Double-stranded RNA-mediated suppression of Period2 expression in the suprachiasmatic nucleus disrupts circadian locomotor activity in rats.

Abstract

Circadian behavioral rhythms in mammals are controlled by a central clock located in the suprachiasmatic nucleus (SCN). PER2, the protein product of the clock gene, Period 2 (Per2), is expressed rhythmically in the SCN [Beaule C, Houle LM, Amir S (2003) Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus: Lack of effect of photic entrainment and disruption by constant light. J Mol Neurosci 21:133-148] and has been implicated in the control of circadian behavioral rhythms based on the evidence that genetic mutations in Per2 abolish free running locomotor activity rhythms in mice [Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, Eichele G, Lee CC, Bradley A (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169-173; Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:525-536]. Such mutations eradicate PER2 expression in the SCN and disrupt the SCN molecular clockwork, however, they also affect PER2 in the rest of the brain and body leaving open the possibility that the changes in behavioral rhythms might be influenced, at least in part, by disruptions in PER2 functioning outside the SCN. We used RNA interference-mediated transient knockdown of Per2 to study the effect of selective suppression of PER2 expression in the SCN, per se, on behavioral circadian rhythms. We found that transient suppression of PER2 in the SCN disrupted free running locomotor activity rhythms for up to 10 days in rats. Infusions of control dsRNA into the SCN or infusions of dsRNA to Per2 immediately dorsal to the SCN had no effect. These results constitute evidence for a direct link between PER2 expression in the SCN and the expression of behavioral circadian rhythms in mammals.

Full text

Page 1

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
1
Section Editor: Menachem Segal
Double-stranded RNA-mediated suppression of PER2 expression in the
suprachiasmatic nucleus disrupts circadian locomotor activity in rats
1Gavrila, A.M., 1Robinson, B., 2Hoy, J., 1Stewart, J., 2Bhargava, A. and 1Amir, S.
1Department of Psychology, Concordia University, Montreal, PQ, CANADA
2Department of Surgery and Physiology, University of California San Francisco,
San Francisco, CA, USA.
Corresponding author: Shimon Amir
Center for Studies in Behavioral Neurobiology
Department of Psychology, Concordia University
7141 Sherbrooke St. West, SP-244
Montréal, QC, Canada H4B 1R6
TEL: 514 848 2424 (EXT 2188)
FAX: 514 848 2817
e-mail: shimon.amir@concordia.ca
Acknowledgements- Supported by grants from the Canadian Institutes of Health
Research, the Natural Sciences and Engineering Research Council of Canada,
the Concordia University Research Chairs Program, and by a NIMH (070650),
NIH grant to AB.
* 3 - Manuscript
Neuroscience 154, 409-414, 2008

Page 2

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
2
List of Abbreviations
ANOVA: Analysis of variance
BNSTov: Oval nucleus of the bed nucleus of the stria terminalis
bp: Base pairs
cDNA: Complementary deoxyribonucleic acid
CEA: Central nucleus of the amygdala
DD: Constant darkness
DG: Dentate gyrus
dsRNA: Double-stranded ribonucleic acid
LD: Light-dark cycle
MMLV-RT: Moloney murine leukemia virus reverse transcriptase
PCR: Polymerase chain reaction
Per2: Period 2
RT: Reverse transcriptase
RNAi: RNA interference
SCN: Suprachiasmatic nucleus
ZT: Zeitgeber time

Page 3

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
3
ABSTRACT
Circadian behavioral rhythms in mammals are controlled by a central clock
located in the suprachiasmatic nucleus (SCN). PER2, the protein product of the
clock gene, Per2, is expressed rhythmically in the SCN (Beaule et al., 2003) and
has been implicated in the control of circadian behavioral rhythms based on the
evidence that genetic mutations in the Per2 abolish free running locomotor
activity rhythms in mice (Zheng et al., 1999, Bae et al., 2001). Such mutations
eradicate PER2 expression in the SCN and disrupt the SCN molecular
clockwork, however, they also affect PER2 in the rest of the brain and body
leaving open the possibility that the changes in behavioral rhythms might be
influenced, at least in part, by disruptions in PER2 functioning outside the SCN.
We used RNAi-mediated transient knockdown of Per2 to study the effect of
selective suppression of PER2 expression in the SCN, per se, on behavioral
circadian rhythms. We found that transient suppression of PER2 in the SCN
disrupted free running locomotor activity rhythms for up to 10 days in rats.
Infusions of control dsRNA into the SCN or infusions of dsRNA to Per2
immediately dorsal to the SCN had no effect. These results constitute evidence
for a direct link between PER2 expression in the SCN and the expression of
behavioral circadian rhythms in mammals.
Keywords: Clock genes, Circadian rhythm, Oval nucleus of the bed nucleus of
the stria terminalis, Central nucleus of the amygdala, Dentate gyrus, RNAi

Page 4

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
4
Studies in mutant mice with targeted disruptions in the Per2 gene have led to the
conclusion that PER2 is essential for the expression of behavioral circadian
rhythms (Zheng et al., 1999, Bae et al., 2001). These rhythms are controlled by
the SCN, however, there is no direct anatomical evidence linking PER2
expression in the SCN to behavioral circadian rhythms. Furthermore, mutations
in Per2 can influence behavioral processes that are independent of the SCN.
Thus changes in behavioral rhythms might be confounded by behavioral
consequences of genetic disruptions of PER2 functioning outside the SCN
(Abarca et al., 2002, Spanagel et al., 2005, Feillet et al., 2006). RNA
interference (RNAi) is a powerful tool to selectively and transiently suppress the
expression of proteins in specific regions of adult, developmentally intact brain, in
vivo (Backman et al., 2003, Bai et al., 2003, Bhargava et al., 2004, Thakker et al.,
2004, Musatov et al., 2006, Lasek et al., 2007). We used long dsRNA to Per2 to
examine the effect of local and highly specific suppression of PER2 in the SCN,
per se, on free running locomotor activity rhythms in rats. We also assessed the
effect of suppression of PER2 in the SCN on the daily fluctuations in PER2
expression in areas of limbic forebrain known to be under the control of the SCN
(Amir et al., 2004, Lamont et al., 2005b).
EXPERIMENTAL PROCEDURES
Animals and housing
Experimental procedures followed the guidelines of the Canadian Council on
Animal Care and were approved by the Animal Care Committee, Concordia

Page 5

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
5
University. Male Wistar rats (300-350g; Charles River Laboratories, St.
Constant, QC, Canada) were individually housed in cages with running wheels
and had free access to food and water. All cages were housed within sound-
and light-proof enclosures equipped with a fluorescent light source and
ventilation system. Running-wheel activity was monitored using VitalView
software (Mini Mitter Co. Inc., Sunriver, OR) and analyzed with Circadia software
(Behavioral Cybernetics, Cambridge, MA) as described (Amir et al., 2004).
dsRNA synthesis for RNAi
dsRNA to Per2 was synthesized using previously described methods (Bhargava
et al., 2004). Briefly, rat brain total RNA (2 µg) was reverse-transcribed using
random hexamers and MMLV-RT in a 20 µl volume (Applied Biosystems,
Branchburg, NJ) as per manufacturer's specifications. Subsequent PCR was
performed using 5 µl RT and Per2-specific primers (derived from GenBank #
NM_031678.1). The primer set used was as follows: Per2 sense primer
5’cactgtaagaaggacgcctt3’ and antisense primer 5'aaggcgtccttcttacagtg3'. The
503 bp PCR products was analyzed by agarose gel electrophoresis, cloned in
pTopo4 (Invitrogen, Carlsbad, CA) and sequenced to confirm identity. The Per2
construct was linearized with SpeI or NotI and sense and antisense RNA were
transcribed from these linearized templates using T7 and T3 RNA polymerases
respectively as described earlier (Bhargava et al., 2004). Rat ß-globin cDNA
sequences were used as nonspecific control dsRNA in order to validate the
sequence-specific effects of dsRNA to Per2 as discussed previously (Bhargava

Page 6

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
6
et al., 2004). For infusions, 20 µg of control or 10 µg of Per2 long dsRNAs (1 µl)
were mixed with 0.5 µl of lipofectAMINE (Gibco, BRL; (Dalby et al., 2004)) and
the mix was incubated at room temperature for 30 minutes before use as
previously described (Bhargava et al., 2004).
Microinfusions
Rats were anaesthetized with a ketamine (100 mg/ml) / xylazine (20 mg/ml)
mixture (1.5 ml/kg, i.p.). A 30 gauge needle was lower into the SCN using the
following stereotaxic coordinates: 1.2 mm posterior to Bregma; 1.8 mm lateral to
the midline; 9.3 mm below the surface of the skull. All rats received bilateral
infusions. Each infusion of dsRNA to Per2 consisted of 6 µg/1.5 µl/side (the
highest concentration possible) or ß-globin (1.5 µl/side) into the SCN was made
over a 10-min interval using an infusion pump. Needle placements were
determined histologically at the end of the study.
Tissue preparation and immunocytochemistry
Rats were anaesthetized with sodium pentobarbital (Somnotol, ~ 100 mg/kg) and
perfused intracardially with 300 ml of cold saline (0.9% NaCl) followed by 300 ml
of cold, 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.3). Brains were
postfixed in 4% paraformaldehyde and stored at 4°C overnight. Serial coronal
sections (50 µm) were collected using a vibratome and stored in Watson’s
cryoprotectant solution. Immunocytochemistry for PER2 was performed as
previously described (Amir et al., 2004) using an affinity purified rabbit polyclonal

Page 7

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
7
antibody raised against PER2 (1:800, Alpha Diagnostics International, San
Antonio, TX). Immunocytochemistry for Fos was performed as described
(Beaule et al., 2001) using a polyclonal cFos antibody raised in rabbit (1:100,000,
Oncogene Sciences, Boston, MA).
Data analysis
Brain sections were examined under a light microscope and images were
captured using a Sony XC-77 video camera, a Scion LG-3 frame grabber, and
Image SXM software (v1.8, S D Barrett, http://www.ImageSXM.org.uk). Cells
immunopositive for PER2 or Fos were counted. The mean number of
immunoreactive cells per region was calculated for each animal from the counts
of 6 unilateral images showing the highest number of labeled nuclei. Data were
analyzed using analysis of variance (ANOVA). Alpha level was 0.05.
RESULTS and DISCUSSION
We first assessed the effect of local infusions of dsRNA on PER2 in the SCN.
Groups of rats (n=4/group) housed under 12:12h light-dark cycle (LD) were given
bilateral infusions of dsRNA to Per2 or ß-globin during the middle of the day and
perfused 3, 6 or 12 days later, at zeitgeber time (ZT) 13, time of peak expression
of PER2 in the SCN (ZT0 indicates time of lights on under LD). Examples of
PER2 expression in the SCN are shown in Fig. 1. Infusions of dsRNA to Per2
significantly suppressed PER2 expression in the SCN at all time intervals tested
(F(1, 18) = 43.04, p < .01). The decrease in PER2 was transient, with maximal

Page 8

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
8
suppression (59% relative to control rats infused with dsRNA to ß-globin) seen
three days after the infusion. After six days the levels of PER2 were 36% below,
whereas 12 days after the infusions they were 26% below those in the SCN of
controls (Fig. 1).
In separate groups of rats, we assessed the effect of bilateral infusions of dsRNA
into the SCN on free-running activity rhythms. Rats housed under LD for two
weeks were given infusions of dsRNA to Per2 or ß-globin during the daytime and
placed in constant darkness (DD) for 45 days. Representative actograms are
shown in Fig. 2. Bilateral infusions of dsRNA to Per2 aimed at the SCN
disrupted circadian wheel-running activity rhythms in 11 of 16 rats. Failure to
affect circadian rhythms in the remaining five rats was associated with one or
both inaccurate injector placements. Infusions of dsRNA to ß-globin into the
SCN (n=8) or infusions of dsRNA to Per2 immediately dorsal to the SCN (n=2)
had no effect. As evident in the actograms, affected rats exhibited fragmented
activity patterns throughout the circadian cycle that lasted up to 9 days. Infusions
of dsRNA into the SCN had no effect on body weight or general health.
Periodgram analysis on the portions of the actograms showing fragmented
wheel-running patterns revealed no rhythms in the circadian range (data not
shown). After this period all affected rats displayed normal free running rhythms
that were indistinguishable from those of control rats, indicating that the
behavioral effects seen were not due to permanent SCN damage. The time
course of loss and recovery of behavioral circadian rhythms following infusions of

Page 9

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
9
dsRNA to Per2 in the SCN closely mirrors that of the loss and recovery of PER2
in the SCN, consistent with the hypothesis that the expression of behavioral
rhythms are linked to the expression of PER2 in the SCN.
In a final experiment we studied the effect of dsRNA infusions into the SCN on
the daily variation in PER2 expression in three limbic forebrain areas, the oval
nucleus of the bed nucleus of the stria terminalis (BNSTov) central nucleus of the
amygdala (CEA) and the dentate gyrus (DG). We have shown previously that
the PER2 rhythms in these areas are controlled by the SCN (Amir et al., 2004,
Lamont et al., 2005b). Accordingly we hypothesized that suppression of PER2
expression in the SCN would disrupt the daily variation in PER2 in the limbic
forebrain. Groups of rats (n=4/group) housed under LD were treated with dsRNA
to Per2 or ß-globin and perfused 6 days later at ZT1 or ZT13, times of minimal
and maximal expression of PER2 in the SCN, BNSTov and CEA, and times of
maximal and minimal expression in the DG. To study the specificity of the
dsRNA, alternate brain sections were stained for Fos. Fos is induced in the core
region of the SCN by light exposure in the morning, whereas in the evening
expression of Fos in the core is low. In the shell region of the SCN Fos
expression is generally high during the day and low at night (Beaule and Amir,
1999, Guido et al., 1999, Beaule et al., 2001).
As shown in Figs. 3 and 4, infusions of dsRNA to Per2 blunted the daily variation
in PER2 in the SCN by suppressing expression at ZT13, consistent with the

Page 10

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
10
findings shown in Fig. 1. The total levels of PER2 in the BNSTov, CEA and DG
were not affected, however, the daily variation was significantly blunted in all
three areas (Fig. 4), further demonstrating that these limbic forebrain PER2
rhythms depend on the integrity of the SCN clock. The results from ANOVAs
carried out on the data from each area are shown in Table 1. It can be seen that
dsRNA Treatment had no effect on the overall levels of PER2 in any region
outside the SCN. The significant effects of Time reflect the daily variation,
whereas the Treatment x Time interactions reflect the blunting of the daily
variation in dsRNA treated groups. Contrary to these effects on PER2
expression, infusions of dsRNA to Per2 into the SCN had no effect on the daily
pattern of expression of Fos in the SCN or limbic forebrain (Figs. 3, 4). In both
the experimental and control groups Fos expression in the core region of the
SCN was high at ZT1 and low at ZT13, whereas in the shell region Fos tended to
be lower at ZT1 than ZT13. Similarly, Fos expression in the BNSTov, CEA and
DG did not vary between treatment groups (Figs. 4). These results show that the
suppression of PER2 in the SCN results from specific action of the dsRNA on
Per2 and is not due to non-specific suppression of gene expression.
The present results demonstrate that local bilateral infusions of dsRNA to Per2,
which was found to partially and transiently suppress PER2 in the SCN, are
sufficient to disrupt free-running locomotor activity rhythms in rats housed in
constant darkness. Importantly, dsRNA reduced PER2 expression in the SCN in
rats housed under a LD cycle showing that it was effective under conditions in

Page 11

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
11
which light might enhance PER2 expression in the SCN. Thus, the effects of
dsRNA in DD may have been even greater, consistent with the complete, but
reversible behavioral disruption in experiment 2. These results extend previous
findings in Per2 mutant mice by providing anatomical evidence for a first direct
link between PER2 expression in the SCN, per se, and circadian behavioral
rhythms (Zheng et al., 1999, Bae et al., 2001). Furthermore, the present results
show that suppression of PER2 in the SCN blunts the expected daily variation in
expression of PER2 in the BNSTov, CEA and DG. This finding is consistent with
our earlier results showing that bilateral lesions of the SCN, which disrupt wheel-
running rhythms, blunt PER2 rhythms in the limbic forebrain in rats (Amir et al.,
2004, Lamont et al., 2005b). Moreover, it is consistent with the evidence that
constant light housing, which blunts the rhythm of PER2 in the SCN, disrupts
locomotor activity rhythms and rhythms of PER2 in the limbic forebrain (Amir et
al., 2004, Lamont et al., 2005a). It is important to point out that the loss of
circadian rhythms of PER2 in the limbic forebrain, per se, could not account for
the disruption of behavior rhythms seen in this study. For example, both
adrenalectomy and thyroidectomy completely blunt the circadian rhythms of
PER2 expression in the BNSTov and CEA without affecting circadian locomotor
activity rhythms (Amir and Robinson, 2006, Segall et al., 2006). Finally, we
found that infusions of dsRNA to Per2 into the SCN had no effect on Fos
induction by light in the core region of the SCN or on the constitutive expression
of Fos in the shell region. Furthermore, it had no effect on expression of Fos in
the BNSTov, CEA and DG. These results not only confirm that the effect of the

Page 12

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
12
dsRNA to Per2 is specific, but also show that the expression of Fos and PER2 in
the SCN and limbic forebrain can be dissociated as we have shown previously
(Beaule and Amir, 2003, Verwey et al., 2007).
PER2 is expressed rhythmically in many brain regions outside the SCN (Shieh,
2003, Sellix et al., 2006) and evidence from Per2 mutant mice indicates that
PER2 participates in behavioral processes that are independent of the SCN,
such as food anticipatory behavior (Feillet et al., 2006), sensitization to the
behavioral activating effects of cocaine (Abarca et al., 2002), and alcohol
preference (Spanagel et al., 2005). The importance of these extra-SCN rhythms
is underscored by the evidence that they can be influenced directly by changes in
circulating hormones (Amir and Robinson, 2006, Perrin et al., 2006, Segall et al.,
2006) and other perturbations that affect behavioral state, such as restricted
feeding (Lamont et al., 2005a) and exposure to drugs (Yamamoto et al., 2005).
There is currently, however, no evidence to link directly the expression of PER2
within specific brain areas to specific behavioral or physiological outputs. The
present experiments show that long dsRNA-mediated RNAi works effectively to
achieve transient, tissue specific knockdown of PER2 expression in the brain of
adult, developmentally intact rats. As such, our dsRNA provides a powerful new
tool to elucidate the behavioral and neural correlates of PER2 within select brain
regions.

Page 13

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
13
REFERENCES
Abarca C, Albrecht U, Spanagel R (2002) Cocaine sensitization and reward are
under the influence of circadian genes and rhythm. Proceedings of the
National Academy of Sciences of the United States of America 99:9026-
9030.
Amir S, Lamont EW, Robinson B, Stewart J (2004) A circadian rhythm in the
expression of PERIOD2 protein reveals a novel SCN-controlled oscillator
in the oval nucleus of the bed nucleus of the stria terminalis. J Neurosci
24:781-790.
Amir S, Robinson B (2006) Thyroidectomy alters the daily pattern of expression
of the clock protein, PER2, in the oval nucleus of the bed nucleus of the
stria terminalis and central nucleus of the amygdala in rats. Neuroscience
letters 407:254-257.
Backman C, Zhang Y, Hoffer BJ, Tomac AC (2003) Short interfering RNAs
(siRNAs) for reducing dopaminergic phenotypic markers. Journal of
neuroscience methods 131:51-56.
Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001)
Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian
clock. Neuron 30:525-536.
Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ (2003) RNAi
reveals doublecortin is required for radial migration in rat neocortex.
Nature neuroscience 6:1277-1283.

Page 14

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
14
Beaule C, Amir S (1999) Photic entrainment and induction of immediate-early
genes within the rat circadian system. Brain Res 821:95-100.
Beaule C, Amir S (2003) The eyes suppress a circadian rhythm of FOS
expression in the suprachiasmatic nucleus in the absence of light.
Neuroscience 121:253-257.
Beaule C, Arvanitogiannis A, Amir S (2001) Light suppresses Fos expression in
the shell region of the suprachiasmatic nucleus at dusk and dawn:
implications for photic entrainment of circadian rhythms. Neuroscience
106:249-254.
Beaule C, Houle LM, Amir S (2003) Expression profiles of PER2
immunoreactivity within the shell and core regions of the rat
suprachiasmatic nucleus: Lack of effect of photic entrainment and
disruption by constant light. J Mol Neurosci 21:133-148.
Bhargava A, Dallman MF, Pearce D, Choi S (2004) Long double-stranded RNA-
mediated RNA interference as a tool to achieve site-specific silencing of
hypothalamic neuropeptides. Brain Res Brain Res Protoc 13:115-125.
Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, Ciccarone VC (2004)
Advanced transfection with Lipofectamine 2000 reagent: primary neurons,
siRNA, and high-throughput applications. Methods (San Diego, Calif
33:95-103.
Feillet CA, Ripperger JA, Magnone MC, Dulloo A, Albrecht U, Challet E (2006)
Lack of food anticipation in Per2 mutant mice. Curr Biol 16:2016-2022.

Page 15

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
15
Guido ME, de Guido LB, Goguen D, Robertson HA, Rusak B (1999) Daily rhythm
of spontaneous immediate-early gene expression in the rat
suprachiasmatic nucleus. Journal of biological rhythms 14:275-280.
Lamont EW, Diaz LR, Barry-Shaw J, Stewart J, Amir S (2005a) Daily restricted
feeding rescues a rhythm of period2 expression in the arrhythmic
suprachiasmatic nucleus. Neuroscience 132:245-248.
Lamont EW, Robinson B, Stewart J, Amir S (2005b) The central and basolateral
nuclei of the amygdala exhibit opposite diurnal rhythms of expression of
the clock protein Period2. Proceedings of the National Academy of
Sciences of the United States of America 102:4180-4184.
Lasek AW, Janak PH, He L, Whistler JL, Heberlein U (2007) Downregulation of
mu opioid receptor by RNA interference in the ventral tegmental area
reduces ethanol consumption in mice. Genes, brain, and behavior 6:728-
735.
Musatov S, Chen W, Pfaff DW, Kaplitt MG, Ogawa S (2006) RNAi-mediated
silencing of estrogen receptor {alpha} in the ventromedial nucleus of
hypothalamus abolishes female sexual behaviors. Proceedings of the
National Academy of Sciences of the United States of America
103:10456-10460.
Perrin JS, Segall LA, Harbour VL, Woodside B, Amir S (2006) The expression of
the clock protein PER2 in the limbic forebrain is modulated by the estrous
cycle. Proceedings of the National Academy of Sciences of the United
States of America 103:5591-5596.