Preventable risk factors for type 2 diabetes can be detected using noninvasive spontaneous electroretinogram signals.

Given the ever-increasing prevalence of type 2 diabetes and obesity, the pressure on global healthcare is expected to be colossal, especially in terms of blindness. Electroretinogram (ERG) has long been perceived as a first-use technique for diagnosing eye diseases, and some studies suggested its use for preventable risk factors of type 2 diabetes and thereby diabetic retinopathy (DR). Here, we show that in a non-evoked mode, ERG signals contain spontaneous oscillations that predict disease cases in rodent models of obesity and in people with overweight, obesity, and metabolic syndrome but not yet diabetes, using one single random forest-based model. Classification performance was both internally and externally validated, and correlation analysis showed that the spontaneous oscillations of the non-evoked ERG are altered before oscillatory potentials, which are the current gold-standard for early DR. Principal component and discriminant analysis suggested that the slow frequency (0.4-0.7 Hz) components are the main discriminators for our predictive model. In addition, we established that the optimal conditions to record these informative signals, are 5-minute duration recordings under daylight conditions, using any ERG sensors, including ones working with portative, non-mydriatic devices. Our study provides an early warning system with promising applications for prevention, monitoring and even the development of new therapies against type 2 diabetes.

Obesity in male volcano mice Neotomodon alstoni affects the daily rhythm of metabolism and thermoregulation.

The mouse N. alstoni spontaneously develops the condition of obesity in captivity when fed regular chow. We aim to study the differences in metabolic performance and thermoregulation between adult lean and obese male mice. The experimental approach included indirect calorimetry using metabolic cages for VO2 intake and VCO2 production. In contrast, the body temperature was measured and analyzed using intraperitoneal data loggers. It was correlated with the relative presence of UCP1 protein and its gene expression from interscapular adipose tissue (iBAT). We also explored in this tissue the relative presence of Tyrosine Hydroxylase (TH) protein, the rate-limiting enzyme for catecholamine biosynthesis present in iBAT. Results indicate that obese mice show a daily rhythm persists in estimated parameters but with differences in amplitude and profile. Obese mice presented lower body temperature, and a low caloric expenditure, together with lower VO2 intake and VCO2 than lean mice. Also, obese mice present a reduced thermoregulatory response after a cold pulse. Results are correlated with a low relative presence of TH and UCP1 protein. However, qPCR analysis of Ucp1 presents an increase in gene expression in iBAT. Histology showed a reduced amount of brown adipocytes in BAT. The aforementioned indicates that the daily rhythm in aerobic metabolism, thermoregulation, and body temperature control have reduced amplitude in obese mice Neotomodon alstoni.

Changes in 24 h Rhythmicity of Spontaneous Locomotor Activity in the Triple Transgenic Mouse for Alzheimer's Disease (3xTg-AD) in a Jet Lag Protocol: Correlations with Retinal Sensitivity.

The progression of amyloid plaques and neurofibrillary tangles in different brain areas is associated with the effects of Alzheimer's disease (AD). In addition to cognitive impairment, circadian alterations in locomotor activity have also been detected, but they have not been characterized in a jet lag protocol. Therefore, the present study aimed to compare 3xTg-AD and non-transgenic mice in changes of 24 h cycles of spontaneous locomotor activity in a jet lag protocol, in an environment without a running wheel, at 3 different states of neuronal damage: early, intermediate and advanced (3, 8 and 13 months, respectively). The 3xTg-AD mice at 3 months presented differences in phase angle and acrophase, and differentially increased activity after advances more than after delays. At 13 months, a shortening of the free-running period in constant darkness was also noted. 3xTg-AD mice showed a significant increase (123%) in global activity at 8 to 13 months and in nighttime activity (153%) at 13 months. In the advance protocol (ADV), 3xTg-AD mice displayed a significant increase in global activity (171%) at 8 and 13 months. The differences in masking effect were evident at 8 months. To assess a possible retinal dysfunction that could interfere with photic entrainment as part of the neurodegenerative process, we compared electroretinogram recordings. The results showed early deterioration in the retinal response to light flashes in mesopic conditions, observed in the B-wave latency and amplitude. Thus, our study presents new behavioral and pathological characteristics of 3xTg-AD mice and reveals the usefulness of non-invasive tools in early diagnosis.

Obese Neotomodon alstoni mice exhibit sexual dimorphism in the daily profile of circulating melatonin and clock proteins PER1 and BMAL1 in the hypothalamus and peripheral oscillators.

Obesity is a global health threat and a risk factor for several metabolic conditions. Though circadian dysfunction has been considered among the multiple causes of obesity, little work has been done to explore the relationship between obesity, circadian dysfunction, and sexual dimorphism. The Neotomodon alstoni mouse is a suitable model for such research. This study employed N. alstoni mice in a chronobiological analysis to determine whether there is circadian desynchronization of relative PER1 and BMAL1 protein levels in the hypothalamus, liver, visceral white adipose tissue, kidney, and heart. It also compared differences between sexes and lean and obese N. alstoni adult mice, by recording behavior and daily circulating serum melatonin as markers of circadian output. We found that obese mice display reduced locomotor activity. Additionally, Cosinor analyses of the relative expression of PER1 and BMAL1 show differences between lean and obese mice in a sex-linked manner. The PER1 24 h rhythm was absent in all tissues of obese males and significant in the tissues of obese females. The BMAL1 24 h rhythm also was significant in most of the tissues tested in lean males, whereas it was significant and shifted the acrophase (peak time of rhythm) in most of the tissues in obese females. Both lean male and female mice showed a rhythmic 24 h pattern of circulating serum melatonin. This daily profile was not only absent in obese mice of both sexes but showed sexual dimorphism. Obese male mice showed lower circulating levels of melatonin compared to lean male mice, but they were higher in obese females compared to lean females. Our results suggest that obesity in N. alstoni is associated with an internal circadian desynchronization in a sex-dependent manner. Overall, this study reinforces the need for further research on the neuroendocrinology of obesity and circadian rhythms using this biological model.

Synchronization of the circadian clock by time-restricted feeding with progressive increasing calorie intake. Resemblances and differences regarding a sustained hypocaloric restriction.

Circadian rhythms are the product of the interaction of molecular clocks and environmental signals, such as light-dark cycles and eating-fasting cycles. Several studies have demonstrated that the circadian rhythm of peripheral clocks, and behavioural and metabolic mediators are re-synchronized in rodents fed under metabolic challenges, such as hyper- or hypocaloric diets and subjected to time-restricted feeding protocols. Despite the metabolic challenge, these approaches improve the metabolic status, raising the enquiry whether removing progressively the hypocaloric challenge in a  time-restricted feeding protocol leads to metabolic benefits by the synchronizing effect. To address this issue, we compared the effects of two time-restricted feeding protocols, one involved hypocaloric intake during the entire protocol (HCT) and the other implied a progressive intake accomplishing a normocaloric intake at the end of the protocol (NCT) on several behavioural, metabolic, and molecular rhythmic parameters. We observed that the food anticipatory activity (FAA) was driven and maintained in both HCT and NCT. Resynchronization of hepatic molecular clock, free fatty acids (FFAs), and FGF21 was elicited closely by HCT and NCT. We further observed that the fasting cycles involved in both protocols promoted ketone body production, preferentially beta-hydroxybutyrate in HCT, whereas acetoacetate was favoured in NCT before access to food. These findings demonstrate that time-restricted feeding does not require a sustained calorie restriction for promoting and maintaining the synchronization of the metabolic and behavioural circadian clock, and suggest that metabolic modulators, such as FFAs and FGF21, could contribute to FAA expression.

Melanin-concentrating hormone peptidergic system: Comparative morphology between muroid species.

Melanin-concentrating hormone (MCH) is a conserved neuropeptide, predominantly located in the diencephalon of vertebrates, and associated with a wide range of functions. While functional studies have focused on the use of the traditional mouse laboratory model, critical gaps exist in our understanding of the morphology of the MCH system in this species. Even less is known about the nontraditional animal model Neotomodon alstoni (Mexican volcano mouse). A comparative morphological study among these rodents may, therefore, contribute to a better understanding of the evolution of the MCH peptidergic system. To this end, we employed diverse immunohistochemical protocols to identify key aspects of the MCH system, including its spatial relationship to another neurochemical population of the tuberal hypothalamus, the orexins. Three-dimensional (3D) reconstructions were also employed to convey a better sense of spatial distribution to these neurons. Our results show that the distribution of MCH neurons in all rodents studied follows a basic plan, but individual characteristics are found for each species, such as the preeminence of a periventricular group only in the rat, the lack of posterior groups in the mouse, and the extensive presence of MCH neurons in the anterior hypothalamic area of Neotomodon. Taken together, these data suggest a strong anatomical substrate for previously described functions of the MCH system, and that particular neurochemical and morphological features may have been determinant to species-specific phenotypes in rodent evolution.

Daily cycle in hepatic lipid metabolism in obese mice, Neotomodon alstoni: Sex differences.

Disruption of circadian rhythms influences the pathogenesis of obesity, particularly with the basic regulation of food intake and metabolism. A link between metabolism and the circadian clock is the peroxisome proliferator-activated receptors (PPARs). The Neotomodon alstoni mouse, known as the "Mexican volcano mouse," may develop obesity if fed a normo-caloric diet. This manuscript documents the changes in part of the hepatic lipid homeostasis in both sexes of lean and obese N. alstoni mice, comparing the daily changes in the BMAL1 clock protein, in regulators of lipid metabolism (PGC-1α, PPARα-γ, SREBP-1c, and CPT-1α) and in free fatty acid (FFA) and hepatic triacylglyceride (TAG) metabolites in light-dark cycles. Hepatic tissue and blood were collected at 5, 10, 15, 19, and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein. The results indicate that obesity affects daily changes in lipid metabolism and the BMAL1 profile in females considerably more than in males. These results suggest that the impact of obesity on lipid metabolism has important differences according to sex.

Visitor effect on the behavior of a group of spider monkeys (Ateles geoffroyi) maintained at an island in Lake Catemaco, Veracruz/Mexico.

We analyzed the effect of human visitors on the behavior of a group of spider monkeys (Ateles geoffroyi) kept on a small tourist island. Although the spider monkey is a common species in zoos, there are very few specific studies on visitor effects on these monkeys. We conducted behavioral observations on the group of spider monkeys to evaluate the effect of visitors. We also used actimetry devices to measure the variations in the spider monkeys' locomotor activity associated with human presence. With regard to the effect on behavior, we found an increase in self-directed behaviors and a decrease in vocalization, both associated with human presence. Moreover, our results suggest that when people feed monkeys, there is an increase in agonistic behaviors. On the other hand, we found that changes in activity levels in response to human presence vary among individuals. We conclude that changes in spider monkeys' behavior could provide evidence of the negative effect of visitors in our study conditions. Although we discuss the differences in activity levels due to differences in social position, further research is required this topic. Our results can be used to inform management plans for this species in captivity. Improving this relationship between humans and non-human primates through tourism education programs would benefit ecotourism and therefore species conservation programs.

Ovariectomy influences the circadian rhythm of locomotor activity and the photic phase shifts in the volcano mouse.

Recently, the relationship between the circadian system and female reproduction has been of great interest; ovarian hormones can modify the amount and distribution of daily activity differently in rodent species. The volcano mouse Neotomodon alstoni is a species in which it is possible to study the circadian rhythm of locomotion, and it offers comparative information about the influence of ovaries on the circadian system. In this study, we used infrared crossings to compare free movement in intact and sham-operated or ovariectomized mice. We analyzed behavioral and endocrine changes related to the estrous cycle and locomotor circadian rhythm in free-running mice and photic phase shifting. Evidence shows that intact mice present a scalloped pattern of daily activity during the estrous cycle. In constant darkness, the ovariectomy reduces the total amount of activity, shortens the free-running circadian period of locomotion and increases photic phase shifts during the early subjective night. During entrainment, the ovariectomized mice increased the amplitude of total activity during the scotophase, and delay the time of activity onset. These results suggest that ovarian hormones in N. alstoni modulate the circadian rhythm of locomotor activity in a species-specific manner.

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling.

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.

Differences in Photic Entrainment of Circadian Locomotor Activity Between Lean and Obese Volcano Mice (Neotomodon alstoni).

Obesity is a growing problem worldwide with a clear impact on health status. It is also a condition that negatively affects circadian rhythms. When the mouse Neotomodon alstoni is fed a regular rodent chow, some individuals develop obesity, representing an opportunity to compare the effects of spontaneous obesity upon the circadian organization in this species with that observed in other rodents with induced obesity. We report differences in the free running circadian locomotor activity rhythm and in the effects of light pulses between lean and obese mice. Also, the photo-induced expression of the c-Fos protein and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN) were examined at circadian time (CT) 14 and 22. We show that obese mice have a larger dispersion of the period of circadian locomotor rhythm in constant darkness. Photic induced phase shifts are nearly 50% shorter at CT 14, and 50% larger at CT 22 than in lean mice. The photoinduction of VIP in the SCN at CT 22 was larger in obese mice, which may be related to the differences observed in photic phase shifting. Our work indicates that the obesity in Neotomodon has effects on the neural mechanisms that regulate the circadian system.

Obesity alters circadian behavior and metabolism in sex dependent manner in the volcano mouse Neotomodon alstoni.

The aim of the present study is to evaluate whether circadian locomotor activity, and the daily profile of plasma parameters related to metabolic syndrome (nutrients: glucose and triacylglycerides, and hormones: insulin and leptin), differ between male and female Neotomodon alstoni mice, both lean and obese. Young adult animals were captured in the field and kept at the laboratory animal facility. After 6 to 7 months feeding the animals ad libitum with a regular diet for laboratory rodents, 50-60% of mice became obese. Comparisons between sexes indicated that lean females were more active than males; however obese females reduced their nocturnal activity either in LD or DD, and advanced the phase of their activity-onset with respect to lights off. No differences in food intake between lean and obese mice, either during the day or night, were observed. Daily profiles of metabolic syndrome-related plasma parameters showed differences between sexes, and obesity was associated with increased values, especially leptin (500% in females and 273% in males) and insulin (150% in both females and males), as compared with lean mice. Our results indicate that lean mice display behavioral and endocrine differences between sexes, and obesity affects the parameters tested in a sex-dependent manner. The aforementioned leads us to propose N. alstoni, studied in captivity, could be an interesting model for the study of sex differences in the effects of obesity.

Time-place learning is altered by perinatal low-protein malnutrition in the adult rat.

Malnutrition produces changes in the central nervous system (CNS) of mammals during development, related to the intensity and timing of the malnutrition insult during the pre- or postnatal period. Protein malnutrition produces irreversible changes in hippocampal formation and some brain stem nuclei. The suprachiasmatic nucleus (SCN) is dramatically altered by low-protein diets during the gestational and perinatal periods. Also, it is known that circadian oscillators regulate physiological, behavioral, and cognitive processes and there is evidence that the time-place learning process exhibits a daily temporal distribution. The aim of this study was to determine the effects of chronic, prenatal, or postnatal malnutrition on daily patterns of the time-place learning process in the adult rat. Forty Sprague-Dawley male 90-day-old rats, were divided into four groups: 10 well nourished controls (Co), 10 chronically (CM), 10 prenatally malnourished (PrM), and 10 postnatally malnourished (PtM) rats. Efficiency in time-place learning was tested by using a behavioral T-maze. Each rat was assayed for 10 trials before considering the final probe of efficiency. Each trial was 60 seconds long, final efficiency was measured by the amount of time the rat took to reach the end of an arm containing a water pot. Each rat was tested in 2-hour spans until completion of a full 24-hour cycle. A Cosinor analysis was used to evaluate acrophase and percentage of rhythmicity. The obtained results suggest that time-place learning process is influenced by the circadian clock. The severity and timing of prenatal or chronic protein malnutrition modifies the acrophase and rhythmicity of the learning circadian pattern, which can impact important cognitive functions.

Deletion of TASK1 and TASK3 channels disrupts intrinsic excitability but does not abolish glucose or pH responses of orexin/hypocretin neurons.

The firing of hypothalamic hypocretin/orexin neurons is vital for normal sleep-wake transitions, but its molecular determinants are not well understood. It was recently proposed that TASK (TWIK-related acid-sensitive potassium) channels [TASK1 (K(2P)3.1) and/or TASK3 (K(2P)9.1)] regulate neuronal firing and may contribute to the specialized responses of orexin neurons to glucose and pH. Here we tested these theories by performing patch-clamp recordings from orexin neurons directly identified by targeted green fluorescent protein labelling in brain slices from TASK1/3 double-knockout mice. The deletion of TASK1/3 channels significantly reduced the ability of orexin cells to generate high-frequency firing. Consistent with reduced excitability, individual action potentials from knockout cells had lower rates of rise, higher thresholds and more depolarized after-hyperpolarizations. However, orexin neurons from TASK1/3 knockout mice retained typical responses to glucose and pH, and the knockout animals showed normal food-anticipatory locomotor activity. Our results support a novel role for TASK genes in enhancing neuronal excitability and promoting high-frequency firing, but suggest that TASK1/3 subunits are not essential for orexin cell responses to glucose and pH.

Lithium and genetic inhibition of GSK3beta enhance the effect of methamphetamine on circadian rhythms in the mouse.

Lithium, a drug commonly used to treat mood disorders, and the psychostimulant methamphetamine are both capable of altering circadian rhythmicity. Although the actions of lithium on the circadian system are thought to occur through inhibition of glycogen synthase kinase-3beta (GSK3beta), the mechanism by which methamphetamine alters circadian rhythms is unknown. We tested the effects of concurrent methamphetamine and lithium treatment on the circadian wheel-running behavior of mice. Methamphetamine alone lengthened both the active duration and the free-running period of locomotor activity in animals housed in constant conditions. Administering lithium enhanced the period-lengthening effects of methamphetamine in animals housed in constant darkness. This effect was even more pronounced when animals were housed in constant light. Lithium increased both methamphetamine intake and serum levels of methamphetamine, possibly contributing to the effects on circadian behavior. We also tested the effect of methamphetamine in mutant mice possessing only one allele for Gsk3beta. These animals, when treated with methamphetamine, responded like wild-type mice treated with a combination of methamphetamine and lithium, displaying long, free-running rhythms. These data, together with many others in the literature, point to a complicated interaction between the circadian system and the development and possible treatment of psychopathologies such as bipolar disorder and drug addiction.

Protein malnutrition and aging affects entraining and intensity of locomotor activity and body temperature circadian rhythms in rats.

Independently, chronic protein malnutrition and aging have been shown to affect locomotor activity (LA) and body temperature (BT) rhythms in mammals. The objective of the present study was to ascertain the combined effects of these two factors by examining period, entrainment and other circadian parameters between LA and BT rhythms. Chronic protein malnourished (PM) and well-nourished (WN) male Sprague-Dawley rats (550-590 days of age) were implanted with activity temperature intraperitoneal radio transmitters (Mini Mitter) and exposed to different lighting protocols during at least 10 days - light-dark cycles (LD 12:12), constant darkness (DD), skeleton photoperiod (SP) and again LD. Results indicate that parametric entrainment, achieved by means of complete photoperiod, is not negatively affected in malnourished rats; however, it is affected under non-parametric entrainment like SP. A different free running period between the LA and BT circadian rhythms was detected for well-nourished and malnourished aged rats.

Interactions between dopamine and melatonin organize circadian rhythmicity in the retina of the green iguana.

Circadian physiology in the vertebrate retina is regulated by several neurotransmitters. In the lateral eyes of the green iguana the circadian rhythm of melatonin content peaks during the night while the rhythm of dopamine peaks during the day. In the present work, the authors explore the interaction of these 2 neurotransmitters during the circadian cycle. They depleted retinal dopamine with intravitreal injections of 6-hydroxydopamine (6-OHDA) and measured ocular melatonin content in vivo throughout 1 circadian cycle. The circadian rhythm of ocular melatonin not only persisted but increased 10-fold in amplitude. This increase was substantially reduced by the intraocular administration of dopamine. 6-OHDA-treated retinas, unlike those from untreated animals, did not express a circadian rhythm of melatonin synthesis in vitro. To deplete retinal melatonin, the authors pinealectomized iguanas and blocked retinal melatonin synthesis by depleting serotonin with intraocular injections of 5,6-dihydroxytryptamine. In animals so treated, they found that the circadian rhythm of retinal dopamine content was abolished, the levels of dopamine were lowered, and the levels of dopamine metabolites were greatly increased. The data suggest that in iguanas, the amplitude of the circadian rhythm of melatonin synthesis in the eye is suppressed by dopamine while the rhythm of dopamine depends, at least in part, on the presence of melatonin.

Period and phase control in a multioscillatory circadian system (Iguana iguana).

The circadian system of the lizard Iguana iguana is composed of several independent pacemakers that work in concert: the pineal gland, retinae of the lateral eyes, and a fourth oscillator presumed to be located in the hypothalamus. These pacemakers govern the circadian expression of multiple behaviors and physiological processes, including rhythms in locomotor activity, endogenous body temperature, electroretinogram, and melatonin synthesis. The numerous, easily measurable rhythmic outputs make the iguana an ideal organism for examining the contributions of individual oscillators and their interactions in governing the expression of overt circadian rhythms. The authors have examined the effects of pinealectomy and enucleation on the endogenous body temperature rhythm (BTR) and locomotor activity rhythm (LAR) of juvenile iguanas at constant temperature both in LD cycles and in constant darkness (DD). They measured the periods (tau) of the circadian rhythms of LAR and BTR, the phase relationships between them in DD (psiAT), and the phase relationship between each rhythm and the light cycle (psiRL). Pinealectomy lengthened tau of locomotor activity in all animals tested and abolished the BTR in two-thirds of the animals. In those animals in which the BTR did persist following pinealectomy, tau lengthened to the same extent as that of locomotor activity. Pinealectomy also delayed the onset of activity with respect to its normal phase relationship with body temperature in DD. Enucleation alone had no significant effect on tau of LAR or BTR; however, after enucleation, BTR became 180 degrees out of phase from LAR in DD. After both pinealectomy and enucleation, 4 of 16 animals became arrhythmic in both activity and body temperature. Their data suggest that rhythmicity, period, and phase of overt circadian behaviors are regulated through the combined output of multiple endogenous circadian oscillators.

Circadian rhythm of iguana electroretinogram: the role of dopamine and melatonin.

The amplitude of the b-wave of the electroretinogram (ERG) varies with a circadian rhythm in the green iguana; the amplitude is high during the day(or subjective day) and low during the night (or subjective night). Dopamine and melatonin contents in the eye are robustly rhythmic under constant conditions; dopamine levels are high during the subjective day, and melatonin levels are high during the subjective night. Dopamine and melatonin affect the amplitude of the b-wave in an antagonistic and phase-dependent manner: dopamine D2-receptor agonists injected intraocularly during the subjective night produce high-amplitude b-waves characteristic of the subjective day, whereas melatonin injected intraocularly during the subjective day reduces b-wave amplitude. Sectioning the optic nerve abolishes the circadian rhythms of b-wave amplitude and of dopamine content. The results of this study suggest that in iguana, a negative feedback loop involving dopamine and melatonin regulates the circadian rhythm of the ERG b-wave amplitude that is at least in part generated in the brain.