Apparent homozygous deletion identified in Alström syndrome patient
ACAMPROSATE IMPLANTATION INTO REWARD AREAS TO SUPPRESS ETHANOL REWARD AND RESTORE NORMAL CIRCADIAN...
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ACAMPROSATE IMPLANTATION INTO REWARD AREAS TO SUPPRESS ETHANOL REWARD AND RESTORE NORMAL CIRCADIAN ACTIVITY HAS DIFFERENTIAL EFFECTS IN WILD-TYPE AND HOMOZYGOUS KNOCKOUT mPER2 MICE Allison J. Brager 1 , Rebecca A. Prosser 2 , and J.David Glass 1 . 1 Dept Biological Sciences, Kent State University, Kent, OH, 2 Dept Biochem and Cell and Molecular Biology, University of Tennessee, Knoxville, TN. INTRODUCTION Acamprosate (trade name Campral) is commonly prescribed to reduce EtOH craving, but has limited clinical efficacy. Reward portals of the brain, including the ventral tegmental area (VTA) and penduculopontine tegmentum (PPT) have dopaminergic and cholinergic projections,of which acamprosate modulates, to the mammalian circadian clock located within the suprachiasmatic nucleus (SCN) of the hypothalamus (Luo et al., 2008; Cain et al., 2004). Electrical stimulation of these reward portals elicits phase-shifts of behavioral circadian rhythms (Guinn et al., 2010). Recently, mice with homozygous deletions of the clock gene, mPer2, which regulates the period of the endogenous circadian oscillator , have potentiated EtOH consumption and preference relative to wild-types (Spanagel et al., 2005) . These experiments, moreover, investigate acamprosate reduction of EtOH preference and consumption in Per2 KO and wild-type mice owing to acamprosate implantation into these reward portals and its rescuing effects on EtOH-induced disruptions of behavioral circadian rhythms . METHODS EtOH Preference and Consumption. Free-choice of 15% EtOH or water was provided ad libitum and daily measured. EtOH preference was equal to EtOH intake/total fluid intake. General Circadian Locomotor Activity. Individually-caged mice entrained to a 12L: 12D photocycle were placed under infrared sensors interfaced with a computerized data acquisition system. Time of activity onset , rhythm period, length of the nocturnal active period, (alpha), and total nighttime activity (product of normalized bout number x bout duration) were calculated. Acamprosate Implantation . Following 3 wk of baseline EtOH consumption and preference and its withdrawal, constant-acamprosate release micropellets (500 µg) or blank wax controls were stereotaxically implanted into the VTA and PPT in separate groups of Per2 KO and wild-type mice. Following two days of post-operative recovery, mice were re-introduced to free-choice of 15% EtOH or water. Figure 3: Using UV spectrometry, constant-release of acamprosate from micropellets (500 µg) immersed in physiological saline was ~ 50 ng/day. Figure 5: Acamprosate implanted into the VTA reduced EtOH preference (top) and consumption (bottom) by 50+3% and 53+2% over 28+ 2 and 26+ 4 days, respectively, in wild-type controls and by 15+ 3% and 17+ 2% over 18+ 2 and 16+ 2 days in Per2 KO mice, respectively. a= PER2 vs. WT; b= WT baseline vs. WT re-introduction; c= PER2 baseline vs. PER2 re-introduction; d= PER2 re-introduction vs. all; p<0.05. DAYS O F EtO H TREATM ENT 0 10 20 30 40 50 60 M EAN EtO H PREFERENCE (% ) 0 20 40 60 80 ACAM P W T ACAM P PER2 W AX W T W AX PER2 DAYS O F EtO H TREATM ENT 0 10 20 30 40 50 60 M EAN EtO H CO NSUM PTIO N (G /KG BO DY W T.) 0 5 10 15 20 25 30 35 40 a a a a a bb b b b bb b b b b b b b b cc c c c c c c c dd d d aa a a a a aa a a aa aa a aa a a b b b b b b b b bb b b b b c c c c c c d d d d a a a aa a a a a ACAM P W T ACAM P PER2 W AX W T W AX PER2 c c c Figure 2: 24-hr systemic EtOH chronopharmacokinetic microdialysis profiles for an individual Per2 KO and WT mouse illustrating the phase-advance in nocturnal EtOH intake concomitant with nocturnal activity in Per2 KO vs WT mice under free-choice of 15% EtOH or water. Estimated tissue levels were ~60 mM and 20 mM per drinking bout in the Per2 KO ZE ITG E B E R TIM E (H R ) 0 5 10 15 20 25 ES TIM A TED [E tO H]IN SYSTEM IC TIS SU E (m M) 0 10 20 30 40 50 60 70 PER2 WT Figure 7: Acamprosate implantation into the VTA (surgery) significantly increased the intensity of locomotor activity across the nocturnal activity period without affecting alpha (A) compared to blank controls (B). Given acamprosate-induced hyperactivity, releasing the animals into constant darkness (shaded region) markedly shortened free-running behavioral rhythms. Figure 4: Photomicrographs of tyrosine- hydroxylase and choline acetyltransferas e stainings for the localization of dopaminergic and cholinergic neurons within the VTA (A) and PPT (B), respectively, and verification of the micropellet implants. P: Figure 1: As shown in above representative actograms, Per2 KO mice (A) had an advanced nocturnal activity onset occurring 2.1+0.2 hr before that of wild-types (B). Length of the nocturnal active period was increased by a similar amount (p<0.01). This phase- advance in activity onset was concomitant with EtOH drinking episodes shown in the representative drinkograms (C; Per2 KO; D; wild-type). The Per2 KO mice preferred and consumed more EtOH and had more EtOH drinking episodes across the 24 hr day vs. wild- Figure 8: Implantation of acamprosate into the VTA in WT mice (A) dramatically increased circadian nocturnal activity by 250% without affecting alpha relative to blank controls (B). This persisted for 27.2+ 2.1 days. RESULTS 1. Per2 KO mice had an advanced nocturnal activity onset that was concomitant with high levels of EtOH consumption (Figures 1 and 2). 2. Acamprosate implantation into the VTA and PPT rescues increased EtOH consumption and preference in Per2 KO and WT mice, but was significantly more efficacious in the WT vs. KO strain (Figures 3- 6). 3. Implanted acamprosate into reward portals markedly potentiated total nocturnal activity and shortened free-running behavioral rhythms in WT mice, but had minimal behavioral effects in Per2 KO (Figures 7-8). Figure 6: Acamprosate implanted into the PPT reduced EtOH preference (top) and consumption (bottom) by 41+2% and 38+2% over 22+ 1 and 16+ 2 days, respectively, in wild-type controls and by 14+1% and 35+ 1% over 4+ 1 and 8+ 3 days in Per2 KO mice, respectively. a= PER2 vs. WT; b= WT baseline vs. WT re-introduction; c= PER2 baseline vs. PER2 re-introduction; d= PER2 re-introduction vs. all; p<0.05. CONCLUSIONS 1. The advanced nocturnal activity chronotype concomitant with EtOH drinking of Per2 KO mice behaviorally elucidates potentiated EtOH consumption in this knockout strain. 2. Acamprosate implantation into reward portals effectively reduces EtOH consumption and preference in the WT type, but is minimally effective in the Per2 KO strain, indicating a strong genetic predisposition to EtOH craving in the Per2 KO strain. 3. Acamprosate restoration of robust behavioral rhythms in the WT strain may additionally A B C D A B Acknowledgements: NIH AA015948 grant to R.A.P and J.D.G DAYS O F SAM PLIN G 0 10 20 30 40 ACAM PRO SATE RELEASE (ng/day) 10 100 1000 A B DAYS O F E tO H TREATM ENT 0 10 20 30 40 M EAN EtO H PREFERENCE (% ) 10 20 30 40 50 60 70 80 ACAM P PER2 W AX PER2 ACAM P W T W AX W T a b c d a a a a a aa a a a a a a a a a a a a a a b b b b b b b b b c d DAYS O F EtO H TREATM ENT 0 10 20 30 40 M EAN EtO H CO NSUM P TIO N (G /KG BO DY W T.) 0 10 20 30 40 ACAM P PER2 W AX PER2 ACAM P W T W AX W T a b d aa a a a a a a a a a a a a a a a a b b b b b b b c d a c d a c d a c
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Transcript of ACAMPROSATE IMPLANTATION INTO REWARD AREAS TO SUPPRESS ETHANOL REWARD AND RESTORE NORMAL CIRCADIAN...
ACAMPROSATE IMPLANTATION INTO REWARD AREAS TO SUPPRESS ETHANOL REWARD AND RESTORE NORMAL CIRCADIAN ACTIVITY HAS DIFFERENTIAL EFFECTS IN WILD-TYPE AND HOMOZYGOUS KNOCKOUT mPER2 MICE
Allison J. Brager1, Rebecca A. Prosser2, and J.David Glass1. 1Dept Biological Sciences, Kent State University, Kent, OH, 2Dept Biochem and Cell and Molecular Biology, University of Tennessee, Knoxville, TN.
INTRODUCTIONAcamprosate (trade name Campral) is commonly prescribed to reduce EtOH craving, but has limited clinical efficacy. Reward portals of the brain, including the ventral tegmental area (VTA) and penduculopontine tegmentum (PPT) have dopaminergic and cholinergic projections,of which acamprosate modulates, to the mammalian circadian clock located within the suprachiasmatic nucleus (SCN) of the hypothalamus (Luo et al., 2008; Cain et al., 2004). Electrical stimulation of these reward portals elicits phase-shifts of behavioral circadian rhythms (Guinn et al., 2010). Recently, mice with homozygous deletions of the clock gene, mPer2, which regulates the period of the endogenous circadian oscillator , have potentiated EtOH consumption and preference relative to wild-types (Spanagel et al., 2005) . These experiments, moreover, investigate acamprosate reduction of EtOH preference and consumption in Per2 KO and wild-type mice owing to acamprosate implantation into these reward portals and its rescuing effects on EtOH-induced disruptions of behavioral circadian rhythms .
METHODSEtOH Preference and Consumption. Free-choice of 15% EtOH or water was provided ad libitum and daily measured. EtOH preference was equal to EtOH intake/total fluid intake.General Circadian Locomotor Activity. Individually-caged mice entrained to a 12L: 12D photocycle were placed under infrared sensors interfaced with a computerized data acquisition system. Time of activity onset , rhythm period, length of the nocturnal active period, (alpha), and total nighttime activity (product of normalized bout number x bout duration) were calculated. Acamprosate Implantation . Following 3 wk of baseline EtOH consumption and preference and its withdrawal, constant-acamprosate release micropellets (500 µg) or blank wax controls were stereotaxically implanted into the VTA and PPT in separate groups of Per2 KO and wild-type mice. Following two days of post-operative recovery, mice were re-introduced to free-choice of 15% EtOH or water.
Figure 3: Using UV spectrometry, constant-release of acamprosate from micropellets (500 µg) immersed in physiological saline was ~ 50 ng/day.
Figure 5: Acamprosate implanted into the VTA reduced EtOH preference (top) and consumption (bottom) by 50+3% and 53+2% over 28+2 and 26+4 days, respectively, in wild-type controls and by 15+3% and 17+2% over 18+2 and 16+2 days in Per2 KO mice, respectively. a= PER2 vs. WT; b= WT baseline vs. WT re-introduction; c= PER2 baseline vs. PER2 re-introduction; d= PER2 re-introduction vs. all; p<0.05.
DAYS OF EtOH TREATMENT0 10 20 30 40 50 60
ME
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EtO
H P
RE
FE
RE
NC
E (
%)
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ACAMP WTACAMP PER2WAX WTWAX PER2
DAYS OF EtOH TREATMENT0 10 20 30 40 50 60
ME
AN
EtO
H C
ON
SU
MP
TIO
N (
G/K
G B
OD
Y W
T.)
0
5
10
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20
25
30
35
40
a aa
aa
b b b
b
b
b bb
b
b
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b
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c cc
c
c c
cc c
d d
d d
a a a aa a a a aa a a a a
aa a a
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bb
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c
cc
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d
a
a aa a aa a
a
ACAMP WTACAMP PER2WAX WTWAX PER2
cc cFigure 2: 24-hr systemic EtOH chronopharmacokinetic
microdialysis profiles for an individual Per2 KO and WT mouse illustrating the phase-advance in nocturnal EtOH intake concomitant with nocturnal activity in Per2 KO vs WT mice under free-choice of 15% EtOH or water. Estimated tissue levels were ~60 mM and 20 mM per drinking bout in the Per2 KO and WT mice, respectively.
ZEITGEBER TIME (HR)
0 5 10 15 20 25
ES
TIM
AT
ED
[E
tOH
] IN
SY
ST
EM
IC T
ISS
UE
(m
M)
0
10
20
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PER2WT
Figure 7: Acamprosate implantation into the VTA (surgery) significantly increased the intensity of locomotor activity across the nocturnal activity period without affecting alpha (A) compared to blank controls (B). Given acamprosate-induced hyperactivity, releasing the animals into constant darkness (shaded region) markedly shortened free-running behavioral rhythms.
Figure 4: Photomicrographs of tyrosine-hydroxylase and choline acetyltransferase stainings for the localization of dopaminergic and cholinergic neurons within the VTA (A) and PPT (B), respectively, and verification of the micropellet implants. P: micropellet; MM: medial mammillary nucleus; Aq: cerebral aqueduct.
Figure 1: As shown in above representative actograms, Per2 KO mice (A) had an advanced nocturnal activity onset occurring 2.1+0.2 hr before that of wild-types (B). Length of the nocturnal active period was increased by a similar amount (p<0.01). This phase-advance in activity onset was concomitant with EtOH drinking episodes shown in the representative drinkograms (C; Per2 KO; D; wild-type). The Per2 KO mice preferred and consumed more EtOH and had more EtOH drinking episodes across the 24 hr day vs. wild-types (Preference: 70+3% vs. 50+3%; Intake: 25+1 g/kg/day vs. 14+1 g/kg/day; Episodes: 14+1 vs. 8+1, respectively; all p<0.01).
Figure 8: Implantation of acamprosate into the VTA in WT mice (A) dramatically increased circadian nocturnal activity by 250% without affecting alpha relative to blank controls (B). This persisted for 27.2+2.1 days.
RESULTS1. Per2 KO mice had an advanced nocturnal activity onset that was
concomitant with high levels of EtOH consumption (Figures 1 and 2).
2. Acamprosate implantation into the VTA and PPT rescues increased EtOH consumption and preference in Per2 KO and WT mice, but was significantly more efficacious in the WT vs. KO strain (Figures 3-6).
3. Implanted acamprosate into reward portals markedly potentiated total nocturnal activity and shortened free-running behavioral rhythms in WT mice, but had minimal behavioral effects in Per2 KO (Figures 7-8).
Figure 6: Acamprosate implanted into the PPT reduced EtOH preference (top) and consumption (bottom) by 41+2% and 38+2% over 22+1 and 16+2 days, respectively, in wild-type controls and by 14+1% and 35+1% over 4+1 and 8+3 days in Per2 KO mice, respectively. a= PER2 vs. WT; b= WT baseline vs. WT re-introduction; c= PER2 baseline vs. PER2 re-introduction; d= PER2 re-introduction vs. all; p<0.05.
CONCLUSIONS1. The advanced nocturnal activity chronotype concomitant with
EtOH drinking of Per2 KO mice behaviorally elucidates potentiated EtOH consumption in this knockout strain.
2. Acamprosate implantation into reward portals effectively reduces EtOH consumption and preference in the WT type, but is minimally effective in the Per2 KO strain, indicating a strong genetic predisposition to EtOH craving in the Per2 KO strain.
3. Acamprosate restoration of robust behavioral rhythms in the WT strain may additionally contribute to the acamprosate’s rescuing effects on EtOH consumption and preference in the WT strain.
A B
C D
A B
Acknowledgements: NIH AA015948 grant to R.A.P and J.D.G
DAYS OF SAMPLING
0 10 20 30 40
AC
AM
PR
OS
AT
E R
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EA
SE
(n
g/d
ay)
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A B
DAYS OF EtOH TREATMENT
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EtO
H P
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%)
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ACAMP PER2WAX PER2ACAMP WTWAX WT
a
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DAYS OF EtOH TREATMENT
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OD
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T.)
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ac d
ac
