RESEARCH ARTICLEDOWN SYNDROME

Restoration of Norepinephrine-Modulated Contextual
Memory in a Mouse Model of Down Syndrome
A. Salehi,1*† M. Faizi,2 D. Colas,3 J. Valletta,1‡ J. Laguna,1 R. Takimoto-Kimura,1
A. Kleschevnikov,1‡ S. L. Wagner,4 P. Aisen,4 M. Shamloo,2 W. C. Mobley1‡(Published 18 November 2009; Volume 1 Issue 7 7ra17)

Down syndrome (trisomy 21) is the most common cause of mental retardation in children and leads to marked
deficits in contextual learning and memory. In rodents, these tasks require the hippocampus and are mediatedby several inputs, particularly those originating in the locus coeruleus. These afferents mainly use norepinephrine as a transmitter. To explore the basis for contextual learning defects in Down syndrome, we
examined the Ts65Dn mouse model. These mice, which have three copies of a fragment of mouse chromosome
16, exhibited significant deficits in contextual learning together with dysfunction and degeneration of locus
coeruleus neurons. However, the postsynaptic targets of innervation remained responsive to noradrenergic
receptor agonists. Indeed, despite advanced locus coeruleus degeneration, we were able to reverse contextual
learning failure by using a prodrug for norepinephrine called L-threo-3,4-dihydroxyphenylserine, or xamoterol,a b1-adrenergic receptor partial agonist. Moreover, an increased gene dosage of App, in the context of Down
syndrome, was necessary for locus coeruleus degeneration. Our findings raise the possibility that restoring
norepinephrine-mediated neurotransmission could reverse cognitive dysfunction in Down syndrome.

INTRODUCTION
Down syndrome (DS) is a complex genetic disorder caused by the presence of a third copy of chromosome 21, resulting in triplication of ~300
genes. It is the most common source of congenital anomalies, with aprevalence of 1 per 733 live births in the United States; 5000 affected
infants are born each year (1). Among several abnormalities in DS, intellectual deficiencies that affect the quality of life for both children andadults are of primary concern. Understanding the neurobiological basisof failed cognition in DS is thus a high priority because deciphering
pathogenesis may lead to effective therapies.
Failed learning and memory is essentially universal in people with DS(2). We have pursued a strategy that emphasizes the initial documentation of phenotypes followed by discovery of underlying gene dose effects
and molecular and cellular mechanisms (3–5). Among the many deficits
present in children with DS, these individuals show severe defects in contextual tasks mediated by the hippocampus. This phenotype is bothrobust and significant, compromising the ability to carry out tasks of daily
life. Cued recall, in which memory is elicited by certain sensory cues, ispartially spared in DS (6, 7); these tasks are modulated by the amygdalaand, unlike the hippocampus, this region shows no change in structure inyoung people with DS (8). The hippocampus is markedly affected in DS(6–8). This brain region is essential for registering events with respect totime and space (9, 10). By modulating contextual discrimination, in which
spatial information is integrated with other salient features of theenvironment, the hippocampus is involved in generating appropriate responses to dynamic changes in milieu (10). Amnesic individuals with hip1
Department of Neurology and Neurological Sciences, Stanford Medical School,
Stanford, CA 94305, USA. 2Behavioral and Functional Neuroscience Laboratory, Stanford
Medical School, Stanford, CA 94305, USA. 3Department of Biology, Stanford Medical
School, Stanford, CA 94305, USA. 4Department of Neurosciences, University of California,
San Diego, La Jolla, CA 92093, USA.
*To whom correspondence should be addressed. E-mail: asalehi@stanford.edu†Present address: Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA.
‡Present address: Department of Neurosciences, University of California, San Diego,
La Jolla, CA 92093, USA.

pocampal damage fail in tests of contextual learning (11). Furthermore,hippocampal and entorhinal cortex damage has been shown to produce
insensitivity to contextual changes in rodents (10, 12), as has transient
inactivation of the hippocampus using g-aminobutyric acid type A(GABAA) agonists (13). Contextual discrimination is made possible byaccessing information from a number of afferent systems, both sensory
and modulatory; sensory information is transmitted from the entorhinal
cortex, whereas modulatory inputs originate in several populations including basal forebrain cholinergic neurons (BFCNs), norepinephrine (NE)–containing neurons of the locus coeruleus (LC), serotoninergic neurons of
the raphe nuclei, and calretinin-positive neurons of the supramammillary
area (4). Modulatory inputs extensively innervate the hippocampus. With
respect to contextual discrimination, the LC, which is the sole source ofNE-positive inputs, appears to play a defining role through the release of
NE to act on b adrenoceptors. Indeed, studies in which the activity of LC
afferents or b1 receptors was selectively inhibited showed that NE neurotransmission is essential for this aspect of hippocampal function (14).
Whether the LC plays a role in contextual discrimination in humans
is yet to be determined. Essential to demonstrating a link would bestudies that dissect hippocampally driven contextual learning fromcued learning, in which the amygdala plays a central role. Examining
disorders in which the LC degenerates is one strategy for exploring LC
function in humans. LC neurons are markedly affected in Alzheimer’s
disease (AD), DS, Parkinson’s disease, Huntington’s disease, dementia
pugilistica, and Wernicke-Korsakov syndrome (15–21). In AD, LC neurons undergo more extensive degeneration than BFCNs (18) and theextensive neurofibrillary degeneration of the LC correlates well with theseverity of cognitive decline (20). Furthermore, NE concentrations are
significantly reduced in the temporal cortex of patients with AD (21).
In DS, individuals show significant hippocampal dysfunction, including
deficits in contextual discrimination (22). Although cued learning remains relatively intact, contextual learning is markedly impaired in bothinfants and adolescents with DS (7, 22); it has been suggested that such
deficits significantly impair learning (6).

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RESULTS
Cognitive deficits in Ts65Dn mice
To explore the pathogenesis of cognitive disabilities in DS, we carriedout studiesgo2sD5odel11.29(i)o20.3(fgo2)-2647(tS)4.4(i).07-324.4(i)-154ssgo2sDisirriedin the Tn

RESEARCH ARTICLE
suggested to be due to phenotypic robustness of TH expression in preexisting neurons during early aspects of aging (30).
The LC innervates most brain regions; its projections are organized
topographically. The anterior pole of the LC innervates the hypothalamus,
whereas the posterior supplies the hippocampus. The neurons between
the subregions innervate the hippocampus, cerebellum, cortex, andspinal cord (Fig. 3D). We examined the possibility that changes in
LC cells would differentiate the different regions of this area. The severity of degeneration in Ts65Dn mice was greatest in the caudal LC (Fig.
3E), a subregion with extensive projections to the hippocampus (31).
To determine whether changes in LC neuron cell bodies are linked tochanges in the hippocampus innervation, we examined monoaminergic
terminals in the hippocampus by examining and quantifying staining
for vesicular monoamine transporter (VMAT2) (32, 33). A majority of
monoaminergic projections to the hippocampus, particularly in thedentate gyrus (DG), contain NE (33). Comparing Ts65Dn and 2N miceat age 3 months, the DG of Ts65Dn mice showed a significant increase
in VMAT2 staining; in particular, there was a marked increase in thenumber of bright puncta. In contrast, by 6 months, the optical density
of VMAT2 staining in Ts65Dn mice showed an overall decrease of~20% relative to 2N mice (Fig. 2D and fig. S2). These observations
are evidence for changes in LC terminals that precede those detected
in neuronal somas.

with age in both 2N and Ts65Dn mice, but only the change in the
Ts65Dn hippocampus was significant (P = 0.019) (Fig. 2F). These findings gave additional evidence for dysfunction in the terminals of the LC
neurons in Ts65Dn mice.
Norepinephrine-mediated neurotransmission
We examined the postsynaptic targets of LC axons in the hippocampus. Noradrenergic neurotransmission is affected in part through b1adrenergic receptors. The b1-adrenergic receptor is present on thepostsynaptic targets of LC axons in the hippocampus and has beenshown to play an important role in cognition, including contextual
learning (14). Regarding hippocampal hilar neurons expressing theb1-adrenergic receptor gene, immunostaining for these receptors isreadily detected on their cell bodies. In Ts65Dn mice at 3 months ofage, there was a marked increase in the size of b1-adrenergic receptor–
immunoreactive cells (P < 0.005) (Fig. 3F). The increase in size was also
seen at age 6 months. At 6 months, we also documented a more than
90% increase in the number of b1-adrenergic receptor–immunoreactive
neurons in the Ts65Dn hippocampus (P = 0.0173) (Fig. 3F). At thesame time, by examining b1 immunoreactive in the entire hippocampus, we noted an overall increase that was significant (P < 0.001). Thesefindings are evidence for changes in the postsynaptic targets of LC neurons. Our data in Ts65Dn mice showing degenerative changes in LC
terminals and reduced NE concentrations suggest that there is a compensatory increase in postsynaptic receptors in response to decreasing
norepinephrinergic transmission.
The continued presence of NE receptors on postsynaptic targets
raised the possibility that postsynaptic mechanisms activated by NE
remained functional even with the presence of LC dysfunction and degeneration. To test whether NE responses are still present in the DG of
Ts65Dn mice, we studied the effect of isoproterenol, an agonist at bothb1- and b2-adrenergic receptors and a widely used agent for studying
the functional integrity of adrenergic receptors. For these studies, weBiochemical changes in the hippocampus of Ts65Dn mice
To determine whether the morphological change in axon terminals in
the hippocampus was correlated with changes in NE, we examined theconcentrations of this neurotransmitter. There was a significant agerelated reduction in NE concentrations. Comparing the hippocampus
in Ts65Dn and 2N mice at 4.5 months of age, there was a 16% reduction in Ts65Dn that was not statistically significant. At 18 months,
however, the decrease in Ts65Dn was significant (P = 0.0007), averaging 31%. Examining absolute values for NE, there was a decrease
2N
Ts65Dn

R

100
80
0

20

40

60

80

100

Time (min)

2000
1500
P=0.0221
1000
500

0

80
70
60
50
40
30
20
10
0

200

P=0.0140

150
100
50
0

3 months 6 months 18 months

3 months 6 months 18 months

Age

Age

E
P=0.0120

NE (ng per gram tissue)

D

P=0.0006

Cell profile area (µm2)

TH-positive total number

Initial EPSP slope (%)

ISO, 10 µM

2N
Ts65DnP=0.0002

S

120

C

B

F
1000

P=0.0190
P=0.0007

800
600
400
200
0

4.5 months

18 months

Age

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Total number of β-adrenergic–
positive neurons

A

VMAT2 (OD)

Fig. 2. (A) Representative field
EPSP responses recorded fromthe granule cell layer after stimulation of the middle molecularlayer before and after application of a b-adrenergic agonist, isoproterenol (ISO) (n = 3 for eachgroup). (B and C) TH-positive neuron number (B) and cell area (C) inthe LC at 3 to 18 months of age.
(D) VMAT2 staining in the DG
of 6-month-old Ts65Dn mice (P =0.0120, n = 3 for each group). (E)Average concentrations of NE in
4.5- and 18-month-old Ts65Dnmice compared with 2N. (F) Total
number of b1-adrenergic–positive
neurons in the polymorphic layer(hilus) of the DG in Ts65Dn miceat age 6 months (P = 0.0173, n = 6
for 2N mice and n = 5 for Ts65Dnmice). 3V, third ventricle; ML, molecular layer; OB, outer blade; IB,
inner blade of the DG.

2N
Ts65DnIB

P=0.0170

3200
ML
OB

1600
3V

0

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2N

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RESEARCH ARTICLE
applied stimulating current to the middle molecular layer of the DG in
acute hippocampal slices, in the absence and presence of isoproterenol,
while recording in the DG granule cell layer. In slices taken from miceat 3 months of age, there were increases in both the slope and the amplitude of excitatory postsynaptic potentials (EPSPs) in both 2N and
Ts65Dn mice. The same was true at age 6 months. At 6 months ofage, the responses to isoproterenol in Ts65Dn slices were consistently
more robust (Fig. 2A and figs. S3 and S4). Thus, responsiveness to NE
was retained despite degenerative changes in LC neurons and theirterminals in the hippocampus.
Restoration of brain norepinephrine concentrations
Continued in vitro responsiveness of the targets of NE-positive innervation suggested that the same might be true in vivo. To test the idea, we
asked whether restoring NE concentrations would rescue contextual fear
conditioning in 6-month-old mice. We used an NE prodrug that readilycrosses the blood-brain barrier (BBB). L-Threo-3,4-dihydroxyphenylserine(L-DOPS) or droxidopa is a synthetic amino acid (34). L-DOPS is metabolized by L-aromatic amino acid decarboxylase within NE-containing
neurons to yield NE. To evoke NE increases in only the central nervous
system (CNS), we administered L-DOPS (1 mg/g) together with carbidopa (CD) (0.125 mg/g), a peripheral L-aromatic amino acid decarboxylase
inhibitor that does not cross the BBB (14, 35). CD administration
alone served as the control. L-DOPS concentrations were measured
6 hours after administration in both 2N and Ts65Dn mice. L-DOPS waspresent in several tissues, particularly the kidneys. In the hippocampus,
no significant differences were found in either L-DOPS (P = 0.5628; fig.

S5) or NE (P = 0.4726) concentrations between 2N and Ts65Dn mice.
In both 2N and Ts65Dn mice, L-DOPS injections markedly increased
hippocampal NE concentrations (fig. S5). Because brain NE reaches
its maximum concentrations within 5 hours of subcutaneous administration (14, 34), mice were tested at this time point. Treatment of
6-month-old Ts65Dn with L-DOPS (1 mg/g; 20 mg/ml) led to a significant improvement in contextual memory (Fig. 1, D and E). In the contextual test, L-DOPS restored fully the difference in freezing between
2N and Ts65Dn mice (Fig. 1, E and F) [P = 0.5247, one-way analysis
of variance (ANOVA)]. In the control group, in contrast, a significant
difference remained (P = 0.0002, one-way ANOVA). When performance was measured on a minute-by-minute basis, L-DOPS treatment
was associated with increased freezing after the first minute, a patternshared with 2N mice treated as controls or with L-DOPS. A similar beneficial effect of L-DOPS was found in nesting behavior. Treating young
adult Ts65Dn mice and their controls with L-DOPS significantly (P =0.0245) improved nesting in Ts65Dn mice (Fig. 1G). With cessation oftreatment, Ts65Dn mice again showed poor nesting behavior (P =0.0121) (Fig. 1G). These findings show that contextual discrimination
can be rescued in a mouse model of DS by increasing tissue concentrations of NE. They provide compelling support for the view that LC dysfunction markedly contributes to failure in contextual learning in thismodel.
The increased b1-adrenergic receptor expression that we observed
raised the possibility that these receptors were involved in transducing
the beneficial effects of L-DOPS in Ts65Dn mice. To test this idea, wetreated Ts65Dn mice with xamoterol, a b1-adrenergic receptor partial

P = 0.00001

P=0.0002

β1- adrenergic receptor–positive neurons(% total neurons)

TH-positive neurons(number/section)

E
D
Fig. 3. (A to C) ImmunocytoA
Cortex andcerebellum
chemical visualization of THIV
Hypothalamus
Hippocampus
positive neurons in the LC region.
1200
Scale bars, 500 mm (A), 200 mm
1000
2N(B), and 20 mm (C). IV, fourth ven800
tricle. (D) Schematic representa600
tion of LC projections to cortical
Ts65Dnand subcortical targets. The ros400
tral part of the LC in rodents
200
P=0.045
projects to the hypothalamus.
0
B
The core of the LC projects to
RostralCaudalRostralCaudalthe hypothalamus, cortex, andspinal cord, and the caudal partof the LC projects exclusively to
Ts65Dnthe hippocampus. (E) Density ofF
6 Months
3 Months
2N
TH-immunoreactive neurons
30
30
across the rostral-caudal axis of
the LC in 6-month-old 2N and
25
25
Ts65Dn mice. Although there20
was a reduction in neuronal den20
Csity across the rostral-caudal axis
15
15of the LC, degeneration of TH
neurons was more severe in the1010
caudal part of the LC. There was5
a significant reduction in the den5
sity of TH-positive neurons in
00Ts65Dn mice relative to the rostral
20
40
60
20
40
60
region of the LC (P = 0.045). (F) FreCell profile area (µm2)
Cell profile area (µm2)
quency distribution of cell area of
b1-positive neurons in the polymorphic layer of the DG in 3- and 6-month-old 2N and Ts65Dn mice. At both 3 months (c2 = 70, P < 0.005, n = 4 for 2N miceand n = 5 for Ts65Dn mice) and 6 months (c2 = 24, P < 0.01, n = 4 for 2N mice and n = 5 for Ts65Dn mice), there was a significant shift to higher values in thefrequency distribution of cell profile area in Ts65Dn mice.
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RESEARCH ARTICLE
agonist. Unlike cued learning, which was not affected in Ts65Dn mice,treatment with xamoterol restored failed contextual learning in
Ts65Dn mice (Fig. 4, A and B) (P = 0.032). These findings are evidence
that b1-adrenergic receptors play a vital role in mediating the effects ofincreasing norepinephrinergic transmission in the Ts65Dn hippocampus. Moreover, they show that pharmacologically targeting functionallyintact postsynaptic neurons can restore contextual learning.
Exploring the genetic basis of LC degeneration
To decipher the underlying genetic basis for LC degeneration in
Ts65Dn mice, we compared mouse models of DS harboring different

P=0.025
P=0.017

40
30
20
10
0

10

1000

*

500

6

E

F

25

+/+
2N

+/+/+

+/+

5

P=0.0025

1500
1000
500

+/+

+/+/+ +/+

2N

Ts65Dn2500
TH-positive neurons(total number)

50

4

P=0.049

2000

0

75

3
Minutes

P=0.7016

App

P=0.0024

0
App

2

2500

18
Age (months)

P=0.0013

100

Saline10

Nestlets (%)

20

0

1500

*

Xam

30

D
2N
Ts65DnTs1Cje

2000

*

40

Saline Xam Saline Xam Saline XamContextual
Cued
Training
memorymemoryC
TH-positive neurons(total number)

B 50
Freezing (% total)

Freezing (% total)

50

TH-positive neurons(total number)

2N
Ts65DnA

triplicated fragments of MMU16 (Fig. 1A). This approach has beenused to identify a role for App in the pathogenesis of BFCN degeneration in Ts65Dn mice (3). Even in old age, Ts1Cje mice showed nosignificant changes in size or number of LC neurons (Fig. 4C). The lackof apparent LC degeneration in Ts1Cje mice suggested that the responsible gene(s) is located on MMU16 fragment between Gabpa
and Sod1 (Fig. 1A); this region contains ~32 genes, including App(23, 24). To test whether increased dose for App contributes to LC degeneration, we examined these neurons in Ts65Dn mice bearing either
two or three copies of App. Deleting the third copy of App in Ts65Dnmice eliminated the decrease in the number of LC neurons, suggestingthat App overexpression is necessary for LC degeneration (Fig. 4D). A
question raised was whether deleting the extra copy of App and normalizing the apparent morphology of LC neurons in Ts65Dn micewould necessarily restore cognition in these mice. To address thispoint, we compared nesting behavior among Ts65Dn mice with two
versus three copies of App. As expected, we found a significant deficit
in nesting behavior in Ts65Dn mice (Fig. 4E) (P = 0.0013). However,deleting an extra copy of App in Ts65Dn mice did not restore nestingin these mice. As a result, no significant difference was detected between Ts65Dn mice with two versus three copies of App (P = 0.6658)(Fig. 4E). To determine whether increased concentrations of APP were
sufficient to cause LC degeneration, we examined mice that overexpress a mutant APPSwe transgene. There was a significant decrease
in LC neurons in these mice (P = 0.0317) (Fig. 4F). This latter finding
is consistent with a recent study showing degeneration of LC neuronsin APP/PS1 transgenic mice (36). Together, the findings are evidence
that APP/App gene dose plays a conspicuous role in the degenerationof LC neurons in these mouse models.

DISCUSSION

P=0.0317

2000
1500
1000
500
0
WT

tg-APPSwe

Ts65DnFig. 4. (A) Contextual learning in Ts65Dn mice treated with saline or
xamoterol (Xam). There was a significant improvement in contextual
learning in Ts65Dn mice treated with xamoterol relative to saline-treated
Ts65Dn mice (P = 0.0175, n = 7 for both groups). (B) A similar pattern was
found in the contextual day (P = 0.032, n = 7 for saline-treated Ts65Dnmice and n = 8 for xamoterol-treated Ts65Dn mice). (C) The LC neuron
degeneration was significant only in Ts65Dn mice (*P <0.05, n = 6 forTs1Cje mice). This suggests that the triplication of the region between
Mrpl39 and Rac must be responsible for degeneration of LC neurons.
(D) There was a significant difference (P = 0.0025) between Ts65Dn witheither two or three copies of App (n = 4 for Ts65Dn/App+/+/– mice andn = 5 for Ts65Dn/App+/+/+ mice). (E) Nesting behavior in Ts65Dn mice with
two or three copies of App. There was a significant deficit in nesting behavior in Ts65Dn mice with three copies of App (P = 0.0013, n = 9 for 2N
mice and n = 8 for Ts65Dn mice). However, deleting the extra copy of App
in these mice did not lead to a significant improvement in nesting behavior(P = 0.6658, n = 11 for Ts65Dn/App+/+/– mice). (F) Number of TH-positive
neurons in LC of 18-month-old APPSwe transgenic (tg-APPSwe) and wildtype (WT) mice (n = 5, P = 0.0317).

In this study, we linked marked defects in hippocampally mediatedcontextual learning in a model of DS to LC dysfunction and demonstrated that these deficits can be restored by treatments targeted atcorrecting deficient NE neurotransmission. The most important implication of our work is that postsynaptic targets of degenerating neurons may remain responsive and functional well after the presence ofadvanced disease in their presynaptic inputs. If so, treatments thattarget still-functional elements of neuronal circuits may restore circuit
function. In particular, treatments targeted to restore the loss of NE
inputs to the hippocampus may prove effective in enhancing cognition in people in whom these neurons are affected.
The age of 6 months was appropriate for testing because of the presence of both degenerative and apparent compensatory processes. Although we found both decreased number and size of LC neurons (Fig.
2, B to D) and reduced VMAT2 expression in presynaptic neurons(Fig. 2D), we detected increased numbers of b1-positive postsynaptic
neurons in 6-month-old Ts65Dn mice (Fig. 2F). Furthermore, it has
already been shown that the hippocampal b-adrenergic receptor transduction is significantly reduced in these mice at a similar age (37).
Our findings reinforce the importance of NE neurotransmission tocontextual learning. It appears that LC modulates the impact of thisinformation on hippocampal function to enhance contextual discrimination. The entorhinal cortex (38) gives rise to the medial perforantpath carrying navigational information and the lateral perforant path

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conveying sensory information. NE, released by the LC, is believed todifferentially affect perforant path inputs. For example, stimulating
NE-positive inputs potentiates the population spike amplitude of themedial perforant path while depressing synaptic potentials in the lateralperforant path (38–42). The net effect is thought to be an enhanced perception of spatial context. NE-containing inputs also appear to modulate directly or indirectly other neuronal systems with efferents to the
hippocampus, including cholinergic neurons of the basal forebrain andserotoninergic neurons of the raphe nuclei (43). Indeed, NE release from
LC axons may play a defining role in cholinergic and serotoninergic
neurotransmission. Given the degeneration of these other neuronalsystems in the Ts65Dn mouse as well as in DS and AD, it might beargued that dysfunction of the LC represents only one of several deficiencies and that rescuing NE concentrations would have no effect on
cognition. Our data argue that this is not the case. Instead, they indicate that restoring NE neurotransmission is effective even when theseother neurons are affected.
The GABAergic system also plays a significant role in cognition.
For instance, we and others recently reported that treatment with GABAA
receptor antagonists is able to improve both synaptic plasticity (44) andcognition (45) in Ts65Dn mice. There is a considerable overlap in the
distribution of GABAergic and catecholaminergic innervation in thebrain (46). NE is able to activate or conversely inhibit (47) GABAergic
neurons, and GABA can augment the release of NE in brain slices (48).
Therefore, it is possible that there is an overlap between mechanisms bywhich GABAA antagonists and L-DOPS improve learning in Ts65Dn
mice.
Unlike Ts65Dn mice, the 18-month-old Swedish APP mutant transgenic mice used in this study show high Ab accumulation in their brain.
In this regard, the relation between App and LC degeneration was investigated in the context of both high concentrations of APP and significant Ab accumulation. On the basis of these results, we can conclude
that although increased gene dose for App is necessary for degenerationof LC neurons in the context of DS, it is not yet possible to state that it isalso sufficient.
In investigating mechanism(s) by which App gene dose affects pathogenesis, it is important to note that the LC appears to be affected more
severely by pathogenic factors than targets of innervation. Indeed,changes in LC axon terminals preceded the observed changes in soma
size and number, a finding reported also for mouse models of AD (34).
Conceivably, other measures may define an even earlier onset of pathogenesis. For example, Ts65Dn mice at age 2 months fail in tests of contextual discrimination, and our own findings are consistent with changes
in nesting behavior as early as 3 months. In view of the findings for LC in
people with DS and those with AD, and the corresponding changes
detected in the mouse models of these disorders, it is possible that
LC dysfunction contributes to cognitive changes in both children andadults.
We found that deleting an extra copy of App in Ts65Dn mice did
not completely restore hippocampus-mediated nesting behavior (Fig.
4E). This finding suggests that although increased App gene dosealone can account for the degenerative changes in LC cell body size
and number, this is not the case for the defects in contextual learning.
The most plausible conclusion is that other genes combine with App toaffect the degeneration of LC neurons. This perspective is quite exciting
because it provides an intellectual paradigm for exploring the neurobiology of DS. In the past, it has been envisioned that one and the samegene would affect both the cell bodies and the terminals of degeneratingneurons. This is not the case, at least as judged by these new data. Thus,
one must now entertain the possibility that different genes affect various aspects of the neurodegenerative phenotype. Moreover, our data
argue that it may be necessary for treatments to address separately themechanisms engaged by different genes. In this regard, the L-DOPS
data suggest that one would be able to therapeutically affect changes
in neurotransmission that are partially independent of treatments
aimed at preventing the degeneration of the neuron cell bodies thatprovide the corresponding efferents.
The mechanism responsible for APP-mediated pathogenesis is yet
to be defined. As one possibility, there may exist a trophic deficiency
whose manifestations arise in the target, a suggestion for which earlier
studies provide support (3, 39). It is possible that local changes in thesynthesis or release of a trophic factor or its ability to signal in a retrograde fashion are impaired (3, 49, 50). Of particular interest is the possibility that brain-derived neurotrophic factor may play a role, especiallyin view of the fact that this protein serves as a trophic factor for LC
neurons (39).
In this study, two agents acted to restore contextual learning. In thecase of L-DOPS, the drug is metabolized by LC terminals to produce
NE. In the case of xamoterol, the drug directly accesses NE receptors.
The fact that both agents were effective suggests that receptor activation is functioning, thus reflecting the need for neuronal LC axonal
terminals. If this circumstance also applies to humans, restoring NE
concentrations in the hippocampus may act to enhance contextual
learning even in patients in which LC degeneration is advanced. Future
clinical trials attempting to increase NE neurotransmission in people
with DS, and possibly AD, may show cognitive benefits. To avoid peripheral nervous system activation, a regimen with drugs that can betargeted specifically to the CNS, as was the case herein for L-DOPS(plus carbidopa), would be preferred. The use of b-adrenergic antagonists with access to the CNS might impair contextual learning (14, 51).
Our findings raise concern for the use of such agents in patients withcognitive difficulties involving the LC and the hippocampus.
Clinical implications
There has been limited success to date in treating cognitive symptoms
in DS. Most efforts have focused on augmenting cholinergic function,
particularly with donepezil, with some limited benefits reported in bothyoung (52) and older (53–55) individuals. Furthermore, memantine,
an antagonist of N-methyl-D-aspartate receptors, is now being tested
in a large group of DS patients, the result of which is yet to be released(56).
Our findings suggest that enhancing NE neurotransmission may
be useful in treating cognitive disability in DS. An important questionis which age group to target. The murine studies reported herein suggest that young adults with DS, in whom pathology is present but notadvanced, may be appropriate. If the status of LC neurons and theirtargets in mice mirrors those in humans, at this stage of the disorder
postsynaptic adrenergic receptors will be present and responsive topharmacologically induced increases in brain NE concentrations.
We envision a plan to test the efficacy of droxidopa in young adults
with DS. The NE prodrug droxidopa is now in a phase III clinical
trial for orthostatic hypotension; in another clinical trial (phase II),
it is being tested, both with and without the peripheral aromatic
amino acid decarboxylase inhibitor carbidopa, for the ability to ameliorate CNS-mediated effects in fibromyalgia. Analogous experimental therapeutic regimens could be applied in DS to test clinically the

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hypothesis that raising NE concentrations in the CNS ameliorates
cognitive impairment in these disorders. Depending on the statusof preclinical toxicology and human studies, it may be possible to advance directly into efficacy studies in DS. Evidence that cerebrospinal
fluid (CSF) NE concentrations are responsive to therapeutic intervention and perhaps correlate with cognitive function encourages thisapproach (57, 58). The initial step would be dose-ranging phase IIpharmacodynamic, safety, and efficacy studies with droxidopa plus
carbidopa. On the basis of experience with cholinergic augmentation
therapies in AD (59), we expect that a 12-week exposure would be
sufficient for assessment of clinical effects. Therefore, it would be appropriate to begin with a 12-week exposure to a range of droxidopa
doses in combination with an optimal dose of carbidopa to evaluate
safety and impact on CSF NE concentrations and to explore efficacy
in terms of cognitive performance, global clinical status, function, andbehavioral measures. An ongoing multicenter trial in aging individuals with and without a dementia diagnosis (60) supports the utilityof a number of assessment tools, including a test of praxis to assess
cognition, a clinician’s global assessment of change utilizing caregiver
input, and behavior and function (by informant questionnaire); thesemeasures are robust across a wide range of intellectual functioning(61). Optimal doses may then be explored in combination, possiblywith and without a standard cholinergic intervention, to assess additive or synergistic benefit. Optimal monotherapy or combined therapy
regimens would then be assessed in pivotal 6-month placebo-controlled
phase III trials.
It appears that the use of L-DOPS in humans has not been associated with life-threatening side effects. The results of multiple clinical
trials in adults have shown that the side effects of L-DOPS include
nausea, headache, increased blood pressure, hallucination, anorexia,
lightheadedness, palpitations, dry mouth, irritability, upset stomach,vomiting, abdominal pain, chest pain, akinesia, laryngeal dyspnea, syn-

RESEARCH ARTICLE
the tone and the shock in each conditional stimulus–unconditional
stimulus pairing. On the second day (tone-cued testing day), mice wereplaced in the novel context (new olfactory and visual cues) for 3 min
and subsequently presented three tone presentations (same as thetraining day) without any shocks. On the last day of the experiment,
mice were placed in the context similar to the training day for 5 minwithout any tones or shocks. Two groups of male Ts65Dn and 2N mice
were injected either with carbidopa (2.5 mg/ml) or with a combinationof L-DOPS (20 mg/ml) and carbidopa (2.5 mg/ml) (50 ml/g subcutaneously) 5 hours before the start in all 3 days of the experiment.
Six-month-old 2N (CD, n = 13; CD + L-DOPS, n = 11) and Ts65Dn(CD, n = 10; CD + L-DOPS, n = 9) mice were used for this study. Because in all behavioral testing(s) we were able to reproducibly detect
deficits in contextual learning Ts65Dn mice, the lack of difference incued learning between 2N and Ts65Dn mice could not be due to insufficient statistical power.
Nesting behavior analysis. The nesting experiment was performed with 10 pairs of 6-month-old male Ts65Dn (CD, n = 10; CD +L-DOPS, n = 10) and 2N (CD, n = 10; CD + L-DOPS, n = 10) mice. The
nesting behavior was analyzed in three different periods (sham, drug,
and no treatment) (see Supplementary Material for details).
Statistical methods for behavioral studies. The data were tested
with one-way ANOVA between the genotypes. The significance of
genotype effects was confirmed by Student’s t test and a nonparametricMann-Whitney test; c2 distribution was used comparing frequency
distributions. All data in the study are presented as means ± SEM.
We performed statistical analyses with Statistica software (version 6.0).
Norepinephrine and L-DOPS determination
Tissue preparation. Mice were deeply anesthetized with sodium
pentobarbital (200 mg/kg intraperitoneally) and brains were extracted
immediately. Mice aged 4.5 months (n = 4 pairs of 2N and Ts65Dn) and18 months (2N, n = 8; Ts65Dn, n = 6) were used for this study.
High-performance liquid chromatography method. An ultraviolet high-performance liquid chromatography machine (Varian)equipped with an autosampler and two pumps was used for thisstudy (65, 66). See the Supplementary Material for details.
Electrophysiological recordings
Three- and 6-month-old 2N and Ts65Dn mice were anesthetized anddecapitated. The brain was quickly removed and immersed for 2 to
3 min in ice-cold artificial CSF (ACSF). The hippocampus was extracted and cut in ice-cold ACSF with a vibratome into 350-mm-thicktransverse slices, which were allowed to recover in oxygenated ACSF
before experimental recordings.

SUPPLEMENTARY MATERIAL
www.sciencetranslationalmedicine.org/cgi/content/full/1/7/7ra17/DC1
Materials and Methods
References
Fig. S1. Photographs showing nesting in 2N and Ts65Dn mice.
Fig. S2. VMAT2 staining in the inner molecular layer of the dentate gyrus in 3- and 6-month-old
mice.
Fig. S3. Recorded responses from the granule cell layer after stimulation of the middle molecularlayer.
Fig. S4. The effects of releasing (10 mM) isoproterenol (ISO) in the middle molecular layer ofdentate gyrus on changes in population spike amplitude of field EPSPs.
Fig. S5. NE concentrations in 6-month-old L-DOPS–treated mice.

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67. Acknowledgments: We thank Chelsea Therapeutics for providing L-DOPS for this study
and B. Barres, C. Epstein, M. Maloney, R. Reeves, and H. Zoghbi for valuable comments.
Funding: NIH grants NS055371 and NS24054, Larry L. Hillblom Foundation, Down Syndrome
Research and Treatment Foundation, Thrasher Research Fund, Adler Foundation, and Alzheimer
Association.
Author contributions: A.S. and W.C.M. designed the experiments and wrote the manuscript;
D.C. designed and performed the nesting experiments; A.K. performed the electrophysiologicalexperiments; J.V. bred the majority of the mice used in this study; A.S., M.F., and R.T.-K. performed the quantitative immunocytochemistry; M.F. and M.S. designed and performed thefear conditioning test(s); J.L. stained and quantified b1-adrenergic receptor staining; P.A. and
S.L.W. were involved in writing the manuscript, particularly the translational section.
Competing interests: A.S., W.C.M., and J.V. have submitted a U.S. patent application related tothis work and entitled “Method of improving cognitive functions in individuals with Down’s
syndrome and Alzheimer’s disease.”
Submitted 9 July 2009
Accepted 21 October 2009
Published 18 November 200910.1126/scitranslmed.3000258
Citation: A. Salehi, M. Faizi, D. Colas, J. Valletta, J. Laguna, R. Takimoto-Kimura, A. Kleschevnikov,
S. L. Wagner, P. Aisen, M. Shamloo, W. C. Mobley, Restoration of norepinephrine-modulated
contextual memory in a mouse model of Down syndrome. Sci. Transl. Med. 1, 7ra17 (2009).

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