Genome-wide analysis in 756,646 individuals provides first genetic evidence that ACE2 expression influences COVID-19 risk and yields genetic risk scores predictive of severe disease.

SARS-CoV-2 enters host cells by binding angiotensin-converting enzyme 2 (ACE2). Through a genome-wide association study, we show that a rare variant (MAF = 0.3%, odds ratio 0.60, P=4.5×10-13) that down-regulates ACE2 expression reduces risk of COVID-19 disease, providing human genetics support for the hypothesis that ACE2 levels influence COVID-19 risk. Further, we show that common genetic variants define a risk score that predicts severe disease among COVID-19 cases.

Global resuscitation alliance utstein recommendations for developing emergency care systems to improve cardiac arrest survival.

INTRODUCTION: The Global Resuscitation Alliance (GRA) was established in 2015 to improve survival for Out- of-Hospital Cardiac Arrest (OHCA) using the best practices developed by the Seattle Resuscitation Academy. However, these 10 programs were recommended in the context of developed Emergency Care Systems (ECS). Implementing these programs can be challenging for ECS at earlier stages of development. We aimed to explore barriers faced by developing ECS and to establish pre-requisites needed. We also developed a framework by which developing ECS may use to build their emergency response capability. METHOD: A consensus meeting was held in Singapore on 1st-2nd August 2017. The 74 participants were key stakeholders from 26 countries, including Emergency Medical Services (EMS) directors, physicians and academics, and two Physicians who sit on the World Health Organisation (WHO) panel for development of Emergency Care Systems. Five discussion groups examined the chain of survival: community, dispatch, ambulance and hospital; a separate group considered perinatal resuscitation. Discussion points were voted upon to reach a consensus. RESULTS: The answers and discussion points from each groupwere classified into a table adapted from WHO's framework of development for Emergency Services. After which, it was used to construct the modified survival framework with the chain of survival as the backbone. Eleven key statements were then derived to describe the pre-requisites for achieving the GRA 10 programs. The participants eventually voted on the importance and feasibility of these 11 statements as well as the GRA 10 programs using a matrix that is used by organisations to prioritise their action steps. CONCLUSION: In this paper, we propose a modified framework of survival for developing ECS systems. There are barriers for developing ECS systems to improve OHCA survival rates. These barriers may be overcome by systematic prioritisation and cost-effective innovative solutions.

Influence of batch-specific biochemical egg characteristics on embryogenesis and hatching success in farmed pikeperch.

Low and variable egg quality remains a major issue in aquaculture impeding a reliable and continuous supply of larvae, particularly in emerging species, such as pikeperch, Sander lucioperca. We assessed the influence of batch-specific egg parameters (fatty acid (FA) profiles, cortisol content) on embryo life-stages until hatching (survival at 2, 24, 48, 72 h post fertilization (hpf), hatching rate) in an integrated study under commercial hatchery conditions (44 egg batches). Embryo mortality was elevated until 48 hpf (average 9.8% mortality between 2 and 48 hpf). Embryos surviving until 48 hpf were very likely (98.5%) to hatch successfully. The inherent egg FA composition was variable in-between batches. Total FA content ranged form 66.1 to 171.7 µg/mg (dry matter) total FA. Whereas specific FA ,18 : 0 and 20 : 5(n-3) (eicosapentaenoic acid) of the polar fraction and the ratio of 22 : 6(n-3) (docosahexaenoic acid) to 20 : 5(n-3) within the neutral fraction, were significantly correlated with early embryo development, contents of the respective FA did not differ between high (>90% hatching rate), mid (70% to 90% hatching rate) and low (<70% hatching rate) quality egg batches. Late embryo development and hatching were relatively independent of the FA profiles highlighting stage-dependent influences especially during early embryogenesis. Cortisol levels ranged from 22.7 to 293.2 ng/ml and did not directly explain for mortalities. However, high cortisol was associated with a lower content of specific FA, in particular highly unsaturated FA. These results demonstrate the magnitude of inter-individual differences in the batch-specific biochemical egg composition under stable hatchery conditions and suggest a stress-mediated lack of essential FA, which in turn affects early embryo survival. Surprisingly, embryos are able to cope well with a broad range of inherent egg parameters, which limits their predictive potential for egg quality in general. Still, specific FA profiles of high quality egg batches have potential for formulating species-specific broodstock diets and improving reproductive management in pikeperch.

Translating animal model research: does it matter that our rodents are cold?

Does it matter that rodents used as preclinical models of human biology are routinely housed below their thermoneutral zone? We compile evidence showing that such rodents are cold-stressed, hypermetabolic, hypertensive, sleep-deprived, obesity-resistant, fever-resistant, aging-resistant, and tumor-prone compared with mice housed at thermoneutrality. The same genotype of mouse has a very different phenotype and response to physiological or pharmacological intervention when raised below or at thermoneutrality.

Long term ablation of protein kinase A (PKA)-mediated cardiac troponin I phosphorylation leads to excitation-contraction uncoupling and diastolic dysfunction in a knock-in mouse model of hypertrophic cardiomyopathy.

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca(2+) decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca(2+)-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca(2+) transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca(2+) content. This abnormal Ca(2+) handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na(+)-Ca(2+) exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.

Pattern recognition in long­term Sooty Shearwater data: applying machine learning to create a harvest index.

Rakiura Maori (New Zealand's southernmost group of indigenous peoples) have harvested the chicks of burrow-nesting Sooty Shearwaters (Titi; Puffinus griseus) for generations. As part of the harvest process, some families have maintained annual harvest diaries, some dating back to the 1950s. We used generalized boosted regression models, a machine-learning algorithm, to calculate a harvest index that takes into account factors that could impact the numbers of birds taken on any given hunt. For predicted vs. observed values, r2 was between 0.59 and 0.90 for the nanao (first half of the season, when chicks are harvested from burrows during the day) and 0.67 and 0.88 for the rama (second half of the season, during which chicks are harvested from the surface at night). Exploration of the controlling factors of the models revealed that "day of season" plays an important role in predicting daily harvest during the second half of the season (the rama). The nightly tally in the rama peaked approximately halfway through (10­15 days in), which is probably related to the timing of birds emerging from burrows to fledge. The models also suggested that data from the rama (when chicks are 100­120 days old) may be the most suitable for long-term monitoring of populations of Sooty Shearwaters due to consistencies in calculated harvest indices between diaries. Nanao harvest indices, although less consistent, showed patterns similar to those of the rama. When comparing these data to the harvest indices calculated by general linear models by Clucas and colleagues, we found that the agreement between both indices was r2 = 0.31 and r2 = 0.59 for the nanao and rama, respectively. The use of machine learning to correct for extraneous factors (e.g., hunting effort, skill level, or weather) and to create standardized measures could be applied to other systems such as fisheries or terrestrial resource management.

Diet-induced obesity resistance of Kv1.3-/- mice is olfactory bulb dependent.

Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3 (Kv1.3-/-), increases olfactory sensitivity and discriminatory ability, and causes resistance to diet-induced obesity (DIO) in mice. The present study aimed to determine whether the enhanced olfactory ability of the Kv1.3-/- mouse contributes to the resistance to DIO. Kv1.3+/+ and Kv1.3-/- mice were subject to bilateral olfactory bulbectomy (OBX) or sham surgery at 9 weeks of age and placed on either a control chow diet or a 32% moderately high-fat diet (MHF). Caloric and water intake, locomotor activity and oxygen consumption were monitored after 5 weeks of diet treatment. At the end of 26 weeks of diet treatment, fat pad weight and blood chemistry were evaluated. Kv1.3+/+ mice exhibited a significant increase in weight, adiposity, fasting glucose and fasting leptin in response to the MHF-diet, with or without OBX. When treated with a MHF-diet, Kv1.3-/- mice gained significantly less weight than Kv1.3+/+ mice and exhibited a significant increase in light phase metabolism. OBX of Kv1.3-/- mice prevented the resistance to DIO and concomitant up-regulation of light phase metabolism at the same time as decreasing dark phase metabolism and total energy expenditure. These findings suggest that pathways activated in Kv1.3-/- that increased energy expenditure and led to resistance to DIO are olfactory bulb dependent. Thus, these findings add to a growing body of evidence suggesting that the olfactory system can modulate the pathways involved in the regulation of energy balance.

Early failure of Class II resin composite versus Class II amalgam restorations placed by dental students.

Using the information from remake request slips in a dental school's predoctoral clinic, we examined the short-term survival of Class II resin composite restorations versus Class II dental amalgam restorations. In the student clinic, resin composite is used in approximately 58 percent of Class II restorations placed, and dental amalgam is used in the remaining 42 percent. In the period examined, Class II resin composite restorations were ten times more likely to be replaced at no cost to the patient than Class II dental amalgam restorations. A total of eighty-four resin composite restorations and six amalgam restorations were replaced due to an identified failure.

Amylin-leptin coadministration stimulates central histaminergic signaling in rats.

Combined amylin+leptin (AMN+LEP) can reduce diet induced obesity and is very effective in combating LEP resistance. The purpose of this study was to evaluate the effect of AMN+LEP on central histaminergic signaling in lean and obese rats. Male rats were administered LEP (300 µg/kg/d), AMN (100 µg/kg/d), AMN+LEP or vehicle (SAL, 0.9% normal saline), via a subcutaneous mini-osmotic pump or single injection (LEP, 300 µg/kg and AMN, 100 µg/kg) for acute studies. AMN+LEP administration increased expression of histamine H1 receptor (HIR) and histidine decarboxylase (HDC) mRNA in the hypothalamus. Increased levels of H1R were seen in arcuate (Arc) and ventromedial hypothalamus (VMH) as well as the area postrema (APOS) and nucleus of solitary tract (NTS) following AMN+LEP administration. APOS and NTS also showed expression of immediate early gene c-FOS in the hindbrain in AMN+LEP-treated rats. We confirmed previous evidence indicating that AMN+LEP increased STAT-3 protein phosphorylation in Arc and VMH. Finally, by in vivo microdialysis, we observed an increase in methyl HIS levels in the VMH of AMN, LEP and AMN+LEP-treated rats. Taken together, these observations are consistent with an important role that neuronal HIS may play in mediating the potent effects of AMN+LEP on food intake and body weight.

Evidence for the role of hindbrain orexin-1 receptors in the control of meal size.

Hypothalamic orexin neurons project to the hindbrain, and 4th-ventricle intracerebroventricular (4th-icv) injection of orexin-A treatment increases food intake. We assessed the effects of hindbrain orexin-A and the orexin-1-receptor antagonist SB334867 on meal pattern in rats consuming standard chow. When injected 4th-icv shortly before dark onset, lower doses of orexin-A increased food intake over a 2-h period by increasing the size of the first meal relative to vehicle, whereas the highest dose increased food intake by causing the second meal to be taken sooner. Conversely, hindbrain SB334867 reduced food intake by decreasing the size of the first meal of the dark phase. We also examined the effects of 4th-icv orexin-A and SB334867 on locomotor activity. Only the highest dose of orexin-A increased activity, and SB334867 had no effect. In addition, hindbrain SB334867 induced c-Fos in the nucleus of the solitary tract. These data support the suggestion that endogenous hindbrain orexin-A acts to limit satiation. Both orexin-A and the pancreatic satiation hormone amylin require an intact area postrema to affect food intake, so we asked whether 4th-icv orexin-A impairs the satiating effect of peripheral amylin treatment. Amylin reduced the size of the first meal of the dark cycle when rats were pretreated with 4th-icv saline, yet amylin was ineffective after 4th-icv orexin-A pretreatment. Using double-label immunohistochemistry, we determined that some orexin-A fibers in the area postrema are located in proximity to amylin-responsive neurons. Therefore, hindbrain orexin-A may increase food intake, in part, by reducing the ability of rats to respond to amylin during a meal.

Long-term caloric restriction reduces metabolic rate and heart rate under cool and thermoneutral conditions in FBNF1 rats.

The long-term metabolic and cardiovascular responses to caloric restriction (CR) are poorly understood. We examined the responses to one year of CR in FBNF1 rats housed in cool (COOL; T(a)=15 °C) or thermoneutral (TMN; T(a)=30 °C) conditions. Rats were acclimated to COOL or TMN for 2 months, instrumented for cardiovascular telemetry and studied in calorimeters. Baseline caloric intake, oxygen consumption (VO(2)), mean arterial blood pressure (MAP), and heart rate (HR) were determined prior to assignment to ad lib (AL) or CR groups (30-40% CR) within each T(a) (n = 8). Groups of rats were studied after 10 weeks CR, one year CR, and after 4 days of re-feeding. Both 10 weeks and one year of CR reduced HR and VO(2) irrespective of T(a). Evaluation of the relationship between metabolic organ mass (liver, heart, brain, and kidney mass) and energy expenditure revealed a clear shift induced by CR to reduce expenditure per unit metabolic mass in both COOL and TMN groups. Re-feeding resulted in prompt elevations of HR and VO(2) to levels observed in control rats. These findings are consistent with the hypothesis that long term CR produces sustained reductions in metabolic rate and heart rate in rats.

Combined amylin-leptin treatment lowers blood pressure and adiposity in lean and obese rats.

OBJECTIVE: To examine the cardiovascular effects of combined amylin (AMN) and leptin (LEP) treatment in lean and obese rats. RESEARCH DESIGN: Rats were instrumented for telemetry and given LEP (300 µg kg(-1) day(-1)), AMN (100 µg kg(-1) day(-1)), AMN+LEP or vehicle (VEH; 0.9% normal saline) via a subcutaneous mini-osmotic pump for 7 days. The VEH group was subdivided into ad libitum fed and pair-fed to the amount of food AMN+LEP animals ate daily. Rats were housed in metabolic chambers for analysis of cardiovascular physiology and metabolism. SUBJECTS: Male Fisher 344 × Brown Norway (FBNF1; Harlan; age=3-5 months; n=72) rats were placed on standard rodent chow (LEAN, n=41) or moderately high-fat diet (OBESE; n=31) to produce obesity. RESULTS: AMN+LEP potently reduced food intake (LEAN: 57% OBESE: 59%) and abdominal fat mass (LEAN: 56% OBESE: 41%). Pair-fed rats displayed bradycardia and metabolic suppression. In contrast, AMN+LEP increased heart rate and oxygen consumption above levels in LEP or AMN-treated rats. LEP reduced blood pressure in both lean and obese rats but AMN had no effect. LEP-induced reductions in blood pressure were not altered by AMN+LEP treatment. Thus, AMN+LEP treatment decreased food intake, body fat and blood pressure in lean and obese rats. CONCLUSION: We conclude that the potent anti-adiposity actions of AMN+LEP are due in part to prevention of the bradycardia and metabolic suppression typically observed with negative energy balance. Furthermore, the hypotensive actions of peripheral LEP treatment are observable in spite of the potent AMN+LEP activation of anorexic and thermogenic mechanisms in the central nervous system.

Phenotyping small animals as models for the human metabolic syndrome: thermoneutrality matters.

It is standard practice in preclinical biomedical research to house mammalian model organisms at an ambient temperature substantially below the thermoneutral zone. These experimental studies are performed using animals that are chronically challenged by mild cold stress. This condition increases food intake, metabolic rate, sympathetic activity, blood pressure and heart rate. Furthermore, this condition alters the behavioral and physiological responses to drug administration, energy restriction and overfeeding. This paper will review these observations, which must be understood in the context of phenotyping small mammals to enhance our understanding of the biology of human disease.

Chemical surface disinfection of eggs of Baltic cod, Gadus morhua L.

The effect of two disinfectants on eggs and larvae of Baltic cod, Gadus morhua, was investigated. The eggs were disinfected for 10 min using various concentrations of either glutaraldehyde (100, 200, 400, 600 and 800 mg L(-1)) or iodophor (10, 50, 100 and 150 mg L(-1)), 1-4-days post-fertilization. Bactericidal effect of disinfection, survival to hatching, hatching success and larval abnormalities were assessed. Larval survival was recorded at 5-, 10- and 15-days post-hatch (dph). Although Baltic cod eggs have an unusually thin chorion, they could tolerate surface disinfection. A reduction in bacterial growth was observed with increased concentrations of disinfectant (3.0 x 10(7)-1.6 x 10(1) CFU mL(-1)). Abnormalities in newly hatched larvae were not related to disinfection. Survival of the yolk sac larvae was significantly better for eggs treated with 400 mg L(-1) glutaraldehyde for 10 min at 10 and 15 dph. Effective disinfection was also recorded using 100 mg L(-1) Actomar K30. Egg batch effect rather than initial bacterial concentration, disinfectant type or incubation method determined the survival of the eggs to hatching and survival of larvae. Because of the carcinogenic effect of glutaraldehyde, iodophor is recommended for routine disinfection of cod eggs.

What is different in operative dentistry?

There have been both large and small changes in operative dentistry in the last 30 years. Extension for prevention is no longer the mantra. The design features of amalgam preparations have moved into the smallest preparations possible to gain full access to the carious dentin. The default Class 2 amalgam or resin composite is a slot preparation with no preparation of the occlusal fissures. Class 1 fissure caries once implied the entire fissure system was to be cut out. Now only the known carious portions of the fissure are cut away, the tooth is restored, and the remaining fissures in that tooth are sealed. Resin composite preparations have no depth requirements and saucer shaped boxes are more favorable for lowering shrinkage strains on the bonded walls. Re-mineralization of proximal lesions that can be seen on a radiograph is now a proven successful service for many lesions that are at or just into the dentin by radiographic interpretation. The largest paradigm shift has been in the decision that in vital teeth with normal pulps soft dentin can be left over a vital asymptomatic pulp with every expectation that the direct restoration will be successful long term.

Female mice and rats exhibit species-specific metabolic and behavioral responses to ovariectomy.

Ovariectomy (OVX) leads to hyperphagia and weight gain in rats, which can be prevented by estradiol (E2) replacement; however, the role of endogenous E2 on feeding and energy homeostasis in female mice has not been well characterized. The primary goal of this study was to assess the relative contribution of increased energy intake and decreased energy expenditure to OVX-induced weight gain in female rats and mice. OVX led to hyperphagia in rats, but did not produce daily, nor cumulative, hyperphagia in mice. OVX decreased mass-specific metabolic rate in mice, but not in rats. OVX decreased home cage locomotor activity in both species. Pair-feeding attenuated OVX-induced weight gain in rats and produced both short- and long-term changes in expression of key hypothalamic genes involved in food intake and energy homeostasis, i.e., the anorexigenic neuropeptide pro-opiomelanocortin (POMC) and the orexigenic neuropeptides: melanin-concentrating hormone (MCH) and agouti-related peptide (AgRP). No differences in hypothalamic gene expression were observed between OVX'd and sham mice. The results suggest that OVX-induced weight gain is mediated by hyperphagia and reduced locomotor activity in rats, but that in mice, it is primarily mediated by reduced locomotor activity and metabolic rate.

Short-term physiological hyperleptinemia decreases arterial blood pressure.

We examined the possibility that chronic, low-dose peripheral leptin infusion would inhibit food intake but not increase blood pressure. Male Fisher Brown Norway (FBNF1) and Sprague-Dawley (SD) rats were instrumented for cardiovascular telemetry, housed in metabolic chambers, and given leptin (LEP: 600 microg/kg/day) or vehicle (SAL: 10 microl/h) via a subcutaneous osmotic pump for seven days. Leptin infusion increased plasma leptin levels to about 40 ng/ml, decreased food intake by 25-35% and stimulated lipolysis in both strains of rats. Leptin infusion for one week decreased mean arterial pressure from baseline. The reduction developed slowly, was generally about 3 to 7 mm Hg, and observed in both strains. The peripheral, hypotensive effect of chronic leptin in FBNF1 rats was prevented by blockade of nitric oxide production with L-NAME treatment. These results indicate that peripheral leptin treatment, at a level which inhibits food intake and induces lipolysis, produces nitric oxide-dependent decreases in blood pressure.

Recommended guidelines for reporting on emergency medical dispatch when conducting research in emergency medicine: the Utstein style.

OBJECTIVES: To establish a uniform framework describing the system and organisation of emergency medical response centres and the process of emergency medical dispatching (EMD) when reporting results from studies in emergency medicine and prehospital care. DESIGN AND RESULTS: In September 2005 a task force of 22 experts from 12 countries met in Stavanger; Norway at the Utstein Abbey to review data and establish a common terminology for medical dispatch centres including core and optional data to be used for health monitoring, benchmarking and future research.

Kv1.3 gene-targeted deletion alters longevity and reduces adiposity by increasing locomotion and metabolism in melanocortin-4 receptor-null mice.

OBJECTIVE: Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3, results in 'super-smeller' mice that have altered firing patterns of mitral cells in the olfactory bulb, modified axonal targeting to glomerular synaptic units, and behaviorally have an increased ability to detect and discriminate odors. Moreover, the Kv1.3-null mice weighed less than their wild-type counterparts, have modified ingestive behaviors, and are resistant to fat deposition following a moderately high-fat dietary regime. In this study, we investigate whether or not gene-targeted deletion of Kv1.3 (Shaker family member) can abrogate weight gain in a genetic model of obesity, the melanocortin-4 receptor-null mouse (MC4R-null). DESIGN: Mice with double gene-targeted deletions of Kv1.3 and MC4R were generated by interbreeding Kv1.3 (Kv)- and MC4R-null mouse lines to homozygosity. Developmental weights, nose to anus length, fat pad weight, fasting serum chemistry, oxygen consumption, carbon dioxide respiration, locomotor activity and caloric intake were monitored in control, Kv-null, MC4R-null and Kv/MC4R-null mice. Physiological and metabolic profiles were acquired at postnatal day 60 (P60) in order to explore changes linked to body weight at the reported onset of obesity in the MC4R-null model. RESULTS: Gene-targeted deletion of Kv1.3 in MC4R-null mice reduces body weight by decreasing fat deposition and subsequent fasting leptin levels, without changing the overall growth, fasting blood glucose or serum insulin. Gene-targeted deletion of Kv1.3 in MC4R-null mice significantly extended lifespan and increased reproductive success. Basal or light-phase mass-specific metabolic rate and locomotor activity were not affected by genetic deletion of Kv1.3 in MC4R-null mice but dark-phase locomotor activity and mass-specific metabolism were significantly increased resulting in increased total energy expenditure. CONCLUSIONS: Gene-targeted deletion of Kv1.3 can reduce adiposity and total body weight in a genetic model of obesity by increasing both locomotor activity and mass-specific metabolism.

Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality.

It is generally accepted that cardiac sympathetic tone dominates the control of heart rate (HR) in mice. However, we have recently challenged this notion given that HR in the mouse is responsive to ambient temperature (T(a)) and that the housing T(a) is typically 21-23 degrees C, well below the thermoneutral zone ( approximately 30 degrees C) of this species. To specifically test the hypothesis that cardiac sympathetic tone is the primary mediator of HR control in the mouse, we first examined the metabolic and cardiovascular responses to rapid changes in T(a) to demonstrate the sensitivity of the mouse cardiovascular system to T(a). We then determined HR in 1) mice deficient in cardiac sympathetic tone ("beta-less" mice), 2) mice deficient in cardiac vagal tone [muscarinic M(2) receptor (M(2)R(-/-)) mice], and 3) littermate controls. At a T(a) of 30 degrees C, the HR of beta-less mice was identical to that of wild-type mice (351 +/- 11 and 363 +/- 10 beats/min, respectively). However, the HR of M(2)R(-/-) mice was significantly greater (416 +/- 7 beats/min), demonstrating that vagal tone predominates over HR control at this T(a). When these mice were calorically restricted to 70% of normal intake, HR fell equally in wild-type, beta-less, and M(2)R(-/-) mice (DeltaHR = 73 +/- 9, 76 +/- 3, and 73 +/- 7 beats/min, respectively), suggesting that the fall in intrinsic HR governs bradycardia of calorically restricted mice. Only when the T(a) was relatively cool, at 23 degrees C, did beta-less mice exhibit a HR (442 +/- 14 beats/min) that was different from that of littermate controls (604 +/- 10 beats/min) and M(2)R(-/-) mice (602 +/- 5 beats/min). These experiments conclusively demonstrate that in the absence of cold stress, regulation of vagal tone and modulation of intrinsic rate are important determinants of HR control in the mouse.