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Abstracts from New Researchers 2006Systems Analysis of Cellular Response to Alcohol Withdrawal
Mary K. McDonald#%, Rishi L. Khan&%, Rajanikanth Vadigepalli%, James S. Schwaber%, Babatunde A. Ogunnaike#
# Department of Chemical Engineering, University of Delaware, Newark, DE 19716
% Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University,
Philadelphia, PA 19107
& Department of Electrical and Computer Engineering, University of Delaware, Newark, DE 19716Alcoholism, a disease that develops as individuals adapt to the effects of alcohol to the point of requiring it for normal function, affects 14 million Americans and accounts for an estimated annual cost of $100 billion in healthcare and related productivity losses [1]. Once individuals are alcohol-adapted, serious consequences arise upon the cessation of alcohol intake, characterized by alcohol withdrawal syndrome (AWS). The symptoms of AWS involve dysregulation of the body’s internal equilibrium, or homeostasis, and include increased blood pressure, increased heart rate, disturbances in the respiratory rhythm, anxiety, seizures, and in some cases cardiac arrhythmias or sudden death [2].
The nucleus tractus solitarius (NTS) is a key brain region in the regulation of homeostasis [3]. It integrates visceral afferents such as blood pressure and heart rate to regulate homeostasis, and it is directly and reciprocally connected to the central nucleus of the amygdala (CeA) [4], which has been identified as a nexus for the regulation of emotions such as fear and anxiety [5]. The NTS and the CeA must therefore be studied jointly for any truly systematic understanding of alcohol withdrawal.
Previous studies of alcoholism and withdrawal have identified localized gene expression changes in the liver [6-9] and brain [10-15], including the NTS [10, 16-17] and CeA [18, 19]. From these studies, it is clear that a large number of genes are involved in the response to alcohol consumption. However, the response of this system to alcohol withdrawal, particularly the coordinated response of the genes underlying the functional disturbances of AWS, is largely unknown. To this end, our strategy is to study global gene expression in the NTS and CeA as observable indicators of the cellular system response to alcohol withdrawal, using microarray technology for the simultaneous collection of expression data for thousands of genes. This allows us to adopt a systems approach, where the network of genes can be studied for interacting responses instead of studying an individual gene. Our primary objective is to identify from such data, the regulatory network involved in withdrawal adaptation and AWS using this systems approach.
In contrast to other systems, physiological responses in the brain typically involve small levels of differential gene expression. These small changes challenge the detectability limits of microarray technologies. Standard microarrays reliably measure two-fold or greater expression differentials, but for gene products with a functional role in the brain, a two-fold change is titanic. Therefore, we have developed and employ in-house technologies that provide the needed precision to detect more subtle gene expression changes [20] reliably. Our microarrays currently include over 8,800 annotated rat genes, and we apply these technologies to detect and quantify the differential gene expression of each of these genes in the NTS and the CeA.
We obtain tissue samples from rat triplets—control, alcohol-adapted, and withdrawal—at various time points following the alcohol withdrawal. This experimental design, along with five biological replicates of each treatment to increase the statistical power of the analysis, provides us with data that adequately captures the gene expression dynamics following alcohol withdrawal. Our presentation will include the development and implications of this microarray time series experimental design.
We will also present results for the 24 hour post-withdrawal time point and contrast the gene expression profiles for each region. We have previously shown that chronic alcoholism in rats produces differential expression in the NTS [10], but here we observe that these changes involve fewer genes and are generally smaller in magnitude than the gene expression changes produced upon alcohol withdrawal. This indicates that the alcohol-adapted state in the NTS and the CeA is quite distinct from the control state, more so than the gene expression profiles suggest. In our presentation, we will discuss these differences and a hypothesis regarding the transcriptional regulation underlying the observed differential expression.
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[12] Stornetta, RL et al. Brain Res. 1993 Oct 8; 624(1-2):19-28.
[13] Lewohl, JM et al. Alcohol Clin Exp Res. 2000 Dec; 24(12): 1873-82.
[14] Lewohl, JM et al. J Biomed Sci. 2001 Jan-Feb; 8(1):28-36.
[15] Lewohl, JM et al. Brain Res. 1997 Mar 14; 751(1):102-12.
[16] Li G et al. Alcohol Clin Exp Res. 2005 Apr; 29(4):639-47.
[17] Wang, X and AA Abdel-Rahman. Alcohol Clin Exp Res. 2004 Aug; 28(8):1264-72.
[18] Lack, AK et al. Alcohol. 2005 Jun;36(2):83-90.
[19] Roberto, M. et al. J Neuroscience 2004. 24(45):10159-10166.[20] Khan, RL et al. Accepted, BMC Genomics
Title: The Alcohol Deprivation Effect (or lack thereof) in female C57BL/6J using the Drinking in the Dark protocol.
Exp#: IB 365
Date: 11/27/2006
PI: John Crabbe (crabbe@ohsu.edu)
Responsible Tech: Lauren Brown (brolaure@ohsu.edu)
Background: The purpose was to determine whether it is possible to see an Alcohol Deprivation Effect (ADE), previously seen in C57BL/6J mice during two-bottle preference drinking, using a shortened initial access period and a modified Drinking in the Dark (DID) procedure. It has been recently shown that a 6 week period of unlimited access to 15% ethanol vs water, followed by a weekly deprivation period (6 days deprivation) and a 1 day, two-bottle reinstatement of choice access, leads to a distinct increase in ethanol consumption by the second one-day test (Melendez et al, Alcohol Clin Exp Res, 2006 Dec;30(12):2017-25). We shortened the initial access period because previously we found that using the DID procedure of 4 hour daily exposures, by just the 4th day, C57BL/6J mice drank enough 20% v/v ethanol to achieve a blood ethanol concentration (BEC) greater than 1 mg/ml (Rhodes et al, Physiol Behav, 2005). With a weekly 6 day deprivation period followed by 1 day of DID 4 hour access, always to a single bottle of 20% ethanol, we hoped to see an escalation in consumption and BEC levels within 2-3 weeks above those of the initial access period.
Methods: We acclimated 30 C57BL/6J female mice to a reverse light-dark cycle (0730-1930) for two weeks prior to the experiment. One week before the start of the experiment the mice were housed individually. On the days of the experiment, three hours after the light went out (10:30AM); the water bottle in each homecage was removed and replaced with a 10 mL drinking tube containing either 20 % v/v ethanol dissolved in tap water or tap water. The ethanol drinking solution was made fresh each day immediately before the drinking tubes were positioned in the cage. (Recall that the animals are not fluid or food deprived at any time, and water is restored at the end of the ethanol access period.) The volumes of the ethanol in the tubes were recorded as rapidly as possible. Volumes were recorded at 2 hour intervals for up to 4 hours. The tubes were removed and a water bottle was replaced in each homecage. For the first week of this experiment, mice were started with a two hour access period to 20% ethanol on day 1, and then the drinking period was extended to four hours per day for another 4 days. At the end of the 4 hr access period, a 20 µl blood sample was obtained from the peri-orbital sinus on the fifth day of the study for blood ethanol concentration determination. Mice were then pseudo-randomly assigned to either the Control group or to the ADE group. For the next six days of the second week of the study, mice in the ADE group had their water bottles replaced with a 10mL drinking tube containing tap water while the control group continued to receive the 20% ethanol during the four hour daily access period. On the seventh day of week 2 all mice were given access to the 20% ethanol for the four hour access period followed by a 20 µl blood sample from the peri-orbital sinus for blood ethanol concentration determination. The procedure for week 2 was repeated through weeks 3 and 4. At the termination of the experiment subjects were euthanized using carbon dioxide asphyxiation.
Results: Results for intake are shown in Figure 1. BEC values on the four test days are given in Figures 2 and 3. In the first week of the study, the initial access period resulted on Day 5 in an overall mean ethanol consumption 8.5 ± 0.2 g/kg in 4 hr and a mean blood ethanol concentration (BEC) of 1.15 ± 0.10 mg/ml. The pseudo-randomly assigned control group and ADE group did not differ statistically on ethanol consumption (p = 0.7368) or BEC (p = 0.6142) for the first week. The second week of the study, the first week of deprivation, resulted in a decrease in ethanol consumption and BEC for both groups. The mean consumption and BEC were not statistically different from the first week and were not statistically different between groups. The third week of the study resulted in a slight increase from week 2 in ethanol consumption but still a lower level than week 2 for the ADE group. BEC remained the same as week 2 for the control group, but lowered in the ADE group to 0.83 ± 0.12 mg/ml which was significantly lower than the mean ADE group BEC from the first week (p=0.03). One animal from the ADE group showed possible signs of an alcohol deprivation effect with a 3.3 g/kg increase in ethanol consumption and a 0.68 mg/ml increase in BEC. The final week of the study resulted in a significant decrease from week 1 in ethanol consumption for both the control group (p=0.0240) and the ADE group (p=0.0076). Both groups saw a slight increase in BEC over Week 3 levels. The two groups were not statistically different in mean consumption or BEC during week 4. One animal from the ADE group showed signs of an alcohol deprivation effect with a 2.7 g/kg increase in consumption and a 0.63 mg/ml increase in BEC. The animal showing ADE during week 3 did not show ADE during week 4. Mean consumption and BEC levels never statistically differed between groups throughout the study.Title: The effect of additional exposures to ethanol during two-bottle choice following a modified drinking in the dark procedure on consumption and blood ethanol content.
Exp#: IB 331
Date: 060816
PI: John Crabbe (crabbe@ohsu.edu)
Responsible Tech: Lauren Brown (brolaure@ohsu.edu)
Background: The purpose of this study was to test whether offering mice a two-bottle choice immediately after the standard ethanol drinking in the dark protocol would lead to increased consumption and to test the effect of concurrent water access on blood ethanol concentration. Previously, we offered mice that had been tested in the drinking in the dark protocol two-bottle choice after several days of water only and found that consumption during the 2 or 4 hour limited access period was approximately equal with and without water choice, but blood ethanol concentrations were less with water choice than without. (Rhodes et al.,2006)
Methods: We acclimated 20 C57BL/6J mice, about half male and half female, to a reverse light-dark cycle (0730-1930) for two weeks prior to the experiment. One week before the start of the experiment the mice were housed individually. All mice received a water bottle in the left side of their cages during the acclimation period and post ethanol exposure periods of this study. Starting on the first test day of the experiment, a solution of 20% v/v ethanol dissolved in tap water was made fresh daily immediately before the test period. Three hours after the lights went out (10:30AM) the water bottle in each home cage was removed and replaced with a 10 mL ethanol drinking tube. The volume of the ethanol in the tube was recorded from the meniscus as rapidly as possible. The volumes were recorded after every two hours of drinking. At the conclusion of the daily ethanol exposure the volumes were recorded, tubes were removed, and the water bottle was placed back in the home cage. For this experiment, mice were acclimated to two hour access to 20% ethanol for 1 day, and then the drinking period was extended to four hours per day for another 3 days. (cf. Drinking in the Dark method in Rhodes et al., 2005). On days 5- 16 animals were given two 10mL drinking tubes, one containing tap water and one containing 20% v/v ethanol dissolved in tap water, for four hours, after which a single water bottle was returned to the cage. Half the mice received their ethanol on the right side and half on the left side of their cage throughout days 5-16 (the side remained constant for each cage). One 20 µl blood sample was obtained from the peri-orbital sinus after four hours of exposure on the final day of the study for blood ethanol concentration determination.
Results: All mice received the single ethanol bottle on the left side of the cage during the first four days of the study. Mice were divided into two groups for the two-bottle choice portion of the study: those receiving ethanol on the left and those receiving ethanol on the right. There was no difference in ethanol consumption between the left and right groups’ intake during the first four days. Starting on day 5, when a second tube was placed during the two-bottle choice, there was an apparent difference between the two groups of mice. Mice that received ethanol on the left side of the cage consumed significantly less fluid overall than the mice with ethanol on the right side (p=0.0001). However, there was never a significant difference in the g/kg ethanol consumption between the groups. In addition, the ethanol consumption in g/kg during limited access 2-bottle preference (days 5-16) was the same as during the single bottle DID procedure (days 1-4) (See Fig. 1). Mice with ethanol on the left preferred the ethanol solution to the water throughout the two-bottle choice portion of the experiment (80.0%±0.02%). Mice receiving ethanol on the right side of the cage preferred the water on the familiar left side over the ethanol solution (62.4%±0.07%) for the first day of the two-bottle choice (day 5) but after acclimating to the new cage set up their preference shifted to the ethanol solution (65.7%±0.17%). However, the right side group’s preference for ethanol was still significantly less than the left group (p=0.0023). There was no difference in the blood ethanol concentrations of the two groups. The mean blood ethanol concentration was 0.73±0.13 mg/ml with a mean ethanol consumption of 5.75±0.25 g/kg on Day 16. (See Fig. 2)
Conclusions and Future Directions: Offering mice a limited access two-bottle choice for 12 days starting 24 hours after the standard ethanol drinking in the dark protocol did not significantly increase consumption in this study, but animals still self-administered g/kg amounts equivalent to the single-bottle period during DID. Average blood ethanol concentration was somewhat lower than seen in previous drinking in the dark studies and this was most likely due to the g/kg ethanol consumption being lower on the final day than previous studies (or, the presence of a water bottle, or both). For future studies mice could be acclimated so that the water bottle is in the center of the cage so that during the two-bottle choice portion the placements of both the tube containing the 20% ethanol mixture and the tube containing the water are equally unfamiliar.Reference List
Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A., and Crabbe, J.C. (2005). Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiology and Behavior 84:53-63.
Rhodes, J.S., Ford, M.M., Yu, C.-H., Brown, L.L., Finn, D.A., Garland Jr., T., and Crabbe, J.C. (2006). Mouse inbred strain differences in ethanol drinking to intoxication. Genes Brain and Behavior. Published on line February 13, 2006Title: The effect of additional exposures to ethanol on the C57BL/6J drinking pattern using an extended Drinking in the Dark protocol.
Exp#: IB 327 Date: 060607
PI: John Crabbe (crabbe@ohsu.edu)
Responsible Tech: Lauren Brown (brolaure@ohsu.edu)
Background: The purpose was to examine whether drinking patterns are altered and/or whether total consumption is increased with increased days of access in the drinking in the dark (DID) procedure. The animals will be trained to expect a two hour access period, then switched to and trained to expect four hour access to evaluate what the additional exposures to the drinking solution have on the drinking pattern of C57BL/6J mice.
Methods: We acclimated 24 C57BL/6J (12 female, 12 male) mice to a reverse light-dark cycle (0730-1930) for two weeks prior to the experiment. One week before the start of the experiment the mice were housed individually. On the days of the experiment three hours after the light went out (10:30AM) the water bottle in each home cage was removed and replaced with a 10 mL drinking tube containing 20 % v/v ethanol dissolved in tap water. The ethanol drinking solution was made fresh each day immediately before the ethanol tubes were positioned in the cage. (Recall that the animals are not fluid or food deprived at any time, and a water tube water is restored at the end of the ethanol access period. See basic DID procedure in (Rhodes et al., 2005). The volume of ethanol in the tubes was recorded as rapidly as possible. Volumes were recorded at 1 hour intervals for two hours, and then the ethanol tubes were removed and a water bottle was replaced in each homecage. For this experiment, mice were acclimated to two hour access to 20% ethanol for 7 days, and then the drinking period was extended to four hours per day for another 13 days. Drinking was measured volumetrically by reading the meniscus at 60 minute intervals. One 20 µl blood sample was obtained from the peri-orbital sinus on the final day of the study from 12 animals after 1 hour of exposure to the drinking solution and from the other 12 after four hours of exposure for blood ethanol concentration determination. At the termination of the experiment subjects were euthanized using carbon dioxide asphyxiation.
Results: Consumption is shown in Figure 1. Drinking was significantly greater during the first hour of exposure to the ethanol solution than the ensuing hours throughout the study. During the four hour exposure periods (days 8-20), male mice drank significantly less than female mice only during the final hour of exposure. During 4 hour exposures female mice maintained their level of intake from the previous two hours while male intake significantly decreased from previous hours. The mean BEC level for bloods taken from mice after one hour of drinking on the final day of the study was 1.08±0.16 mg/ml and 1.33±.16 mg/ml for bloods taken after 4 hours of exposure (see Fig. 2). BECs did not differ in hr 1 vs hr 4 or between sexes.
Conclusions and Future Directions: C57BL/6J drinking patterns remained stable and did not escalate even after nearly 2 weeks of daily access to ethanol for 4 hours.Title: Mice Selectively Bred for Drinking in the Circadian Dark: Results from 6 Generations of Selection
Exp #: DDS 01, DDS 03, DDS 04, DDS 05, DDS 06, DDS 08, DDS 09
Date: 111406
PI: John Crabbe (crabbe@ohsu.edu)
Responsible Tech: Chia-Hua Yu (yuc@ohsu.edu)
Background: Previously, we presented a novel method for inducing ethanol drinking in C57BL/6J mice, drinking in the dark (DID), where subjects consume pharmacologically significant amounts of ethanol during a limited period of access shortly after the onset of the circadian dark phase. Corollary studies demonstrated significant, reliable differences among inbred strains using this procedure, implicating some degree of genetic influence.(Rhodes et al., 2005; Rhodes et al., 2006).
Methods: To explore the genetic underpinnings of this behavior further and to provide a genetic animal model for mechanistic studies, we are developing a line of mice selectively bred for high drinking in the dark (HDID-1) starting with the genetically heterogenous HS/Npt stock. The mice are given limited access to a 20% v/v ethanol solution in tap water starting three hours into their dark cycle by replacing their water bottle with an ethanol tube. Mice are neither food or fluid restricted, and are given ethanol access for 2 hrs the first day, and 4 hrs the second day. A retroorbital sinus blood sample is drawn at the end of the second day to ascertain blood ethanol concentration (BEC). Within-family selection is used to choose the mice with the highest BECs to produce the next generation. The unselected HS/Npt colony serves as a genetic control group.
Results: After six generations of selection we have seen a two-fold increase in mean BEC with respect to the founding population. We have also seen a concomitant increase in ethanol consumed. (see Figure 1). Response to selection in early generations, when starting from an 8-way cross, can be expected to be slow for a few generations, and then to accelerate as genes with initially very low frequencies are recruited. In an attempt to accelerate this process further, we switched to mass selection (excluding common grandparents) with the matings to produce S7 offspring. This will have the effect of increasing the intensity of selection.
Conclusions and Future Directions: Response to selection thus far corroborates the conclusion drawn from inbred strain studies using this paradigm: the drinking in the dark behavior is mediated in part genetically. With continued selection, this line should continue to increase in self-administered BEC, and an increasing proportion of mice should attain BECs leading to intoxication. We plan to initiate a second replicate selected line in December, 2006.
In our opinion, at S6, the HDID-1 and HS/Npt control lines do not differ markedly enough to warrant studies comparing them for other (correlated) responses. Although it is impossible to predict with certainty, we expect that another 1-3 generations of selection will increase the divergence of the lines, making mice available for other studies in spring or summer of 2007.Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A., and Crabbe, J.C. (2005). Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiology and Behavior 84:53-63.
Rhodes, J.S., Ford, M.M., Yu, C.-H., Brown, L.L., Finn, D.A., Garland Jr., T., and Crabbe, J.C. (2006). Mouse inbred strain differences in ethanol drinking to intoxication. Genes Brain and Behavior , published online Feb 13, 2006.Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A., and Crabbe, J.C. (2005). Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiology and Behavior 84:53-63.