A novel method for measurements of sleep/wake states, feeding and drinking behaviors using the tracking technique of 3D positions in freely moving mice.

We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.

Rapid fluctuations in brain oxygenation during glucose-drinking behavior in trained rats.

Proper inflow of oxygen into brain tissue is essential for maintaining normal neural functions. Although oxygen levels in the brain's extracellular space depend upon a balance between its delivery from arterial blood and its metabolic consumption, the use of high-speed electrochemical detection revealed rapid increases in brain oxygen levels elicited by various salient sensory stimuli. These stimuli also increase intrabrain heat production, an index of metabolic neural activation, but these changes are slower and more prolonged than changes in oxygen levels. Therefore, under physiological conditions, the oxygen inflow into brain tissue exceeds its loss due to consumption, thus preventing any metabolic deficit. Here, we used oxygen sensors coupled with amperometry to examine the pattern of real-time oxygen fluctuations in the nucleus accumbens during glucose-drinking behavior in trained rats. Following the exposure to a glucose-containing cup, oxygen levels rapidly increased, peaked when the rat initiated drinking, and relatively decreased during consumption. Similar oxygen changes but more episodic drinking occurred when Stevia, a calorie-free sweet substance, was substituted for glucose. When water was substituted for glucose, rats tested the water but refused to consume all of it. Although the basic pattern of oxygen changes during this water test was similar to that with glucose drinking, the increases were larger. Finally, oxygen increases were significantly larger when rats were exposed to concealed glucose and made multiple unsuccessful attempts to obtain and consume it. Based on these data, we discuss the mechanisms underlying behavior-related brain oxygen fluctuations and their functional significance.NEW & NOTEWORTHY Oxygen sensors coupled with high-speed amperometry were used to examine brain oxygen fluctuations during glucose-drinking behavior in trained rats. Oxygen levels rapidly increased following presentation of a glucose-contained cup, peaking at the initiation of glucose drinking, and relatively decreasing during drinking. Oxygen increases were larger when rats were exposed to concealed glucose and made multiple attempts to obtain it. We discuss the mechanisms underlying behavior-related brain oxygen fluctuations and their functional significance.

Neural circuits underlying thirst and fluid homeostasis.

Thirst motivates animals to find and consume water. More than 40 years ago, a set of interconnected brain structures known as the lamina terminalis was shown to govern thirst. However, owing to the anatomical complexity of these brain regions, the structure and dynamics of their underlying neural circuitry have remained obscure. Recently, the emergence of new tools for neural recording and manipulation has reinvigorated the study of this circuit and prompted re-examination of longstanding questions about the neural origins of thirst. Here, we review these advances, discuss what they teach us about the control of drinking behaviour and outline the key questions that remain unanswered.

Post-diagnostic coffee and tea consumption and breast cancer survival.

BACKGROUND: We examined the role of post-diagnostic coffee and tea consumption in relation to breast cancer-specific and all-cause mortality among women with breast cancer in prospective cohort studies. METHODS: We identified 8900 women with stage I-III breast cancer from 1980 through 2010 in the Nurses' Health Study (NHS) and from 1991 through 2011 in the NHSII. Post-diagnostic coffee and tea consumption was assessed by a validated food frequency questionnaire every 4 years after diagnosis. RESULTS: During up to 30 years of follow-up, we documented 1054 breast cancer-specific deaths and 2501 total deaths. Higher post-diagnostic coffee consumption was associated with a lower breast cancer-specific mortality: compared with non-drinkers, >3 cups/day of coffee was associated with a 25% lower risk (hazard ratio (HR) = 0.75, 95% confidence interval (CI) = 0.59-0.96; Ptrend = 0.002). We also observed a lower all-cause mortality with coffee consumption: compared with non-drinkers, >2 to 3 cups/day was associated with a 24% lower risk (HR = 0.76, 95% CI = 0.66-0.87) and >3 cups/day was associated with a 26% lower risk (HR = 0.74, 95% CI = 0.63-0.87, Ptrend < 0.0001). Post-diagnostic tea consumption was associated with a lower all-cause mortality: compared with non-drinkers, >3 cups/day was associated with a 26% lower risk (HR = 0.74, 95% CI = 0.58-0.95; Ptrend = 0.04). CONCLUSIONS: Among breast cancer survivors, higher post-diagnostic coffee consumption was associated with better breast cancer and overall survival. Higher post-diagnostic tea consumption may be related to better overall survival.

Molecular mechanisms underlying alcohol-drinking behaviours.

The main characteristic of alcohol use disorder is the consumption of large quantities of alcohol despite the negative consequences. The transition from the moderate use of alcohol to excessive, uncontrolled alcohol consumption results from neuroadaptations that cause aberrant motivational learning and memory processes. Here, we examine studies that have combined molecular and behavioural approaches in rodents to elucidate the molecular mechanisms that keep the social intake of alcohol in check, which we term 'stop pathways', and the neuroadaptations that underlie the transition from moderate to uncontrolled, excessive alcohol intake, which we term 'go pathways'. We also discuss post-transcriptional, genetic and epigenetic alterations that underlie both types of pathways.

Thirst driving and suppressing signals encoded by distinct neural populations in the brain.

Thirst is the basic instinct to drink water. Previously, it was shown that neurons in several circumventricular organs of the hypothalamus are activated by thirst-inducing conditions. Here we identify two distinct, genetically separable neural populations in the subfornical organ that trigger or suppress thirst. We show that optogenetic activation of subfornical organ excitatory neurons, marked by the expression of the transcription factor ETV-1, evokes intense drinking behaviour, and does so even in fully water-satiated animals. The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus. In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals. These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

Influence of anterior midcingulate cortex on drinking behavior during thirst and following satiation.

In humans, activity in the anterior midcingulate cortex (aMCC) is associated with both subjective thirst and swallowing. This region is therefore likely to play a prominent role in the regulation of drinking in response to dehydration. Using functional MRI, we investigated this possibility during a period of "drinking behavior" represented by a conjunction of preswallow and swallowing events. These events were examined in the context of a thirsty condition and an "oversated" condition, the latter induced by compliant ingestion of excess fluid. Brain regions associated with swallowing showed increased activity for drinking behavior in the thirsty condition relative to the oversated condition. These regions included the cingulate cortex, premotor areas, primary sensorimotor cortices, the parietal operculum, and the supplementary motor area. Psychophysical interaction analyses revealed increased functional connectivity between the same regions and the aMCC during drinking behavior in the thirsty condition. Functional connectivity during drinking behavior was also greater for the thirsty condition relative to the oversated condition between the aMCC and two subcortical regions, the cerebellum and the rostroventral medulla, the latter containing nuclei responsible for the swallowing reflex. Finally, during drinking behavior in the oversated condition, ratings of swallowing effort showed a negative association with functional connectivity between the aMCC and two cortical regions, the sensorimotor cortex and the supramarginal gyrus. The results of this study provide evidence that the aMCC helps facilitate swallowing during a state of thirst and is therefore likely to contribute to the regulation of drinking after dehydration.

Impact of Reduced Deuterium Intake on Thermoregulation.

The role of stable hydrogen isotopes in the thermoregulation and its regulation is poorly studied. We analyzed fluctuations in body temperature and changes in thermoregulation parameters in mice under conditions of reduced deuterium intake. The study was performed on male C57BL/6 mice that consumed water with a low (10 ppm) and normal (146 ppm) deuterium content. In 7 days, fluctuations of body temperature, locomotor activity, and oxygen uptake were assessed. Deuterium depletion in the body reduced the mean value of minute fluctuations of body temperature and the mean spectral density of minute fluctuations in body temperature in the 2-20-min periods. This attested to a stabilizing effect of deuterium depletion on the rhythms of body temperature fluctuations, without significant shifts in the thermogenesis parameters. Thus, drinking water with reduced deuterium content makes them less sensitive to external influences.

A Young man with Peri-ictal water drinking.

Neuro-vegetative features have been linked to epilepsy arising from the temporal lobe, which can be seen during ictal events and play an important role in determining the focal side of the lesion. Among the rare known features is peri-ictal water drinking (PIWD). Here, we present the case of a 31-year-old male with refractory temporal epilepsy, having episodes of PIWD. The patient was considered a candidate for resective surgery and was investigated accordingly, including long-term video electroencephalogram (EEG), brain magnetic resonance imaging (MRI), and neuropsychology assessment, which pointed towards a lesioned temporal lobe over the non-dominant hemisphere. The patient had an excellent outcome following right anterior temporal lobectomy. The lateralization significance of PIWD has not yet been established definitively in the literature, partly due to a limited number of published cases. This case highlights the previously observed association between PIWD and the involvement of the non-dominant hemisphere in epilepsy patients.

A novel regulator of thirst behavior: phoenixin.

There are examples of physiological conditions under which thirst is inappropriately exaggerated, and the mechanisms for these paradoxical ingestive behaviors remain unknown. We are interested in thirst mechanisms across the female life cycle and have identified a novel mechanism through which ingestive behavior may be activated. We discovered a previously unrecognized endogenous hypothalamic peptide, phoenixin (PNX), identified physiologically relevant actions of the peptide in brain and pituitary gland to control reproductive hormone secretion in female rodents, and in the process identified the previously orphaned G protein-coupled receptor Gpr173 to be a potential receptor for the peptide. Labeled PNX binding distribution in brain parallels areas known to be important in ingestive behaviors as well in areas where gonadal steroids feedback to control estrous cyclicity (Stein LM, Tullock CW, Mathews SK, Garcia-Galiano D, Elias CF, Samson WK, Yosten GLC, Am J Physiol Regul Integr Comp Physiol 311: R489-R496, 2016). We have demonstrated upregulation of Gpr173 during puberty, fluctuations across the estrous cycle, and, importantly, upregulation during the last third of gestation. It is during this hypervolemic, hyponatremic state that both vasopressin secretion and thirst are inappropriately elevated in humans. Here, we show that central administration of PNX stimulated water drinking in both males and females under ad libitum conditions, increased water drinking after overnight fluid deprivation, and increased both water and 1.5% NaCl ingestion under fed and hydrated conditions. Importantly, losartan pretreatment blocked the effect of PNX on water drinking, and knockdown of Gpr173 by use of short interfering RNA constructs significantly attenuated water drinking in response to overnight fluid deprivation. These actions, together with the stimulatory action of PNX on vasopressin secretion, suggest that this recently discovered neuropeptide may impact the recruitment of critically important neural circuits through which ingestive behaviors and endocrine mechanisms that maintain fluid and electrolyte homeostasis are regulated.

Tea consumption and risk of diabetes in the Chinese population: a multi-centre, cross-sectional study.

The aim of the present study was to explore the influence of tea consumption on diabetes mellitus in the Chinese population. This multi-centre, cross-sectional study was conducted in eight sites from south, east, north, west and middle regions in China by enrolling 12 017 subjects aged 20-70 years. Socio-demographic and general information was collected by a standardised questionnaire. A standard procedure was used to measure anthropometric characteristics and to obtain blood samples. The diagnosis of diabetes was determined using a standard 75-g oral glucose tolerance test. In the final analysis, 10 825 participants were included and multiple logistic models and interaction effect analysis were applied for assessing the association between tea drinking with diabetes. Compared with non-tea drinkers, the multivariable-adjusted OR for newly diagnosed diabetes were 0·80 (95 % CI 0·67, 0·97), 0·88 (95 % CI 0·71, 1·09) and 0·86 (95 % CI 0·67, 1·11) for daily tea drinkers, occasional tea drinkers and seldom tea drinkers, respectively. Furthermore, drinking tea daily was related to decreased risk of diabetes in females by 32 %, elderly (>45 years) by 24 % and obese (BMI > 30 kg/m2) by 34 %. Moreover, drinking dark tea was associated with reduced risk of diabetes by 45 % (OR 0·55; 95 % CI 0·42, 0·72; P < 0·01). The results imply that drinking tea daily was negatively related to risk of diabetes in female, elderly and obese people. In addition, drinking dark tea was associated with decreased risk of type 2 diabetes mellitus.

Exclusively drinking sucrose or saline early in life alters adult drinking behavior by laboratory rats.

Proper fluid balance is critical for life. Learning plays an important role in shaping the appetitive behaviors required for drinking. Children often forego drinking plain water and instead consume beverages such as milk or juice. What effect this may have on adult thirst responses remains an open question. To model aspects of the human condition, we bred Sprague-Dawley rats and prevented the pups from obtaining fluid other than from nursing. Pups were weaned onto either tap water, 5% sucrose, or 0.45% saline, and given access to only that fluid for at least 7 weeks. We then measured intake of water or sucrose/saline in one-bottle tests after mild hypertonic saline (HS) injection, or overnight fluid deprivation, and in two-bottle tests after HS injection while rats were maintained on their respective fluids, and after all subjects had only water to drink for a week. We found that sucrose- and saline-maintained rats drank less water than did controls after the HS challenge. After overnight fluid deprivation, rats maintained on saline drank less water and more saline, but there was no difference in intake between water-maintained and sucrose-maintained rats. Differences in licking patterns, including more licks/burst for sucrose by sucrose-maintained rats were detected, even in cases when total intake was not different. These data provide evidence that adult rat water intake can be reduced by exclusively drinking sucrose or saline early in life.

Challenges to Body Fluid Homeostasis Differentially Recruit Phasic Dopamine Signaling in a Taste-Selective Manner.

The internal environment of an organism must remain stable to ensure optimal performance and ultimately survival. The generation of motivated behaviors is an adaptive mechanism for defending homeostasis. Although physiological state modulates motivated behaviors, the influence of physiological state on phasic dopamine signaling, an underlying neurobiological substrate of reward-driven behavior, is underexplored. Here, we use sodium depletion and water restriction, manipulations of body fluid homeostasis, to determine the flexibility and specificity of dopamine responses. Changes in dopamine concentration were measured using fast-scan cyclic voltammetry in the nucleus accumbens shell of male rats in response to intraoral infusions of fluids that either satisfied or did not satisfy homeostatic need. Increases in dopamine concentration during intraoral infusions were observed only under conditions of physiological deficit. Furthermore, dopamine increases were selective and limited to those that satisfied the need state of the animal. Thus, dopamine neurons track fluid balance and respond to salt and water stimuli in a state- and taste-dependent manner. Using Fluoro-Gold tracing and immunohistochemistry for c-Fos and Foxp2, a marker of sodium-deprivation responsive neurons, we revealed brainstem populations of neurons that are activated by sodium depletion and project directly to the ventral tegmental area. The identified projections may modulate dopamine neuron excitability and consequently the state-specific dopamine release observed in our experiments. This work illustrates the impact of physiological state on mesolimbic dopamine signaling and a potential circuit by which homeostatic disruptions are communicated to mesolimbic circuitry to drive the selective reinforcement of biologically-required stimuli under conditions of physiological need.SIGNIFICANCE STATEMENT Motivated behaviors arise during physiological need and are highly selective for homeostasis-restoring stimuli. Although phasic dopamine signaling has been shown to contribute to the generation of motivated behaviors, the state and stimulus specificity of phasic dopamine signaling is less clear. These studies use thirst and sodium appetite to show that dopamine neurons dynamically track body fluid homeostasis and respond to water and salt stimuli in a state- and taste-dependent manner. We also identify hindbrain sodium deprivation-responsive neurons that project directly to the ventral tegmental area, where dopamine neuron cell bodies reside. This work demonstrates command of homeostasis over dopamine signaling and proposes a circuit by which physiological need drives motivated behavior by state- and taste-selective recruitment of phasic dopamine signaling.

Anterior cingulate cortex modulates the affective-motivative dimension of hyperosmolality-induced thirst.

Neuroimaging studies have shown that the anterior cingulate cortex (ACC) is consistently activated by thirst and may underlie the affective motivation of drinking behaviour demanded by thirst. But direct evidence for this hypothesis is lacking. The present study evaluated potential correlations between ACC neuronal activity and drinking behaviour in rats injected with different concentrations of saline. We observed an increased number of c-Fos-positive neurons in the ACC after injection of hypertonic saline, indicating strong ACC neuronal activation under hyperosmotic thirst. Increased firing rates of putative ACC pyramidal neurons preceded drinking behaviour and positively correlated with both the total duration of drinking and the total amount of water consumed. Chemogenetic inhibition of ACC pyramidal neurons changed drinking behaviour from an explosive and short-lasting pattern to a gradual but more persistent pattern, without affecting either the total duration of drinking or the total amount of water consumed. Together, these findings support a role of the ACC in modulating the affective-motivative dimension of hyperosmolality-induced thirst.

Adolescent social stress and social context influence the intake of ethanol and sucrose in male rats soon and long after the stress exposures.

Social instability stress in adolescent rats (SS; postnatal day 30-45, daily 1 hr isolation +new cage partner) alters behavioural responses to psychostimulants, but differences in voluntary consumption of natural and drug rewards are unknown. SS also is associated with an atypical behavioural repertoire, for example reduced social interactions. Here, we investigated whether SS rats differ from control (CTL) rats in ethanol (EtOH) or sucrose intake in experiments involving different social contexts: alone, in the presence of an unfamiliar peer, in the presence of its cage partner, or in competition against its cage partner. SS rats drank more EtOH than CTL rats irrespective of social context, although the effects were driven primarily by those tested soon after the test procedure rather than weeks later in adulthood. SS and CTL rats did not differ in sucrose intake, except in adulthood under conditions of competition for limited access (SS>CTL). Adolescent rats drank more sucrose than adults, in keeping with evidence that adolescents are more sensitive to natural rewards than adult animals. Overall, adolescent SS modified the reward value of EtOH and sucrose, perhaps through stress/glucocorticoids modifying the development of the mesocorticolimbic system.

Developing and evaluating threshold-based algorithms to detect drinking behavior in dairy cows using reticulorumen temperature.

In this study, we assessed for the first time the use of a reticuloruminal temperature bolus and a thresholding method to detect drinking events and investigated different factors that can affect drinking behavior. First, we validated the detection of drinking events using 16 cows that received a reticuloruminal bolus. For this, we collected continuous drinking behavior data for 4 d using video recordings and ambient and water temperature for the same 4 d. After all the data were synchronized, we performed 2 threshold algorithms: a general-fixed threshold and a cow-day specific threshold algorithm. In the general-fixed threshold, a positive test was considered if the temperature of any cow fell below a fixed threshold; in the cow-day specific threshold, a positive test was considered when the temperature of specific cows fell below the threshold value deviations around the mean temperature of the cow for that day. The former was evaluated using a threshold varying between 35.7 and 39.5°C, and the latter using the formula µ-n10σ, where µ = mean of the temperature of each cow for one day, n = 1, 2, …, 20, and σ = standard deviation of the temperature of each cow on that day. The performance of the validation of detection using each of the threshold types was computed using different metrics, including overall accuracy, precision, recall (also known as sensitivity), F-score, positive predictive value, negative predictive value, false discovery rate, false omission rate, and Cohen's kappa statistic. The findings of the first study showed that the cow-day specific threshold of n = 10 performed better (true positives = 466; false positives = 167; false negatives = 165; true negatives = 8,416) than using a general-fixed threshold of 38.1°C (true positives = 449; false positives = 181; false negatives = 182; true negatives = 8,402). With the information gained in this first study, we investigated the different factors associated with temperature drop characteristics per cow: number of drops, mean amplitude of the drop, and mean recovery time. For this, we used data from 54 cows collected for almost 1 yr to build a mixed-effect multilevel model that included days in milk, parity, average monthly milk production, and ambient temperature as explanatory variables. Cow characteristics and ambient temperature had significant effects on drinking events. Our results provide a platform for automated monitoring of drinking behavior, which has potential value in prediction of health and welfare in dairy cattle.

Estimated Sweat Loss, Fluid and Carbohydrate Intake, and Sodium Balance of Male Major Junior, AHL, and NHL Players During On-Ice Practices.

Several previous studies have reported performance decrements in team sport athletes who dehydrated approximately 1.5-2% of their body mass (BM) through sweating. This study measured on-ice sweat loss, fluid intake, sodium balance, and carbohydrate (CHO) intake of 77 major junior (JR; 19 ± 1 years), 60 American Hockey League (AHL; 24 ± 4 years), and 77 National Hockey League (NHL; 27 ± 5 years) players. Sweat loss was calculated from pre- to post-exercise BM plus fluid intake minus urine loss. AHL (2.03 ± 0.62 L/hr) and NHL (2.02 ± 0.74 L/hr) players had higher sweat rates (p < .05) than JR players (1.63 ± 0.58 L/hr). AHL (1.23 ± 0.69%; p = .006) and NHL (1.29% ± 0.63%; p < .001) players had ∼30% greater BM losses than JR players (0.89% ± 0.57%). There was no difference in fluid intake between groups (p > .05). Sodium deficits (sodium loss - intake) were greater (p < .05) in AHL (1.68 ± 0.74 g/hr) and NHL (1.56 ± 0.84 g/hr) players compared with JR players (1.01 ± 0.50 g/hr). CHO intake was similar between groups (14-20 g CHO/hr), with 29%, 32%, and 40% of JR, AHL, and NHL players consuming no CHO, respectively. In summary, sweat rates were high in all players, but the majority of players (74/77, 54/60, and 68/77 of JR, AHL, and NHL, respectively) avoided mild dehydration (>2% BM) during 60 min of practice. However, ∼15%, 41%, and 48% of the JR, AHL, and NHL players, respectively, may have reached mild dehydration and increased risk of performance decrements in a 90-min practice.

Prenatal androgen receptor activation determines adult alcohol and water drinking in a sex-specific way.

Alcohol use disorders are major psychiatric disorders. Correlational studies in humans suggested organizational hormonal effects during embryonic development as a risk factor for adult alcohol dependence. Permanent changes can be induced by the activity of sex hormones, like testosterone. Here, we demonstrate a relationship between prenatal androgen receptor (AR)-activation and adult alcohol as well as water drinking in mice in a sex-dependent fashion. Prenatal AR inhibition using the antagonist flutamide decreased adult male alcohol consumption. In contrast, prenatal AR activation by dihydrotestosterone (DHT) led to an increase in adult alcohol consumption in females. These effects were different in adult water drinking, flutamide increased water consumption in females and DHT increased water consumption in males. Prenatal flutamide reduced locomotion and anxiety in adult males but was ineffective in females. We found that prenatal AR activation controls adult levels of monoaminergic modulatory transmitters in the brain and blood hormone levels in a sex-specific way. RNA-Seq analysis confirmed a prenatal AR mediated control of adult expression of alcohol drinking-related genes like Bdnf and Per2. These findings demonstrate that prenatal androgen activity is a risk factor for the establishment of alcohol consumption in adults by its organizational effects.

Short communication: Tick-borne disease is associated with changes in feeding behavior in automatically fed weaned dairy calves.

Tick-borne disease is a hemolytic disease complex that causes extensive losses to the dairy industry in tropical regions. Its timely diagnosis is challenging and labor intensive. Our objective was to investigate whether tick-borne disease was associated with changes in ingestive behavior of weaned dairy calves. Weaned calves (n = 35) enrolled at 108 ± 8 d of age were group housed (n = 12, 12, and 11/group) for 4 wk in the experimental pen, which had 12 electronic feed bins and 2 electronic water bins. Data were automatically collected at each feed or water bin visit via radio frequency identification ear tags. Water and total mixed ration were provided ad libitum. Calves were examined weekly for tick-borne disease detection. A disease bout was confirmed if packed cell volume was ≤25% at any exam. Diseased calves received antimicrobial and antipyretic treatment. Data were summarized by day within bin type (feed or water) as intake (kg/d; as-fed basis), frequency of visits (visits/d), and total duration of visits (min/d). Day of detection was set as d 0; a 9-d behavioral screening period for evaluation of behavioral changes was set according to d 0 (d -4 to +4). A within-calf mean was calculated for the healthy period (HP; mean of all days between 2 consecutive negative exams) for each response variable. Data were analyzed as within-calf differences between HP and each day of the behavioral screening period. Tick-borne disease was detected in 12 calves; thus, only data referent to these animals were included in the analyses. Compared with HP, daily feed intake was reduced on d -1, 0, and +1, and daily frequency and total duration of feed bin visits were reduced from d -3 to d +4. Daily feed intake was reduced by 35% on d -1 (3.5 ± 0.4 vs. 5.4 ± 0.5 kg/d for HP), and daily frequency and duration of visits were reduced by 27% (56.3 ± 7.8 vs. 76.7 ± 8.1 visits/d for HP) and 24% (27.0 ± 3.8 vs. 35.4 ± 3.9 min/d for HP) on d -3, respectively. Daily water intake on d 0 (9.1 ± 1.4 kg/d) was lower than at HP (12.9 ± 1.6 kg/d), but other drinking behaviors were not different from healthy means during the screening period. Feeding behavior, but not drinking behavior, was different from HP means before detection at weekly exams. Therefore, feeding behavior could be further explored for the development of algorithms for tick-borne disease detection.