Partitioning and kinetics of methylmercury among organs in captive mink (Neovison vison): A stable isotope tracer study.

Despite the importance of methylmercury (MeHg) as a neurotoxin, we have relatively few good data on partitioning and kinetics of MeHg among organs, particularly across the blood-brain barrier, for mammals that consume large quantities of fish. The objective of this study was to determine the partition coefficients between blood and brain, liver and kidney and fur for MeHg under steady-state conditions and to measure the half-lives for MeHg in these organs. Captive mink (Neovison vison) were fed a diet enriched with two stable isotopes of Hg, Me(199)Hg and Me(201)Hg for a period of 60 days. After a period of 10 days the diet was changed to contain only Me(201)Hg so that, between days 10 and 60, we were able to measure both uptake and elimination rates from blood, brain, liver kidney and fur. Liver and kidney response was very rapid, closely following changes in blood concentrations but there was a small lag time between peak blood concentrations and peak brain concentrations. Half-lives for MeHg were 15.4, 10.2 and 13.4 days for brain, liver and kidney, respectively. There was no measurable conversion of the MeHg to inorganic Hg (IHg) in the brain over the 60 day period, unlike in liver and kidney.

De novo lipogenesis is suppressed during fasting but upregulated at population decline in cyclic voles.

Arvicolines are susceptible to the development of fatty liver during short-term fasting. We examined the potential role of de novo lipogenesis (DNL) (i) in the development of fasting-induced fatty liver and (ii) during a population cycle by measuring the mRNA expression of acetyl-CoA carboxylase-1 (ACC1) and fatty acid synthase (FAS). Laboratory voles (Microtus oeconomus and Microtus arvalis) were fed or fasted for 12 or 18 h and their liver mRNA levels were determined. Both species showed decreased mRNA expression of ACC1 and FAS during fasting. This suggests that DNL does not participate in the development of fatty liver in voles, different from human non-alcoholic fatty liver disease. In wild bank voles (Myodes glareolus), the mRNA levels of the genes of interest were higher during the population decline compared to the increase phase. In conclusion, DNL was suppressed during acute fasting but upregulated during a long-term population decline-a period of purported scarcity of high-quality food.

The Role of Food Peptides in Lipid Metabolism during Dyslipidemia and Associated Health Conditions.

Animal and human clinical studies have demonstrated the ability of dietary food proteins to modulate endogenous lipid levels during abnormal lipid metabolism (dyslipidemia). Considering the susceptibility of proteins to gastric proteolytic activities, the hypolipidemic functions of proteins are possibly due, in part, to their peptide fragments. Food-derived peptides may directly modulate abnormal lipid metabolism in cell cultures and animal models of dyslipidemia. The peptides are thought to act by perturbing intestinal absorption of dietary cholesterol and enterohepatic bile acid circulation, and by inhibiting lipogenic enzymatic activities and gene expression in hepatocytes and adipocytes. Recent evidence indicates that the hypolipidemic activities of some peptides are due to activation of hepatic lipogenic transcription factors. However, detailed molecular mechanisms and structural requirements of peptides for these activities are yet to be elucidated. As hypolipidemic peptides can be released during enzymatic food processing, future studies can explore the prospects of combating metabolic syndrome and associated complications using peptide-rich functional food and nutraceutical products.

Methylmercury accumulation and elimination in mink (Neovison vison) hair and blood: results of a controlled feeding experiment using stable isotope tracers.

Concentrations of metals in hair are used often to develop pharmacokinetic models for both animals and humans. Although data on uptake are available, elimination kinetics are less well understood; stable isotope tracers provide an excellent tool for measuring uptake and elimination kinetics. In the present study, methylmercury concentrations through time were measured in the hair and blood of mink (Neovison vison) during a controlled 60-d feeding experiment. Thirty-four mink were fed a standard fish-based diet for 14 d, at the end of which (day 0), 4 mink were sacrificed to determine baseline methylmercury (MeHg) concentrations. From day 0 to day 10, the remaining mink were fed a diet consisting of the base diet supplemented with 0.513 ± 0.013 µg Me(199) Hg/g and 0.163 ± 0.003 µg Me(201) Hg/g. From day 10 to day 60, mink were fed the base diet supplemented with 0.175 ± 0.024 µg Me(201) Hg/g. Animals were sacrificed periodically to determine accumulation of Me(201) Hg in blood and hair over the entire 60-d period and the elimination of Me(199) Hg over the last 50 d. Hair samples, collected from each mink and cut into 2.0-mm lengths, indicate that both isotopes of MeHg appeared in the hair closest to the skin at approximately day 10, with concentrations in the hair reaching steady state from day 39 onward. The elimination rate of Me(199) Hg from the blood was 0.05/d, and the ratio of MeHg in the hair to blood was 119. A large fraction of MeHg (22% to >100%) was stored in the hair, suggesting that in fur-bearing mammals the hair is a major route of elimination of MeHg from the body.

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis.

We investigated the presence of inflammatory signs in the progression of fatty liver disease induced by fasting. Sixty standard black American mink (Neovison vison) were fasted for 0, 1, 3, 5, or 7 days and one group for 7 days followed by re-feeding for 28 days. Liver sections were evaluated histologically and liver mRNA levels indicating endoplasmic reticulum (ER) stress, adipogenic transformation, and inflammation were assessed by quantitative real-time PCR. After 3 days of fasting, the mink had developed moderate liver steatosis. Increased hyaluronan reactivity in lymphocytic foci but no Mallory-Denk bodies were seen in livers of the mink fasted for 5-7 days. Up-regulation of glucose-regulated protein, 78 kDa was observed on day 7 indicating ER stress, especially in the females. Liver lipoprotein lipase and monocyte chemoattractant protein 1 mRNA levels increased in response to 5-7 days of food deprivation, while tumor necrosis factor α (TNF-α) was the highest in the mink fasted for 5 days. The expression of the genes of interest, except for TNF-α, correlated with each other and with the liver fat content. The mRNA levels were found to change more rapidly below n-3/n-6 polyunsaturated fatty acid ratio threshold of 0.15. Following re-feeding, hepatocyte morphology and mRNA abundance returned to pre-fasting levels. Within the studied timeframe, evidence for ER stress, adipogenic transformation, and liver inflammation suggested incipient transition from steatosis to steatohepatitis with potential for development of more severe liver disease. This may present a possibility to influence disease progression before histologically observable steatohepatitis.

Ideal body condition improves reproductive performance and influences genetic health in female mink.

Selection for large body size in mink (Neovison vison) can result in obesity, which is associated with poor reproduction and metabolic disorders. Caloric restriction is effective in diminishing oxidative stress and delaying aging-related diseases. This study investigated the effects of moderate diet restriction on body condition, health, and reproductive success of mink breeder females. One-hundred control females were fed according to conventional feeding practice, while the feed allowance of their 100 sister-pair females was restricted in order to maintain an ideal body condition during the fall and eliminate the need for drastic slimming prior to breeding. Repeated measures analyses revealed that body weight gain during the fall and weight loss prior to breeding was significantly less for the restricted females. The restricted females had significantly larger live litters (5.88 kits) than the control dams (4.62 kits; P<0.05). They were also able to maintain their body weight and condition during early lactation and were able to regain weight and condition post-lactation, unlike their control sisters. Based on their comet scores (restricted: 88; control: 116), the restricted primiparous females experienced less DNA damage (P<0.05), while no significant differences were apparent for the multiparous females (restricted: 170; control: 153). No changes in telomere length were observed among the dams. Moderate diet restriction of mink breeder females during the fall eliminated extreme fluctuations in body weight and condition throughout the seasonal production cycle and improved their litter size, and in primiparous females, lessened DNA damage.

High feeding intensity increases the severity of fatty liver in the American mink (Neovison vison) with potential ameliorating role for long-chain n-3 polyunsaturated fatty acids.

BACKGROUND: Rapid body fat mobilization, obesity, and an inadequate supply of n-3 polyunsaturated fatty acids (PUFA) have been suggested to play roles in the etiology of fatty liver in the American mink (Neovison vison). This study examined the effects of feeding intensity and dietary fat source on fatty liver induced by fasting. In a multi-factorial design, 3 different fat sources (herring oil, rich in n-3 PUFA, soya oil, rich in n-6 PUFA, and canola oil, rich in n-9 monounsaturated fatty acids) were fed to mink at a low and high feeding intensity for 10 weeks, followed by an overnight or a 5-day fasting treatment to induce fatty liver. RESULTS: Fasting led to the development of fatty liver with increased severity in the mink fed at the high feeding intensity. The herring oil diet, high in long-chain n-3 PUFA, was found to decrease the severity of fatty liver in the mink at the high feeding intensity. CONCLUSION: Preventing excessive weight gain and increasing dietary intake of n-3 long-chain PUFA may help prevent excessive lipid accumulation during prolonged periods of fasting or inappetence by promoting hepatic fatty acid oxidation.

Manifestations of fasting-induced fatty liver and rapid recovery from steatosis in voles fed lard or flaxseed oil lipids.

Long-chain n-3 polyunsaturated fatty acids (PUFA) can have beneficial effects against fat deposition, cardiovascular diseases, and liver steatosis. We investigated how diets based on lard (predominantly saturated and monounsaturated fatty acids) or flaxseed oil (rich in 18:3n-3) affect liver fat-% and fatty acid profiles of tundra voles (Microtus oeconomus). We also studied potential participation of hyaluronan (HA) in the pathology of fatty liver and whether the development and recovery of fasting-induced steatosis are influenced by n-3 PUFA. The dietary fatty acid composition was manifested in the liver fatty acid signatures. Fasting for 18 h induced macrovesicular steatosis and the liver fat-% increased to 22% independent of the preceding diet. Fasting-induced steatosis did not involve inflammation or connective tissue activation indicated by the absence of both leukocyte accumulation and increased HA. Food deprivation modified the liver fatty acid signatures to resemble more closely the diets. Fasting reduced the proportions of long-chain n-3 PUFA in both dietary regimes and n-3/n-6 PUFA ratios in the lard-fed voles. Decreases in long-chain n-3 PUFA may promote lipid accumulation by modulating the expression of lipid-metabolizing genes. Dietary 18:3n-3 did not prevent the development or attenuate the manifestation of steatosis in the fasted voles or promote the recovery.

Physiological adaptations to prolonged fasting in the overwintering striped skunk (Mephitis mephitis).

Wintertime physiology of captive striped skunks (Mephitis mephitis) in response to cold ambient temperature (Ta) and fasting was investigated with body temperature (Tb) and activity recordings and analyses of hematology, plasma biochemistry and tissue fatty acids (FA). After 105 days of food deprivation, the skunks were in phase II of fasting indicated by the elevated plasma nonesterified FA and glycerol but no accumulation of nitrogen end products. Shorter-chain saturated and monounsaturated FA together with C18-20 n-3 polyunsaturated FA were preferentially mobilized. Individual amino acids responded to fasting in a complex manner, while essential and nonessential amino acid sums remained stable. Increases in hemoglobin and hematocrit suggested dehydration. The activity levels were lower in mid-January-early March, and the activity bouts were mostly displayed between 17:00-23:00 h. Daily torpor was observed in two females with 29 and 46 bouts. The deepest torpor (Tb<31 °C) occurred between dawn and early afternoon and lasted for 3.3 ± 0.18 h. The average minimum Tb was 29.2 ± 0.15 °C and the lowest recorded Tb was 25.8 °C. There was significant relation between the average 24-h Tb and Ta. Increases in wintertime Ta, as predicted by climate change scenarios, could influence torpor patterns in the species.

Effects of methylmercury on epigenetic markers in three model species: mink, chicken and yellow perch.

We previously reported that methylmercury (MeHg) exposure is associated with DNA hypomethylation in the brain stem of male polar bears. Here, we conveniently use archived tissues obtained from controlled laboratory exposure studies to look for evidence that MeHg can disrupt DNA methylation across taxa. Brain (cerebrum) tissues from MeHg-exposed mink (Neovison vison), chicken (Gallus gallus) and yellow perch (Perca flavescens) were analyzed for total Hg levels and global DNA methylation. Tissues from chicken and mink, but not perch, were also analyzed for DNA methyltransferase (DNMT) activity. In mink we observed significant reductions in global DNA methylation in an environmentally-relevant dietary exposure group (1 ppm MeHg), but not in a higher group (2 ppm MeHg). DNMT activity was significantly reduced in all treatment groups. In chicken or yellow perch, no statistically significant effects of MeHg were observed. Dose-dependent trends were observed in the chicken data but the direction of the change was not consistent between the two endpoints. Our results suggest that MeHg can be epigenetically active in that it has the capacity to affect DNA methylation in mammals. The variability in results across species may suggest inter-taxa differences in epigenetic responses to MeHg, or may be related to differences among the exposure scenarios used as animals were exposed to MeHg through different routes (dietary, egg injection), for different periods of time (19-89 days) and at different life stages (embryonic, juvenile, adult).

Role of hepatic de novo lipogenesis in the development of fasting-induced fatty liver in the American mink (Neovison vison).

American mink (Neovison vison) develop fatty liver quickly in response to food deprivation, which results in preferential mobilisation of n-3 PUFA. The altered n-3:n-6 PUFA ratio in the liver may activate the endocannabinoid system resulting in increased lipid synthesis. The objective of the present study was to investigate the effects of feeding intensity (80 or 120% RDA), dietary fat source (n-3, n-6 or n-9 fatty acids (FA)) and short-term fasting (1-7 d) on hepatic de novo lipogenesis (DNL) and the development of fatty liver in mink. Significantly elevated expression of mRNA encoding for acetyl-CoA carboxylase-1 (ACC-1) and FA synthase (FAS) was observed in the liver of mink fasted for 5-7 d, while upon re-feeding for 28 d after a 7 d food deprivation, DNL returned to pre-fasting levels. The females had a higher expression of ACC-1 and FAS mRNA than the males. In the non-fasted animals, dietary fat source and feeding intensity had significant effects on ACC-1 mRNA. The highest levels were observed in the mink fed the rapeseed oil (n-9) diet at 80% RDA, while the lowest levels were seen when the same diet was fed at 120% RDA. For FAS, the highest gene expression was seen in the fasted mink fed at 80% RDA and the lowest in the non-fasted mink fed at 80%. It is concluded that short-term food deprivation induces hepatic lipidosis in mink and that during this process, hepatic DNL further exacerbates liver fat accumulation.

Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis.

Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7d of food deprivation and 28d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology.

A survey of Aleutian mink disease virus infection of feral American mink in Nova Scotia.

Spleen samples from 14 mink that were trapped in 4 counties of Nova Scotia were tested for the presence of the Aleutian mink disease virus (AMDV) by polymerase chain reaction. Viral DNA was not detected in samples from Kings County (n = 2), but was detected in all the mink sampled from Colchester (n = 2) and Halifax (n = 6) counties, and 3 of 4 mink from Yarmouth County. The high level of AMDV-infected mink in Colchester and Halifax counties may pose a serious threat to the captive mink and wild animal populations. Because treatment of infected free-ranging mink is not an option, AMDV control strategies for the captive mink should be primarily focused on bio-security to protect clean ranches.

In vitro and whole animal evidence that methylmercury disrupts GABAergic systems in discrete brain regions in captive mink.

The effects of mercury (Hg) on key components of the GABAergic system were evaluated in discrete brain regions of captive juvenile male American mink (Neovison vison) using in vitro and in vivo (whole animal) experimental approaches. In vitro studies on cortical brain tissues revealed that inorganic Hg (HgCl(2); IC50=0.5+/-0.2microM) and methyl Hg (MeHgCl; IC50=1.6+/-0.2microM) inhibited glutamic acid decarboxylase (GAD; EC 4.1.1.15) activity. There were no Hg-related effects on [(3)H]-muscimol binding to GABA(A) receptors (IC50s>100microM). HgCl(2) (IC50=0.8+/-0.3microM) but not MeHgCl (IC50>100microM) inhibited GABA-transaminase (GABA-T; EC 2.6.1.19) activity. In a whole animal study, neurochemical indicators of GABAergic function were measured in brain regions (occipital cortex, cerebellum, brain stem, and basal ganglia) of captive mink fed relevant levels of MeHgCl (0 to 2microg/g feed, ppm) daily for 89d. No effects on GAD activity were measured. Concentration-dependent decreases in [(3)H]-muscimol binding to GABA(A) receptors and GABA-T activity were found in several brain regions, with reductions as great as 94% (for GABA(A) receptor levels) and 71% (for GABA-T activity) measured in the brain stem and basal ganglia. These results show that chronic exposure to environmentally relevant levels of MeHg disrupts GABAergic signaling. Given that GABA is the main inhibitory neurotransmitter in the mammalian nervous system, prolonged disruptions of its function may underlie the sub-clinical impacts of MeHg at relevant levels to animal health.

Variation of cholinergic biomarkers in brain regions and blood components of captive mink.

Studies are increasingly using cholinergic parameters as biomarkers of early neurotoxicity, but few have characterized this system in ecologically relevant model organisms. In the present study, key neurochemicals in the cholinergic pathway were measured and analyzed from discrete parts of brain and blood from captive mink (Mustela vison). Similar to other mammals, the regional distribution of cholinergic parameters in the brain could be ranked from highest to lowest as: basal ganglia > occipital cortex > brain stem > cerebellum (F (3,192) = 172.1, p < 0.001). Higher variation in cholinergic parameters was found in the cerebellum (coefficient of variation = 34.9%), and the least variation was measured in the brain stem (19.7%). Variation was also assessed by calculating the difference between the lowest and highest measures among individual animals: choline acetyltransferase (1.6x fold difference), cholinesterase (2.0x), muscarinic receptor levels (2.4x), acetylcholine (3.7x), nicotinic receptor levels (3.9x), and choline transporter (5.0x). In blood samples, activity and inter-individual variation of cholinesterase was highest in whole blood and lowest in plasma and serum. By using captive mink of a common genetic source, age, gender, and rearing conditions, these data help establish normal levels, ranges, and variations of cholinergic biomarkers among brain regions, blood components, and individual animals. Such information may better enable the utility of cholinergic biomarkers in environmental assessments.

Rapid development of fasting-induced hepatic lipidosis in the American mink (Neovison vison): effects of food deprivation and re-alimentation on body fat depots, tissue fatty acid profiles, hematology and endocrinology.

Hepatic lipidosis is a common pathological finding in the American mink (Neovison vison) and can be caused by nutritional imbalance due to obesity or rapid body weight loss. The objectives of the present study were to investigate the timeline and characterize the development of hepatic lipidosis in mink in response to 0-7 days of food deprivation and liver recovery after 28 days of re-feeding. We report here the effects on hematological and endocrine variables, body fat mobilization, the development of hepatic lipidosis and the alterations in the liver lipid classes and tissue fatty acid (FA) sums. Food deprivation resulted in the rapid mobilization of body fat, most notably visceral, causing elevated hepatosomatic index and increased liver triacylglycerol content. The increased absolute amounts of liver total phospholipids and phosphatidylcholine suggested endoplasmic reticulum stress. The hepatic lipid infiltration and the altered liver lipid profiles were associated with a significantly reduced proportion of n-3 polyunsaturated FA (PUFA) in the livers and the decrease was more evident in the females. Likewise, re-feeding of the female mink resulted in a more pronounced recovery of the liver n-3 PUFA. The rapid decrease in the n-3/n-6 PUFA ratio in response to food deprivation could trigger an inflammatory response in the liver. This could be a key contributor to the pathophysiology of fatty liver disease in mink influencing disease progression.

Response to fasting in an unnaturally obese carnivore, the captive European polecat Mustela putorius.

The European polecat (Mustela putorius) is a naturally lean carnivore prone to excessive weight gain in captivity. This study assessed its suitability to investigate the natural history of the obese phenotype displayed in overweight humans, domestic animals, and seasonally obese wild mammals. Ten farm-bred polecats were subjected to a 5-day fast with 10 controls. Obesity (40% body fat) was associated with an unfavorable plasma lipid profile and high glucose and insulin concentrations. The polecats were in phase II of fasting with normoglycemia, low liver carbohydrate stores, and decreased plasma concentrations of urea and most amino acids. Although the plasma nonesterified fatty acid (NEFA) levels were elevated, the adipose tissue lipase activities suggested a blunted lipolytic response. Lipid mobilization was more efficient from intraabdominal fat. The animals developed hepatic lipidosis with elevated NEFA influx into the liver and losses of n-3 polyunsaturated fatty acids and arginine as hypothetical etiological factors. The plasma leptin, insulin, and triiodothyronine levels decreased but were not accompanied by reduced sex steroid or increased stress hormone concentrations. The blunted lipolytic response often encountered in obesity suggests that the organism is trying to defend the obese phenotype. Liver lipidosis and decreased insulin and triiodothyronine levels seem to be among the most consistent responses to fasting manifested in diverse mammalian orders and different levels of body fatness. The polecat could be recommended as an easily accessible carnivorean model to study the natural history of the obese phenotype and its comorbidities.

Molecular characterization of the Himalayan mink.

A rare color variant of the American mink (Neovison vison), discovered on a ranch in Nova Scotia and referred to as the "marbled" variety, carries a distinctive pigment distribution pattern resembling that found in some other species, e.g., the Siamese cat and the Himalayan mouse. We tested the hypothesis that the color pattern in question-light-colored body with dark-colored points (ears, face, tail, and feet)-is due to a mutation in the melanin-producing enzyme tyrosinase (TYR) that results in temperature-sensitive pigment production. Our study shows that marbled mink carry a mutation in exon 4 of the TYR gene (c.1835C > G) which results in an amino acid substitution (p.H420Q). The location of this substitution corresponds to the amino acid position that is also mutated in the TYR protein of the Himalayan mouse. Thus, the marbled variant is more aptly referred to as the Himalayan mink.

Fatty acid composition and development of hepatic lipidosis during food deprivation--mustelids as a potential animal model for liver steatosis.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome characterized by asymptomatic hepatic steatosis. It is present in most cases of human obesity but also caused e.g., by rapid weight loss. The patients have decreased n-3 polyunsaturated fatty acid (PUFA) proportions with decreased percentages of 18:3(n-3), 20:5(n-3) and 22:6(n-3) and an increased n-6/n-3 PUFA ratio in liver and/or white adipose tissue (WAT). The present study examined a new experimental model to study liver steatosis with possible future applications to NAFLD. Ten European polecats (Mustela putorius), the wild form of the domestic ferret, were food-deprived for 5 days with 10 fed animals as controls. The food-deprived animals showed micro- and macrovesicular hepatic steatosis, decreased proportions of 20:5(n-3), 22:6(n-3) and total n-3 PUFA and increased n-6/n-3 PUFA ratios in liver and WAT. At the same time, the product/precursor ratios decreased in liver. The observed effects can be due to selective fatty acid mobilization preferring n-3 PUFA over n-6 PUFA, decreased Delta5 and Delta6 desaturase activities, oxidative stress, decreased arginine availability and activation of the endocannabinoid system. Hepatic lipidosis induced by food deprivation was manifested in the fatty acid composition of the polecat with similarities to human NAFLD despite the different principal etiologies.

The effects of mercury on muscarinic cholinergic receptor subtypes (M1 and M2) in captive mink.

A combination of in vitro (competitive binding assays) and in vivo (tissues from animals exposed to dietary methyl mercury, MeHg) experimental procedures was employed to assess the effects of mercury (MeHg, HgCl(2)) on the two-key muscarinic cholinergic (mACh) receptor subtypes (M1, M2) in two brain regions (occipital cortex, brain stem) of captive mink (Mustela vison). In vitro, HgCl(2) and MeHg were equipotent in inhibiting [(3)H]-pirenzipine binding to the M1 receptor in the occipital cortex, but in the brain stem, MeHg was about 65x more potent than HgCl(2). For the M2 receptor, both HgCl(2) and MeHg were more potent at inhibiting [(3)H]-AFDX-384 binding in the occipital cortex than in the brain stem. Within each brain region, HgCl(2) was more potent at inhibiting [(3)H]-AFDX-384 binding than MeHg. In vivo exposure of captive mink to MeHg (0.5, 1, and 2ppm MeHg in the diet for 89 days) resulted in greater binding of radioligands to the M1 and M2 receptor in the occipital cortex, but not in the brain stem, when compared to control animals. Based on the in vitro results, we could not conclude which mACh receptor subtype or brain region was most sensitive to Hg, but the in vivo findings suggest that Hg preferentially affects mACh receptor subtype (M1 and M2) levels in the occipital cortex. By studying distinct mACh receptors, these results extend upon previous studies in laboratory rodents and wildlife that showed Hg to affect the global population of mACh receptors.