Increased ethanol drinking after repeated chronic ethanol exposure and withdrawal experience in C57BL/6 mice

BACKGROUND: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self-administer ethanol. METHODS: With the use of a modified sucrose-fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8-hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5-day test period for ethanol drinking. RESULTS: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air-exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels. CONCLUSIONS: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2-fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).     

Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol

Background: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling‐type transition from moderate to escalated use, leading to dependence. Intermittent 24‐hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats. Methods: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short‐term, limited access “binge” drinking procedure, similar to drinking‐in‐the‐dark (DID). B6 mice were also assessed for ethanol dependence with handling‐induced convulsion, a reliable measure of withdrawal severity. Results: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2‐hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h). Discussion: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.     

Effects of food availability and administration of orexigenic and anorectic agents on elevated ethanol drinking associated with drinking in the dark procedures

Background: Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations reaching levels that have measurable affects on physiology and/or behavior. The present study determined if increased ethanol drinking associated with DID procedures may be motivated by caloric need rather than by the postingestive pharmacological effects of ethanol. To this end, food availability was manipulated or mice were given peripheral administration of orexigenic or anorectic agents during DID procedures. Methods: C57BL/6J had 2‐hours of access to the 20% (v/v) ethanol solution beginning 3‐hours into the dark cycle on days 1 to 3, and 4‐hours of access to the ethanol bottle on day 4 of DID procedures. In Experiment 1, the effects of food deprivation on ethanol consumption during DID procedures was assessed. In Experiments 2 and 3, mice were given intraperitoneal (i.p.) injection of the orexigenic peptide ghrelin (0, 10 or 30 mg/kg) or the anorectic protein leptin (0 or 20 μg/g), respectively, before access to ethanol on day 4 of DID procedures. In Experiment 4, hourly consumption of food and a 0.05% saccharin solution were assessed over a period of hours that included those used with DID procedures. Results: Consistent with previous research, mice achieved blood ethanol concentrations (BECs) that ranged between 100 and 150 mg% on day 4 of DID experiments. Neither food deprivation nor administration of orexigenic or anorectic compounds significantly altered ethanol drinking with DID procedures. Interestingly, mice exhibited their highest level of food and saccharin solution consumption during hours that overlapped with DID procedures. Conclusions: The present observations are inconsistent with the hypothesis that C57BL/6J mice consume large amounts of ethanol during DID procedures in order to satisfy a caloric need.     

Naltrexone Effects on Ethanol Drinking Acquisition and on Established Ethanol Consumption in C57BL/6J Mice

Naltrexone's success as a treatment agent for alcoholism seems to be due to its ability to reduce craving in abstinent, dependent individuals and to reduce the pleasure associated with subsequent intake. However, more study is needed to establish the optimal amount of time that naltrexone treatment should be continued. Little information seems to have been collected regarding the most effective dosing regimen for reducing alcohol craving and consumption, and the usefulness of opiate antagonists in the prevention of alcohol dependence in nonaddicts, rather than just as a treatment agent in addicted individuals, also deserves further study. The alcohol-preferring C57BL/6J (B6) mice were used to: (1) study naltrexone effects on consumption in established drinkers using an increasing dosing regimen, (2) study naltrexone effects on the acquisition of ethanol drinking, and (3) study the effects of chronic naltrexone from timed-release pellets on drinking in alcohol-naive mice. Naltrexone reduced ethanol preference in established drinkers, but its effects waned at increasing doses. Naltrexone slowed the acquisition of ethanol drinking, but was ineffective when readministered after a phase when ethanol was offered in the absence of naltrexone. Mice with chronic naltrexone pellets consumed greater amounts of ethanol and showed higher ethanol preference than did placebo-pelleted animals. The observed reduced efficacy of naltrexone with increasing dosage and chronic treatment may have been due to naltrexone-induced opiate receptor changes. Such changes are presumably more likely to occur when naltrexone doses remain high or perhaps accumulate. Thus, dose and frequency of administration may be important factors in determining naltrexone's effectiveness in treating alcohol dependence.     

Blockade of the corticotropin releasing factor type 1 receptor attenuates elevated ethanol drinking associated with drinking in the dark procedures

Background: Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations (BECs) reaching levels that have measurable affects on physiology and/or behavior. The present experiments determined whether the increased ethanol drinking caused by DID procedures can be attenuated by pretreatment with CP‐154,526; a corticotropin releasing factor type‐1 (CRF₁) receptor antagonist. Methods: In Experiment 1, male C57BL/6J mice received ethanol (20% v/v) in place of water for 4 hours, beginning with 3 hours into the dark cycle. On the fourth day, mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle. In Experiment 2, C57BL/6J mice had 2 hours of access to the 20% ethanol solution, beginning with 3 hours into the dark cycle on days 1 to 3, and 4 hours of access to the ethanol bottle on day 4 of DID procedures. Mice were given an intraperitoneal injection of one of the 4 doses of CP‐154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle on day 4. Tail blood samples were collected immediately after the 4‐hour ethanol access period on the fourth day of each experiment. Additional control experiments assessed the effects of CP‐154,526 on 4‐hour consumption of a 10% (w/v) sucrose solution and open‐field locomotor activity. Results: In Experiment 1, the vehicle‐treated group consumed approximately 4.0 g/kg/4 h of ethanol and achieved BECs of approximately 30 mg%. Furthermore, pretreatment with the CRF₁ receptor antagonist did not alter ethanol consumption. On the other hand, procedures used in Experiment 2 resulted in vehicle‐treated mice consuming approximately 6.0 g/kg/4 h of ethanol with BECs of about 80 mg%. Additionally, the 10 mg/kg dose of CP‐154,526 significantly reduced ethanol consumption and BECs to approximately 3.0 g/kg/4 h and 27 mg%, respectively, relative to vehicle‐treated mice. Importantly, the 10 mg/kg dose of the CRF₁R antagonist did not significantly alter 4‐hour sucrose consumption or locomotor activity. Conclusions: These data indicate that CRF₁R signaling modulates high, but not moderate, levels of ethanol drinking associated with DID procedures.     

Increased consumption but not operant self-administration of ethanol in mice lacking the RIIbeta subunit of protein kinase A

BACKGROUND: Accumulating evidence indicates that adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is involved in the neurobiological responses to ethanol. Previous reports indicate that mice lacking the RIIbeta subunit of PKA (RIIbeta(-/-)) voluntarily consume more ethanol than wild-type controls (RIIbeta(+/+)) using 2-bottle testing procedures. Although such procedures primarily measure consummatory behavior, operant self-administration procedures allow analysis of consummatory as well as appetitive or "ethanol-seeking" behavior (i.e., lever pressing is required to gain access to the ethanol solution). Therefore, we determined whether the high ethanol consumption characteristic of RIIbeta(-/-) mice would be complemented by increased appetitive ethanol-seeking behavior in an operant paradigm. METHODS: RIIbeta(-/-) (n=8) and RIIbeta(+/+) (n=8) mice were initially sucrose-faded until they were lever responding for nonsweetened ethanol (10, 14, and 18%). Following the self-administration testing, RIIbeta(+/+) and RIIbeta(-/-) mice were given access to 2 bottles, one containing water and the other ethanol to replicate the voluntary ethanol drinking data previously from our laboratory. Finally, immediately after voluntary consumption all mice were again tested for self-administration of 10% ethanol. Alterations in the reinforcement schedule were also explored as RIIbeta(+/+) and RIIbeta(-/-) mice were tested for self-administration of 10% ethanol at FR-3 and FR-5 schedules. RESULTS: The RIIbeta(-/-) mice displayed lower operant responding for ethanol and food reinforcement compared with RIIbeta(+/+) controls. However, this effect was driven by a significant increase in lever responses made by female RIIbeta(+/+) mice. When the excessive lever responses of the female RIIbeta(+/+) mice are accounted for, the RIIbeta(-/-) mice show ethanol lever responses comparable to controls. Following operant self-administration testing, RIIbeta(-/-) mice of both sexes consumed more ethanol solution compared with RIIbeta(+/+) mice during 2-bottle testing. CONCLUSIONS: Increased ingestion of ethanol by RIIbeta(-/-) mice is likely the result of altered PKA activity within neuronal pathways that control ethanol-consummatory behaviors. Conversely, the RIIbeta subunit of PKA appears not to play a critical role in neuronal pathways that regulate appetitive behaviors directed at obtaining ethanol. Finally, increased operant self-administration of food and ethanol by female wild-type mice was absent in female RIIbeta(-/-) mice, suggesting that normal PKA signaling may be part of a general, and sex-dependent, mechanism involved with reinforcement-seeking behavior.     

Assessment of voluntary ethanol consumption and the effects of a melanocortin (MC) receptor agonist on ethanol intake in mutant C57BL/6J mice lacking the MC-4 receptor

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent evidence shows that chronic exposure to ethanol significantly blunts central MC peptide immunoreactivity and MC receptor (MCR) agonists protect against high ethanol intake characteristic of C57BL/6J mice. Here, we assessed the role of the MC‐4 receptor (MC4R) in voluntary ethanol intake and in modulating the effects of the nonselective MCR agonist melanotan‐II (MTII) on ethanol consumption. Methods: To assess the role of the MC4R, MC4R knockout (Mc4r^?/?) and littermate wild‐type (Mc4r^+/+) mice on a C57BL/6J background were used. Voluntary ethanol (3, 5, 8, 10, 15, and 20%, v/v) and water intake were assessed using standard two‐bottle procedures. In separate experiments, Mc4r^?/? and Mc4r^+/+ mice were given intracerebroventricular (i.c.v.) infusion of MTII (0, 0.5, or 1.0 μg/1 μl) or intraperitoneal (i.p.) injection of MTII (0 or 5 mg/kg/5 ml). The effects of MTII (0 or 0.5 μg/1 μl, i.c.v.) on 10% sucrose and 0.15% saccharin intake were assessed in C57BL/6J mice. Results: Mc4r^?/? mice showed normal consumption of ethanol over all concentrations tested. I.c.v. infusion of MTII significantly reduced ethanol drinking in Mc4r^+/+ mice, but failed to influence ethanol intake in Mc4r^?/? mice. When administered in an i.p. injection, MTII significantly reduced ethanol drinking in both Mc4r^?/? and Mc4r^+/+ mice. MTII attenuated consumption of caloric (ethanol, sucrose, and food) and noncaloric (saccharin) reinforcers. Conclusions: When given centrally, the MCR agonist MTII reduced ethanol drinking by signaling through the MC4R. On the other hand, MTII‐induced reduction of ethanol drinking did not require the MC4R when administered peripherally. Together, the present observations show that the MC4R is necessary for the central actions of MCR agonists on ethanol drinking and that MTII blunts the consumption natural reinforcers, regardless of caloric content, in addition to ethanol.     

Lesions of the extended amygdala in C57BL/6J mice do not block the intermittent ethanol vapor-induced increase in ethanol consumption

Background: The central extended amygdala (cEA) which includes the central nucleus of the amygdala (CeA) and the lateral posterior bed nucleus of the stria terminalis (BNSTLP), has been proposed to play a key role in excessive ethanol consumption in humans (Koob and Le Moal, 2005 Nat Neurosci 8:1442). To examine this relationship, we used a murine model of ethanol dependence (Becker and Lopez, 2004 Alcohol Clin Exp Res 28:1829; Lopez and Becker, 2005 Psychopharmacology (Berl) 181:688) and compared animals with sham lesions and electrolytic lesions of the CeA and BNSTLP. Methods: Male C57BL/6J (B6) mice were first acclimated to a limited‐access 2‐bottle‐choice preference procedure. The access period began 3 hours into the dark phase of the light‐dark cycle and continued for 2 hours. Once acclimated (1 week), mice underwent chronic exposure to and intermittent withdrawal from ethanol vapor. The animals were then retested in the limited‐access 2‐bottle‐choice preference procedure. In some experiments, electrolytic and sham lesions of the CeA or BNSTLP were performed prior to initiating the 2‐bottle choice procedure. Results: In a series of 5 preliminary experiments, mice were randomly assigned either to the standard intermittent ethanol vapor procedure or to the standard procedure but with air in the vapor chamber (control). The air‐control procedure produced no change in ethanol intake when compared to baseline consumption. In contrast, intermittent ethanol vapor exposure increased ethanol consumption by almost 50%. The increase in consumption was associated with an increase in total fluid volume consumed and no change in ethanol preference. Lesions of both the BNSTLP and CeA significantly decreased baseline ethanol consumption, the former by decreasing fluid consumption and the latter by decreasing ethanol preference. Intermittent ethanol vapor exposure significantly increased consumption in both the BNSTLP‐ and CeA‐lesioned animals, largely by increasing the total volume of fluid consumed. Conclusions: The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited‐access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor‐induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.     

Interleukin-12 Therapy Restores Cell-Mediated Immunity in Ethanol-Consuming Mice

Previous studies from our laboratory show that ethanol consumption impairs antigen-specific, cell-mediated, but not, humoral immune responses of C57BL/6, BALB/c, and D011.10 T-cell receptor transgenic mice. This ethanol-associated deficit is associated with decreased interleukin (IL)-12 and interferon–γ (IFN-γ) production, but not IL-2 or antigen-specific T-cell proliferation by explanted leukocytes from ethanol-consuming mice. IL-12 expression by macrophage/monocytes is viewed as a requirement for the production of IFN-γ by Th1 lymphocytes that mediate cellular immunity. In this study, we restored antigen-specific, cell-mediated immunity, delayed hypersensitivity, to ethanol-consuming C57BL/6 or BALB/c mice with a single 100 ng of intravenous injection of recombinant IL-12 at the time of immunization. The addition of exogenous recombinant IL-12 to co-cultures of antigen-presenting cells derived from ethanol-consuming mice and purified T cells derived from ethanol-nonconsuming DO11.10 repairs the ability of Th1 cells to make IFN-γ in response to antigen. Administration of recombinant IL-12 opens a potential for restoring cell-mediated immune function to ethanol-consuming individuals.     

The Alcohol Deprivation Effect in C57BL/6J Mice is Observed Using Operant Self-Administration Procedures and is Modulated by CRF-1 Receptor Signaling

Background: The alcohol deprivation effect (ADE) is characterized by transient excessive alcohol consumption upon reinstatement of ethanol following a period of ethanol deprivation. While this phenomenon has been observed in rats using both bottle drinking (consummatory behavior) and operant self‐administration (consummatory and appetitive “ethanol‐seeking” behavior) procedures, ADE studies in mice have primarily relied on bottle drinking measures. Furthermore, the neurochemical pathways that modulate the ADE are not well understood. Therefore, we determined whether the ADE can be observed in C57BL/6J mice using operant self‐administration procedures and if expression of the ADE is modulated by the corticotropin releasing factor‐1 (CRF‐1) receptor. Methods: C57BL/6J mice were trained in a 2‐hour operant self‐administration paradigm to lever press for 10% ethanol or water on separate response keys. Between operant sessions, mice had access to ethanol in their homecage. Once stable responding occurred, mice were deprived of ethanol for 4 days and were then retested with ethanol in the operant paradigm for 3 consecutive days. Next, to assess the role of the CRF‐1 receptor, mice were given intraperitoneal (i.p.) injection (0, 10, or 20 mg/kg) of the CRF‐1 receptor antagonist CP‐154,526 30 minutes before ADE testing. Additional experiments assessed (i) ADE responding in which the alternate response lever was inactive, (ii) the effects of CP‐154,526 on self‐administration of a 1% sucrose solution following 4 days of deprivation, and (iii) ADE responding in which mice did not received i.p. injections throughout the experiment. Results: Mice exhibited a significant increase in postdeprivation lever responding for ethanol with either a water reinforced or inactive alternate lever. Interestingly, i.p. injection of a 10 mg/kg dose of CP‐154,526 protected against the ADE while not affecting lever responding for a sucrose solution. Finally, baseline and deprivation‐induced increases of ethanol reinforced lever responding were greater in mice not given i.p. injections. Conclusions: The ADE in C57BL/6J mice can be modeled using the operant self‐administration paradigm and increased ethanol self‐administration associated with the ADE is modulated by CRF‐1 receptor signaling.     

Alteration of ethanol drinking in mice via modulation of the GABA(A) receptor with ganaxolone, finasteride, and gaboxadol

Background: Neurosteroids and other γ‐aminobutyric acid_A (GABA_A) receptor–modulating compounds have been shown to affect ethanol intake, although their mechanism remains unclear. This study examined how patterns of 24‐hour ethanol drinking in mice were altered with the synthetic GABAergic neurosteroid ganaxolone (GAN), with an inhibitor of neurosteroid synthesis (finasteride [FIN]), or a GABA_A receptor agonist with some selectivity at extrasynaptic receptors (gaboxadol HCL [THIP]). Methods: Male C57BL/6J mice had continuous access to a 10% v/v ethanol solution (10E) or water. Using lickometer chambers, drinking patterns were analyzed among mice treated in succession to GAN (0, 5, and 10 mg/kg), FIN (0 or 100 mg/kg), and THIP (0, 2, 4, 8, and 16 mg/kg). Results: GAN shifted drinking in a similar but extended manner to previous reports using low doses of the neurosteroid allopregnanolone (ALLO); drinking was increased in hour 1, decreased in hours 2 and 3, and increased in hours 4 and 5 postinjection. THIP (8 mg/kg) and FIN both decreased 10E drinking during the first 5 hours postinjection by 30 and 53%, respectively, while having no effect on or increasing water drinking, respectively. All 3 drugs altered the initiation of drinking sessions in a dose‐dependent fashion. FIN increased and GAN decreased time to first lick and first bout. THIP (8 mg/kg) decreased time to first lick but increased time to first bout and attenuated first bout size. Conclusions: The present findings support a role for the modulation of ethanol intake by neurosteroids and GABA_A receptor–acting compounds and provide hints as to how drinking patterns are shifted. The ability of THIP to alter 10E drinking suggests that extrasynaptic GABA_A receptors may be involved in the modulation of ethanol intake. Further, the consistent results with THIP to that seen previously with high doses of ALLO suggest that future studies should further examine the relationship between neurosteroids and extrasynaptic GABA_A receptors, which could provide a better understanding of the mechanism by which neurosteroids influence ethanol intake.     

Topiramate does not affect the acquisition or expression of ethanol conditioned place preference in DBA/2J or C57BL/6J mice

BACKGROUND: Topiramate, an anticonvulsant, has been reported to increase the number of abstinent days and decrease craving in alcohol-dependent individuals. However, the neurobiological basis for topiramate's effect is unknown. To assess topiramate's effect on ethanol's rewarding and conditioning rewarding effects, the present experiments examined the effects of topiramate on the acquisition and expression of ethanol-induced conditioned place preference (CPP) in DBA/2J and C57BL/6J mice. METHODS: A biased apparatus and subject assignment were used. Mice received ethanol (2 g/kg) or saline paired with an initially nonpreferred floor (CS+) and saline paired with an initially preferred floor (CS-) for 5-minute conditioning trials. During the acquisition experiments, mice received a pretreatment of topiramate (0, 5, 10, 20, 50, or 100 mg/kg) 1 hour before the CS+ trials. On intervening CS- trials, mice received a pretreatment of saline. For the preference test, all mice received saline injections and were placed on a split floor for a 30-minute test. During the expression experiments, mice received no drug pretreatment on conditioning trials, but were pretreated with topiramate (0, 10, 50, or 100 mg/kg) 1 hour before the test session. RESULTS: Ethanol-induced CPP was observed in both strains, but topiramate did not affect the acquisition or expression of ethanol-induced CPP in either strain. Despite its failure to alter CPP, topiramate produced dose-dependent locomotor activating effects in both strains. These effects were observed both in the presence and in the absence of ethanol. CONCLUSIONS: These findings indicate that topiramate has no effect on ethanol's rewarding or conditioned rewarding effects as indexed by the place conditioning procedure. Thus, these studies raise the possibility that topiramate's efficacy in the treatment of alcoholism results from its impact on brain areas other than those that mediate ethanol's rewarding or conditioned rewarding effects. One alternative possibility is that topiramate decreases withdrawal-induced negative affective states that normally contribute to relapse.     

Period determination in the food-entrainable and methamphetamine-sensitive circadian oscillator(s)

Daily rhythmic processes are coordinated by circadian clocks, which are present in numerous central and peripheral tissues. In mammals, two circadian clocks, the food-entrainable oscillator (FEO) and methamphetamine-sensitive circadian oscillator (MASCO), are "black box" mysteries because their anatomical loci are unknown and their outputs are not expressed under normal physiological conditions. In the current study, the investigation of the timekeeping mechanisms of the FEO and MASCO in mice with disruption of all three paralogs of the canonical clock gene, Period, revealed unique and convergent findings. We found that both the MASCO and FEO in Per1(-/-)/Per2(-/-)/Per3(-/-) mice are circadian oscillators with unusually short (～21 h) periods. These data demonstrate that the canonical Period genes are involved in period determination in the FEO and MASCO, and computational modeling supports the hypothesis that the FEO and MASCO use the same timekeeping mechanism or are the same circadian oscillator. Finally, these studies identify Per1(-/-)/Per2(-/-)/Per3(-/-) mice as a unique tool critical to the search for the elusive anatomical location(s) of the FEO and MASCO.     

Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis

GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.     

Changes of the intestinal endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma （3LL）： An immunohistochemical quantitative study

AIM: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL). METHODS: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28~(th) d after implantation of 3LL cells (1×10~5 cell/mouse). RESULTS: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition, glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham. CONCLUSION: Implantation of tumor cell mass (3LL) induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.     

Ethanol‐Induced Increase of Agouti‐Related Protein (AgRP) Immunoreactivity in the Arcuate Nucleus of the Hypothalamus of C57BL/6J,but not 129/SvJ,Inbred Mice

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor, pro‐opiomelanocortin (POMC). Previous research has shown that MC receptor (MCR) agonists reduce, and MCR antagonists increase, ethanol consumption in rats and mice. Consistently, genetic deletion of the endogenous MCR antagonist, agouti‐related protein (AgRP), causes reductions of ethanol‐reinforced lever pressing and binge‐like ethanol drinking in C57BL/6J mice. Ethanol also has direct effects on the central MC system, as chronic exposure to an ethanol‐containing diet causes significant reductions of α‐melanocyte stimulating hormone (α‐MSH) immunoreactivity in specific brain regions of Sprague‐Dawley rats. Together, these observations suggest that the central MC system modulates neurobiological responses to ethanol. To further characterize the role of the MC system in responses to ethanol, here we compared AgRP and α‐MSH immunoreactivity in response to an acute injection of saline or ethanol between high ethanol drinking C57BL/6J mice and moderate ethanol drinking 129/SvJ mice. Methods: Mice received an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg or 3.5 g/kg; mixed in 0.9% saline) or an equivolume of 0.9% saline. Two hours after injection, animals were sacrificed and their brains were processed for AgRP and α‐MSH immunoreactivity. Results: Results indicated that acute ethanol administration triggered a dose‐dependent increase in AgRP immunoreactivity in the arcuate (ARC) of C57BL/6J mice, an effect that was not evident in the 129/SvJ strain. Although acute administration of ethanol did not influence α‐MSH immunoreactivity, C57BL/6J mice had significantly greater overall α‐MSH immunoreactivity in the ARC, dorsomedial, and lateral regions of the hypothalamus relative to the 129/SvJ strain. In contrast, C57BL/6J mice displayed significantly lower α‐MSH immunoreactivity in the medial amygdala. Conclusions: The results show that acute ethanol exposure has direct effects on endogenous AgRP activity in ethanol preferring C57BL/6J mice. It is suggested that ethanol‐induced increases in AgRP may be part of a positive feedback system that stimulates excessive binge‐like ethanol drinking in C57BL/6J mice. Inherent differences in α‐MSH immunoreactivity may contribute to differences in neurobiological responses to ethanol that are characteristically observed between the C57BL/6J and 129/SvJ inbred strains of mice.     

Establishment of an Aging Farm of F344/N Rats and C57BL/6 Mice at the National Institute for Longevity Sciences (NILS)

In 1996 the National Institute for Longevity Sciences (NILS) started an aging/aged farm (Aging Farm), an animal farm for producing aging/aged laboratory rodents on inbred strains, F344/N rats and C57BL/6 mice at its Experimental Animal Facility Wing, based on plans prepared by the Laboratory Animal Research Facilities (LARF). The NILS Aging Farm, being well established, began internal supply of aging/aged laboratory rodents in 1999 to promote both aging and longevity science. This report describes development of the NILS Aging Farm under NILS Aging Farm Guide and the effectiveness of the Guide.     

Ethanol exposure and withdrawal sensitizes the rat hippocampal CA1 pyramidal cell region to beta-amyloid (25-35)-induced cytotoxicity: NMDA receptor involvement

Abstract : Background: Millions of Americans suffer from Alzheimer's Disease (AD), which is characterized by significant neurological impairment and an accumulation in brain tissue of senile plaques consisting of beta amyloid (Aβ) peptide. The hippocampus, a region primarily responsible for learning and memory, appears to be particularly susceptible to AD‐related injury and chronic alcohol abuse. Although certain risk factors for AD are known, it is unclear if alcohol abuse or dependence may contribute to neuropathology in AD. Recent research suggests that low‐to‐moderate consumption of alcohol may protect against development of AD, while alcohol dependence may increase risk of developing AD. Therefore, the current studies aimed to investigate the effects of exposure to 50 or 100 mM ethanol (EtOH) and withdrawal on hippocampal injury induced by Aβ peptide treatment. Methods: The present studies exposed organotypic hippocampal slice cultures to 50 or 100 mM ethanol (EtOH) for 10 days, after which the slices underwent ethanol withdrawal (EWD) in the presence of varying concentrations of Aβ 25‐35 (0.1, 1, 10 μM), or 35‐25 (200 μM), a negative control reverse sequence peptide. Cellular injury, as evidenced by uptake of propidium iodide (PI), was assessed for each subregion of the hippocampal complex (CA1, CA3, and dentate gyrus). Results: Cellular injury in the CA1 pyramidal cell layer was significantly increased during withdrawal from exposure to 100 mM, but not 50 mM, EtOH. Exposure to Aβ in ethanol‐naïve cultures did not produce significant cytotoxicity. However, exposure to Aβ during EWD from 100 mM produced marked increases in CA1 pyramidal cell region cytotoxicity, effects reversed by cotreatment with a nontoxic concentration of the NMDA receptor channel blocker MK‐801 (20 μM). Conclusions: These data suggest that withdrawal from exposure to a high concentration of EtOH produces marked cellular injury in the hippocampus, particularly the CA1 subregion. Further, this EtOH exposure and withdrawal regimen sensitizes the hippocampus to the toxic effects of Aβ treatment in a manner reflecting over activity of NMDA receptor function.     

Measurements of insulin activities in pancreas and serum of mice with spontaneous (“obese” and “New Zealand obese”) and induced (goldthioglucose) obesity and hyperglycemia,with considerations on the pathogenesis of the spontaneous syndrome

Summary 1. Insulin-like activity (ILA) and immunoreactive insulin (IRI) of pancreatic extracts and of serum have been measured in lean and obese (obob) mice from the Jackson Memorial Laboratory in Bar Harbor, Maine; in New Zealand obese mice; and in goldthioglucose-obese Swiss mice, and their lean controls. In all three types of mice the relative amounts of suppressible and nonsuppressible insulin activities of serum have also been established. — 2. Pancreatic content of IRI in (obob) mice is much greater than in their lean sibblings at five months of age, but smaller at 5 weeks, whereas serum IRI and ILA of (obob) mice are greatly in excess of the same activities of serum from their lean littermates at both ages. It is suggested that increased peripheral demand for insulin preceeds increased insulin synthesis and storage in this type of obesity. — 3. In all three strains of mice, obese or lean, the ratio of biologic to immunologic activity of pancreatic extracts was constant and close to unity. In serum, this ratio was also constant but approximately five times greater than unity. These findings do not support the notion that differences in insulin structure might account for differences in the biological activity of insulin in obese and non-obese animals; they are in keeping with the concept of a potentiation of serum insulin activity by some component of serum in all mice. — 4. Approximately 90% of serum ILA in all three types of obese mice studied was suppressed in the presence of anti-insulin serum, although the absolute levels of non-suppressible ILA were similar to those reported for other species. It is unlikely, therefore, that bound insulin accounts for a significant part of the increased ILA of serum of any of the three types of obese animals studied. — 5. Present data support the concept of a marked tissue-bound muscular resistance to insulin action as an important, possibly primary pathogenetic feature in (obob) mice. A similar, but much less marked muscular resistance can be demonstrated in NZO mice, whereas muscular resistance to insulin action is totally absent in goldthioglucose obese Swiss mice after a 24 hour fast.This work was supported by the Fonds National Suisse de la Recherche Scientifique (Grant No. 3618), Berne, Switzerland, the Fondation Emil Barell pour le développement de la recherche médico-scientifique, Basel, Switzerland, and the Diabetes Foundation, Inc., Boston, Massachusetts. Dr.Lambert is aspirant of the Fonds National Belge de la Recherche Scientifique.