
time points after PrP depletion compared to performance
ANOVA analysis of all data sets for group effects also re-
sulted in p < 0.0001. Uninfected mice examined at repre-
sentative time points retained exploratory capacity
(Figure S2), ruling out the possibility that PrPCdepletion it-
self might alter object-recognition memory.
The loss of preferential exploration of a novel object in
tg37 mice was not due to prion-related changes in motor
function. Indeed, wefoundthatactivityin the openfieldin-
creased, rather than decreased, with disease progression
up to 12 wpi in tg37 animals (Figure 1D) (p = 0.013 for
activity of tg37 mice at 12 wpi compared to 8 wpi, and
p = 0.015 compared to 10 wpi), but not in NFH-Cre/tg37
mice or in uninfected animals (Figure S3). This is consis-
tent with previous observations in prion-infected mice of
several strains (Cunningham et al., 2005; Deacon et al.,
2001; Dell’Omo et al., 2002; Guenther et al., 2001). Limb
power, assessed by testing grip strength, was unaffected
in all mice, and coordinated movement, assessed here by
the ability of the mice to reorientate themselves on a verti-
cal wire grid after inversion, was also maintained (data not
Loss of PrP expression due to Cre-mediated recombi-
nation in the hippocampi of NFH-Cre/tg37 mice after
8 wpi (or 9 weeks of age for uninfected mice) was con-
firmed by immunohistochemistry and RT-PCR for PrP
mRNA expression at representative time points (Figure 2).
Burrowing Declines in Early Prion Infection
We also tested the mice for changes in burrowing and
nesting previously described in early prion infection. In
the burrowing task, mice actively burrow in a container
that is filled with objects, pushing or carrying them out
into the cage until the container is almost emptied
(Figure 3A). We found that all mice burrowed actively, dis-
placing 70%–80% of the pellets in a 24 hr period at 6
(mean 78% ± 5 SEM) and 7 wpi (73% ± 4 SEM), respec-
tively (Figure 3B). Mice burrowed less from 8 wpi (to
a mean of 66% ± 6 for tg37 and 54% ± 7 for NFH-Cre/
tg37 mice, falling to 42% ± 6 and 45% ± 6, respectively,
at 9 wpi). The timing of onset of the impairment is consis-
tent with observations in other prion-infected mouse
strains (Cunningham et al., 2003, 2005; Deacon et al.,
2001; Guenther et al., 2001). The decline was significant
for burrowing at 8 and 9 wpi compared to activity at 6
and 7 wpi for both tg37 mice and NFH-Cre/tg37 mice
(p = 0.001 in each case). In tg37 mice, burrowing con-
tinued to decline until the mice reached onset of clinical
disease, when they displaced just 21%–28% of pellets.
PrP Depletion Reverses Burrowing Deficits
In contrast, in mice with Cre-mediated PrP knockout,
there was recovery of burrowing behavior by 10 wpi,
Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc. 327
Reversal of Prion-Induced Cognitive Defects
which was significant when compared to control tg37
mice at this time point (p = 0.004) and highly significant
at 12 wpi (p = 0.0001). The increase in burrowing activity
in mice 10–14 wpi (post-PrP depletion) compared with
the reduction seen in mice at the earlier time points of
8 and 9 wpi (prior to/around time of PrP depletion) was
also significant (p = 0.001). Uninfected mice of both geno-
types showed sustained burrowing activity when tested at
representative time points pre- and post-PrP depletion
(Figure S4). ANOVA analysis of all data to account for
group effects confirmed significance of data; p < 0.0001.
The later recovery of burrowing (at 10 wpi) compared
to that of novel-object recognition (at 9 wpi) likely reflects
the fact the animals were tested at the beginning of
the week for burrowing and at the end for novel-object
recognition, so that the actual difference in age at testing
is ?2–3 days.
All mice constructed nests from nestlets up to 10 wpi,
when tg37 mice then stopped shredding their nestlets.
Mice with Cre-mediated PrP depletion did not show any
change in this behavior at any stage. This is probably
due to the fact that in this model nesting is affected
relatively later than burrowing behavior in the disease
process, when Cre-mediated recombination and reversal
of early pathology has already occurred.
As motor impairment is not a factor here, these data
support a motivational—rather than a motor—impairment
as a basis for loss of burrowing activity, as proposed by
Deacon et al. (Deacon et al., 2001).
Behavioral Recovery Parallels Reversal of Pathology
Histological examination of age-matched RML prion-
infected mice confirmed absence of detectable prion
pathological change at 6 and 7 wpi, when all mice showed
normal learning, memory, and burrowing behavior (Fig-
ures 1C, panels A and D and Figures 4A–4C and 4J–4L).
Loss of memory and burrowing correlated with the devel-
opment of spongiform change in neurons at 8 wpi (Fig-
ure 1C, panels B and E), and recovery of these behaviors
correlated with the reversal of spongiosis by 10 wpi in
mice with PrP depletion (Figure 1C, panel F). Reduced
levels of synaptophysin 1 in the dorsal hippocampus
have been correlated with reduced burrowing behavior
in prion-infected mice (Cunningham et al., 2003). We
Figure 2. Cre-Mediated PrP Depletion Occurs after 8 wpi or
9 Weeks of Age in NFH-Cre/tg37 Mice
(A) Immunohistochemical detection of PrP using ICSM 35 antibody
shows loss of PrP expression after 8 wpi (panels [b] and [c]). Box indi-
cates CA1 region where neurophysiological recordings were made. (B)
RT-PCR confirms reduction of PrP mRNA in hippocampal mRNA
extracts from NFH-Cre/tg37 mice after 8 wpi (or 9 weeks of age in
uninfected mice). Total transcript is reduced by ?30%–40%, consis-
tent with its absence within neurons in the hippocampus at this
stage. The reduction is equivalent to the fraction of recombined
DNA detected by Southern blotting previously described (Mallucci
et al., 2002, 2003). RNA was pooled from five mice at each time
point, and each reaction was performed in triplicate. Error bars repre-
sent standard deviation. Arrow indicates onset of Cre-mediated
Figure 3. Burrowing Behavior Declines Early in Prion Infec-
tion but Recovers in Mice following Neuronal PrP Depletion
(A) Healthy rodents ‘‘burrow’’ food pellets, carrying them out of a con-
tainer and into their cage. (B) Early in prion infection, all mice burrowed
equally. Burrowing then declined significantly in both tg37 and
NFH-Cre/tg37 mice, respectively, at 9 wpi and continued to decline
in tg37 mice. In mice with Cre-mediated PrP depletion, burrowing
behavior resumed by 10 wpi and remained normal up to 20–30 wpi.
Numbers and symbols are as for Figure 1 legend.
328 Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc.
Reversal of Prion-Induced Cognitive Defects
found marked loss of synaptophysin immunoreactivity
throughout the hippocampus in tg37 mice from 10 wpi,
most notable in regions CA1-3 (Figure 4, panel I). We
also found reduction in total synaptophysin levels in scra-
pie sick tg37 animals on Western blotting of hippocampal
homogenates (Figures S5A), but we did not detect
reduced levels at earlier time points in correlation with
behavioral change here (Figure S5B), suggesting that
these behavioral deficits occur before there is significant
loss of synapses structurally. Importantly, normal levels
of synaptophysin immunoreactivity were maintained in
infected NFH-Cre/tg37 mice up to 40 wpi (Figure 4, panel
U), suggesting that synaptic integrity and density remains
intact long term despite the extensive accumulation of
non-neuronal PrPSc(Figure 4, panel T).
CA1 Synaptic Responses Are Impaired but Rapidly
Recover after PrP Depletion
Wefound thatbehavioral changes were paralleled byneu-
rophysiological changes (Figure 5). We tested synaptic
responses and plasticity in the stratum radiatum of the
CA1 region of hippocampal slices. At 8 wpi, both prion-
infected tg37 and NFH-Cre/tg37 mice showed similar
significant reductions in evoked excitatory postsynaptic
field potentials (EPSPs) at ?50% of values seen in both
uninfected mice and in recovered NFH-Cre/tg37 animals
(Figures 5A–5C). Cre-mediated neuronal PrP depletion in
NFH-Cre/tg37 mice resulted in rapid recovery of EPSPs
to control levels, a recovery sustained in mice examined
up to 18 wpi. In contrast, synaptic responses in the
prion-infected tg37 mice continued to decline, and at
9 wpi were 22% of those of uninfected control mice. The
stimulus-response curves differed significantly between
groups at different time points (p < 0.001, general linear
model [GLM]; pair-wise differences described above as
significant had p < 0.001 with Bonferroni correction for
multiple comparisons). Action potentials in the presynap-
tic axons giverise to ‘‘fiber volleys’’(Winegarand MacIver,
2006) that can be seen immediately after the stimulus
artifact. We found that the fiber volleys’ amplitudes were
linearly related to the EPSPs (correlation coefficient 0.97;
Figure 6A), suggesting that the change in EPSP was
directly due to a change in the presynaptic action poten-
tials, rather than at a synaptic level.
Figure 4. Prion-Infected Mice with Neu-
ronal PrP Depletion Have Preserved
Synaptic Density in the Hippocampus
Hippocampal sections of prion-infected mice
were stained for synaptophysin, PrPSc, and
glial fibrillary acidic protein (GFAP) immunore-
activity. There are no pathological signs of
prion infection in any mice at 6 wpi when learn-
ing and behavior are normal. By 10 wpi, tg37
mice have marked loss of synaptophysin
immunoreactivity in the dorsal hippocampus,
PrPScaccumulation, and gliosis, but mice
with Cre-mediated PrP depletion have pre-
served synaptic density despite accumulating
extraneuronal PrPScand gliosis up to 40 wpi.
Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc. 329
Reversal of Prion-Induced Cognitive Defects
We also measured long-term potentiation (LTP) (Fig-
ure 6C and Figure S6), a form of synaptic plasticity
linked with some forms of learning and memory. In all
mice, LTP was sustained for at least 30 min despite differ-
ences in baseline (half-maximal) EPSPs between the ex-
perimental groups and the particularly low baseline values
for tg37 mice at 9 wpi. As the EPSPs are expressed as
a percentage of the mean preconditioning control values;
the apparently greater LTP in the 9 wpi tg37 mice
(Figure 6C) may be a result of this normalization (see
also Figure S6). Overall, we found no reduction in LTP in
prion-infected mice with or without PrP depletion.
Our combined behavioral and neurophysiological analy-
ses provide the first direct evidence (to our knowledge)
for early neuronal dysfunction producing functional cogni-
hippocampal pathology in prion-infected mice is associ-
ated with impaired synaptic responses and with cognitive
and behavioral deficits in hippocampal tasks. These defi-
cits precede neuronal and synaptic loss, and they are rap-
idly reversed when PrPCis depleted, demonstrating the
potential for recovery of neuronal function at this stage.
Figure 5. Synaptic Responses Are Depressed in Prion-
Infected Mice but Recover when Neuronal PrPCIs Depleted
(A) Field excitatory postsynaptic potential (EPSP) slope input-output
curves show marked differences between the experimental groups.
At 8 wpi, both tg37 (blue squares) and NFH-Cre/tg37 mice (red
squares) have smaller synaptic responses than age-matched uninoc-
ulated control mice of both genotypes (gray diamonds). In prion-
infected tg37 mice at 9 wpi (blue circles), synaptic responses are fur-
ther reduced. However, after Cre-mediated PrP depletion, synaptic
responses return to control levels in NFH-Cre/tg37 mice at 9 wpi (red
circles) and are sustained at this level up to 18 wpi (red triangles).
p < 0.001, general linear model analysis (GLM). (B) Means for the
experimental groups in the GLM analysis reveal significantly smaller
EPSPs in scrapie-infected tg37 mice (blue bars) than in both NFH-
Cre/tg37 mice (red bars) after PrP depletion at 9 wpi and uninfected
control mice (gray bar). p < 0.001, pairwise comparisons, using
the Bonferroni correction for multiple comparisons; dotted line repre-
sents mean EPSP value and dashed lines represent 95% confidence
intervals of the control group. Error bars represent SEM. (C) Represen-
tative sample traces for each group. The amplitude of response in
uninfected control mice is shown at the top (gray diamond). This
amplitude is reduced at 8 wpi in tg37 and NFH-Cre/tg37 mice (blue
and red squares, respectively) and is further diminished in tg37 mice
at 9 wpi (blue circles). The response recovers in mice with PrP deple-
tion at 9 wpi (red circle), which is sustained in mice up to 18 wpi (red
Figure 6. Deficits in Synaptic Responses Correlate with
Reduction in the Presynaptic Axonal Fiber Volley, but Synap-
tic Plasticity Is Preserved in Prion-Infected Mice, and They
Display Normal Long-Term Potentiation
(A) The relationship between EPSP slope and fiber volley (FV) ampli-
tude is unaltered during scrapie infectivity (linear regression, solid
line, R2= 0.97). (B) Representative fiber volleys for each group (indi-
cated by arrows) are shown in the sample traces of the synaptic
responses. For clarity, the stimulus artifact was removed as indicated
by the break in the traces. The amplitude of response in uninfected
control mice is shown at the top (gray diamond). This amplitude is
reduced at 8 wpi in tg37 and NFH-Cre/tg37 mice (blue and red
squares, respectively) and is further diminished in tg37 mice at 9 wpi
(blue circles). The response recovers in mice with PrP depletion at
9 wpi (red circle), which is sustained in mice up to 18 wpi (red triangle).
(C) Plot of mean population synaptic potential (PSP) slope versus time
nificant potentiation of EPSP. A theta-burst protocol was applied at
time 0 (see Experimental Procedures for details) and resulted in signif-
icant potentiation of the PSP slope. Symbols represent mean PSP, er-
ror bars represent SEM. n = 7–12 slices from at least four animals per
group, except for 14 wpi and 18 wpi mice, for which data were pooled,
thus n = 10 animals for this group.
330 Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc.
Reversal of Prion-Induced Cognitive Defects
Further, both their occurrence and recovery are inde-
pendent of PrPScaccumulation, with novel implications
for both mechanisms of neurotoxicity and therapeutic
Wefoundthatrelatively early in prioninfection, by8wpi,
mice lost the capacity for novel-object recognition, a test
of nonspatial memory, and showed significantly reduced
burrowing behavior (Figures 1 and 3). These were not
impaired cognitive and motivational function. Further, PrP
depletion itself had no effect on behavior and cognition in
we observed reduced synaptic responses in the CA1
region at the same time points (Figure 5). Remarkably,
by 9 wpi, soon after neuronal PrP depletion in NFH-Cre/
tg37 mice both synaptic and cognitive/behavioral deficits
recovered, and this recovery was sustained up to 30 wpi.
The development of phenotypic deficits thus paralleled
the development of early spongiform change, and recov-
ery of these deficits in mice with PrP depletion paralleled
its reversal. We found no objective evidence of synapse
loss in the early stages of infection associated with early
cognitive failure (Figure 4 and Figure S5), in contrast to
observations made by others in association with burrow-
ing decline in prion infection (Cunningham et al., 2003),
which may reflect differences in the strains of mice and
of infectious prions used. The relationship between spon-
giform change and the functional deficits we observed is
not clear. Spongiform degeneration in prion disorders in-
volves the formation of membrane-bound vacuoles within
neurons and neuropil—predominantly within dendrites—
and intradendritic distensions and dendritic spine loss
colocalize with vacuolar pathology in the hippocampus
(Jeffrey et al., 1997). The deficits we observe may thus
reflect the earliest neuronal and dendritic dysfunction,
although more detailed neurophysiological analysis impli-
cates impaired function at the level of the presynaptic
Thus, we found that synaptic responses were de-
pressed in our mouse model, which can be explained
by the proportional loss of the presynaptic fiber volley
(Figure 6B): the correlation between reduced field synap-
sion of presynaptic fiber volleys has been reported late in
the progression of another scrapie model (Chiti et al.,
2006), where it was tentatively attributed to loss of the
presynaptic neurons (CA3 pyramidal cells). The present
loss of the fiber volley was rapidly reversible and therefore
cannot be due to neuronal loss; more likely it was due to
Synaptic plasticity appears to be maintained, however:
all stages of disease implies that those synapses still
remaining in prion-infected tg37 mice have the intact
molecular apparatus both to release transmitter and to
sustain LTP, for review see Collingridge et al., 2004. It
appears that here memory is impaired due to reduction
in effective synaptic integration itself, implicating mecha-
nisms other than CA1 LTP in deficient memory. Dissocia-
of information and the endpoint of such changes in
expressed behavior have been reported elsewhere (Frag-
kouli et al., 2005), and learning and memory have been
found associated with impaired synaptic responses and
intact LTP in other rodent models (Brace et al., 1985).
However, LTP alterations have been described in prion-
infected mice, of different mouse strains than those
used here, at a relatively later stage in the disease process
and infected with different prion strains and using
more intense conditioning stimulation to induce LTP
(Chiti et al., 2006).
Both cognitive and neurophysiological impairment and
recovery in our model appeared to be independent of
PrPScaccumulation. Impaired object-recognition mem-
ory, burrowing behavior, and reduced postsynaptic
potentials in CA1 occur at 8 wpi in both mouse lines,
before extensive deposits of PrPScare apparent. Further,
both the loss and recovery of memory and species-typical
behaviors appear to be independent of aggregated PrPSc
deposition, up to 20+ wpi, as is synaptic function up to
Indeed, early synaptic pathology and dendritic dysfunc-
tion precede PrPScaccumulation (Jamieson et al., 2001),
consistent withour observations on behavioral and neuro-
physiological changes in the absence of extensive aggre-
gated PrPScdeposits here.
Our data are also consistent with findings in other
models of neurodegenerative disorders: that neuronal
dysfunction and cognitive defects occur before cellular
degeneration and independently of pathological aggrega-
tion of disease-associated proteins (Lambert et al., 1998;
Santacruz et al., 2005; Yamamoto et al., 2000), and they
can result from synaptic dysfunction rather than neuronal
loss (Buttini et al., 2002; Mucke et al., 2000; Walsh et al.,
2002; Wang et al., 2002). In several models of Alzheimer’s
disease there is increasing evidence that this may be due
to soluble Ab oligomers rather than amyloid (Billings et al.,
2005; Dodart et al., 2002; Kotilinek et al., 2002), explaining
plaque-independent cognitive failure in these animals.
Consistent with these studies, our findings support the
concept that a transient—as yet unidentified—neurotoxic
species is generated within neurons when PrPCis con-
verted to PrPSc, which rapidly impairs neuronal function
and synaptic responses. Depleting neuronal PrPCwould
halt formation of such an intermediate, whether it be
a new molecular species or an oligomeric form of PrPSc,
allowing rapid recovery of neuronal function.
Targeting PrPCtherapeutically remains somewhat con-
troversial, however, as the physiological role of the native
protein is still unclear. While both embryonic (Bueler et al.,
1992; Manson et al., 1994) and postnatal (Mallucci et al.,
2002) PrP knockout mice are viable and phenotypically
essentially normal, there is conflicting evidence for a role
for PrPCin some learning tasks (Criado et al., 2005) but
not others (Roesler et al., 1999). Our data suggest that at
Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc. 331
Reversal of Prion-Induced Cognitive Defects
least nonspatial memory tasks are independent of PrPC
tegrity and effective synaptic transmission. Also, we know
that PrPCdepletion does affect certain intrinsic properties
of CA1 cells, causing reduction in the late afterhyperpola-
rization potential (AHP) (Colling et al., 1996; Mallucci et al.,
2002). However, the only known phenotypic correlate of
late AHP reduction in transgenic mice is loss of social
transference of food preference between mice, which
can be reversed by environmental enrichment (Giese
et al., 1998).
Recent work implicates PrP expression in neurogenesis
and in the the proliferation of multipotent neuronal precur-
sor cells in the dentate gyrus in vivo. Despite having
reduced numbers of proliferating precursor cells com-
pared to wild-type or PrP-overexpressing animals, mice
constitutively lacking in PrP nonetheless have equal final
numbers of neurons in the dentate gyrus as PrP-express-
ing mice (Steele et al., 2006). Consistent with these data,
we also found that numbers of granule cell neurons in
the dentate gyrus were unaltered after PrP depletion in in-
fected and uninfected NFH-Cre/tg37 mice (Figure S7).
Overall, we conclude that the dramatic benefits to neuro-
nal function and survival in prion-infected mice we have
shown here support targeting neuronal PrPCdirectly as
a therapeutic approach.
Our findings of early reversible neurophysiological and
cognitive deficits occurring prior to neuronal loss open
new avenues in the prion field. To date, prion infection in
mice has conventionally been diagnosed when motor
deficits reflect advanced neurodegeneration. Now the
identification of earlier dysfunction helps direct the study
of mechanisms of neurotoxicity and therapies to earlier
stages of disease, when rescue is still possible. Eventually
it may also enable preclinical testing of therapeutic strate-
gies through cognitive endpoints. These data now lead to
the hope that early intervention in human prion disease,
will not only halt clinical progression but allow reversal of
early behavioral and cognitive abnormalities.
tg37 and NFH-Cre transgenic mice (Mallucci et al., 2002) were bred in-
house and were housed in a temperature- and light-controlled mouse
free access to food and water. All cages contained Perspex boxes
(‘‘igloos’’), cylindrical cardboard tubes, and folded sheets of tissue
paper in view of the positive effects of such enrichment on cognition
in rodents. All animal work conformed to the United Kingdom regula-
tions and institutional guidelines and was performed under Home
Office project license. Generation and characterization of these
mice, including onset and timing of recombination and genotyping
by PCR screening of DNA from tail biopsy samples are described
(Mallucci et al., 2002). The mice were generated on a Prnp0/0back-
ground so that all PrP expression is from the PrP (MloxP) transgene.
The genetic background is predominantly FVB after ten generations
of backcrossing. All animals are hemizygous for one or both trans-
genes (MloxP alone or also with NFH-Cre).
Inoculation with Prions
One-week-old mice were anesthetized and inoculated intracerebrally
into the right parietal lobe with 20 ml of 1% brain homogenate of
Laboratories) and subsequently examined daily for signs of scrapie, as
described (Mallucci et al., 2003).
All testing took place in a room with sombre lighting and constant
background noise. Mice were handled for 10 min a day for several
days and habituated to the test environment prior to testing. Only
group-housed males were used. Groups of eight to ten mice were
tested sequentially over time for each paradigm, unless otherwise
stated. Mice were handled, habituated, trained, and tested at the
same time for each experiment.
Novel Object Recognition Task
This was performed as described (Bozon et al., 2003). Briefly, mice
were tested in a dark cylindrical arena (69 cm diameter) mounted
with a 96 LED cluster infra-red light source and Watec video camera
(both Track-sys). Various plastic objects were constructed from inter-
locking plastic building blocks and were used in all experiments. (Pilot
studies confirmed all objects were of equal inherent interest.) On day 1
of each experiment (learning phase), two objects were placed in the
center of two 15 cm diameter circles inside the arena. Each mouse
was placed in the arena for two blocks of 10 min for exploration of
the objects with an intertrial interval of 5 min. Forty-eight hours later,
a novel object was randomly exchanged for one of the familiar ones,
and retention was tested by placing the mice back in the arena for
a 5 min session (test phase). The amount of time spent exploring all
objects was measured for each animal with the examiner blind to
genotype and time point for the animals. All objects and arena were
cleansed thoroughly between trials to ensure the absence of olfactory
cues. Criteria for exploration were based strictly on active exploration,
where mice had at least both forelimbs within a circle of 15 cm around
an object, head oriented toward it or touching it with their noses. Mice
with overt motor symptoms were not used. New sets of objects were
used at each time point.
Burrowing and Nesting
Two hours before the start of the dark period, mice (which had not
been food deprived) were placed in individual plastic cages containing
a gray plastic tube 20 cm long 3 6.8 cm diameter, filled with 200 g of
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.
Each mouse was placed in the corner of an empty, clear plastic arena
(50 3 32 3 20cm) divided into 10 3 10 cm squares and observed for
3 min. The number of squares entered (all four paws inside) and num-
ber of rears was recorded.
Inverted Screen Test of Muscular Strength and Reorientation
timefor themouse tofall offwasmeasured upto1min.Fororientation,
the mouse was first placed facing upward on the vertical mesh, which
was then rotated through 180?in the vertical plane so that the mouse
faced the floor. The time taken for the mouse to reorientate itself and
return to its original position was recorded.
Mice were anesthetized by intraperitoneal injection of a mixture of
medetomide (1 mg/kg) and ketamine (76 mg/kg) prior to being killed
332 Neuron 53, 325–335, February 1, 2007 ª2007 Elsevier Inc.
Reversal of Prion-Induced Cognitive Defects
by cervical dislocation, their brains removed for preparation of 400 mm
horizontal slices ofventral hippocampus,usingaremote control Vibro-
slice (Campden Instruments, Loughborough, UK) housed in a HEPA-
filtered thin-film isolator to contain contaminated aerosols. Slices
were kept in an interface holding chamber in the isolator until trans-
ferred to an interface chamber for recording. ACSF comprised (in
mM) 135 NaCl, 16 NaHCO3, 1.25 NaH2PO4, 3 KCl, 2 CaCl2, 1 MgCl,
and 10 glucose, pH 7.4, gassed with 95% O2, 5% CO2, maintained
at 32?C, and directed to a container containing 2 M NaOH after single
passage through the chamber.
Stimuli were applied through a bipolar stimulating electrode placed
in the stratum radiatum approximately 150 mm from the CA1 pyramidal
cell layer. Extracellular recordings of EPSPs were made from the same
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
response curves used a fixed set of stimulus strengths, and the field
EPSPs were measured as the initial slope (between 20%–80% of
maximum). LTP was induced by a high-intensity theta-burst protocol
(Morgan and Teyler, 2001) comprising five trains of four pulses at
100 Hz separated by 200 ms, repeated six times with an interburst in-
terval of 10 s, delivered after at least 30 min of recording half-maximal
stimuli delivered every 30 s. The same test stimuli were then delivered
for a further 45 min.
Neuropathological Examination of Brain Sections
PrPScin brain tissue was detected after denaturation of fixed sections
35 (Asante et al., 2002), and an automated immunostaining system
(www.ventanamed.com) as described (Joiner et al., 2002). Astrocyto-
sis was detected using anti-GFAP polyclonal antiserum (Dako; 1:500
dilution), and synaptophysin was detected with polyclonal rabbit anti-
serum (Zymed; prediluted) using the same system. Neu N staining was
performed using a mouse monoclonal antibody (Chemicon Interna-
tional; 1:2000 dilution). Sections of brains from all animals culled at
each time point and from those succumbing to scrapie were examined
by the same person, blinded to the identity of the animal and time of
culling. Sections were scored for spongiosis, neuronal loss, gliosis,
PrPScdeposition, and synaptophysin-signal intensity.
Immunohistochemistry in Frozen Sections
This was performed as described (Mallucci et al., 2002). PrP was
detected using monoclonal antibody ICSM 35, as above, at 1:100
dilution for 1 hr at room tempertature followed by incubation for
45 min in HRP-conjugated secondary antibody (Sigma; 1:10,000 dilu-
tion). Sections were counterstained with hematoxylin (Harris) for 5 min.
Hippocampi were dissected from freshly culled mice and stored
at ?20?C in five volumes of RNAlater (QIAGEN). RNA was extracted
with an RNeasy midi kit according to manufacturer’s instructions
(QIAGEN). One-step RT-PCR amplification of the MloxP transgene
mRNA and b-actin was performed on a PRISM 7000 Taqman machine
(ABI). Primers and probes for the Prnp transgene were as follows.
Forward primer, 50-GGCCCATGATCCATTTTGG-30; reverse primer,
50-GCGGTACATGTTTTCACGGTAGT-30; probe, 50-FAM-AACGACTG
GGAGGACC-30. b-actin was amplified using a commercially available
ABI assay: Mouse ACTB Endogenous control, VIC labeled MGB
probe. All reactions were performed in triplicate, and negative controls
sion was normalized to b-actin. Cycling conditions were 50?C for
20 min, 95?C for 15 min, then 40 cycles of 94?C for 45 s followed by
60?C for 45 s.
Individual data sets were tested for normality using the goodness of
fit test, Lilliefor’s test of normality (http://home.ubalt.edu/ntsbarsh/
Business-stat/otherapplets/Normality.htm). Student’s t tests were
applied to all data sets with two tails (two samples; unequal var-
iance). ANOVA testing was performed using one-way analysis for
group effects (http://www.physics.csbsju.edu/stats/anova.html). Mann
Whitney U tests (Wilcoxon rank sum tests) were performed where
data was not normally distributed; notably for motor testing. Analysis
of neurophysiological data were performed using general linear model
(GLM) in SPSS version 13.
The Supplemental Data for this article, including seven figures and
Supplemental Experimental Procedures, can be found online at
We thank Julie Underwood, Michelle Hainsworth, and Jackie Linehan
fortechnical assistance; RayYoungforhelpwithgraphics; SamCooke
and Tim Bliss (National Institute of Medical Research, Mill Hill) and
Colm Cunningham (University of Southampton) for advice and guid-
ance on behavioral testing. This work was funded by the Medical Re-
search Council of Great Britain. The neurophysiological experiments
were funded by a Physiological Society summer studentship (H.K.)
and the Biotechnology and Biological Sciences Research Council
Received: September 28, 2006
Revised: December 11, 2006
Accepted: January 5, 2007
Published: January 31, 2007
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