Sympathetic overactivity precedes metabolic dysfunction in a fructose model of glucose intolerance in mice

De Angelis K, Senador DD, Mostarda C, Irigoyen MC, Morris M. Sympathetic overactivity precedes metabolic dysfunction in a fructose model of glucose intolerance in mice. Am J Physiol Regul Integr Comp Physiol 302: R950-R957, 2012. First published February 8, 2012; doi: 10.1152/ajpregu.00450.2011.-Consumption of high levels of fructose in humans and animals leads to metabolic and cardiovascular dysfunction. There are questions as to the role of the autonomic changes in the time course of fructose-induced dysfunction. C57/BL male mice were given tap water or fructose water (100 g/l) to drink for up to 2 mo. Groups were control (C), 15-day fructose (F15), and 60-day fructose (F60). Light-dark patterns of arterial pressure (AP) and heart rate (HR), and their respective variabilities were measured. Plasma glucose, lipids, insulin, leptin, resistin, adiponectin, and glucose tolerance were quantified. Fructose increased systolic AP (SAP) at 15 and 60 days during both light (F15: 123 +/- 2 and F60: 118 +/- 2 mmHg) and dark periods (F15: 136 +/- 4 and F60: 136 +/- 5 mmHg) compared with controls (light: 111 +/- 2 and dark: 117 +/- 2 mmHg). SAP variance (VAR) and the low-frequency component (LF) were increased in F15 (>60% and >80%) and F60 (>170% and >140%) compared with C. Cardiac sympatho-vagal balance was enhanced, while baroreflex function was attenuated in fructose groups. Metabolic parameters were unchanged in F15. However, F60 showed significant increases in plasma glucose (26%), cholesterol (44%), triglycerides (22%), insulin (95%), and leptin (63%), as well as glucose intolerance. LF of SAP was positively correlated with SAP. Plasma leptin was correlated with triglycerides, insulin, and glucose tolerance. Results show that increased sympathetic modulation of vessels and heart preceded metabolic dysfunction in fructose-consuming mice. Data suggest that changes in autonomic modulation may be an initiating mechanism underlying the cluster of symptoms associated with cardiometabolic disease.

Experimental gestational hypothyroidism evokes hypertension in adult offspring rats

Gestational hypothyroidism is a prevalent disorder in pregnant women. We aimed to investigate the impact of experimental gestational hypothyroidism (EGH) on cardiovascular and autonomic nervous systems (ANS) in the offspring of rats. EGH was induced with methimazole (MMI) 0.02% in drinking water from day 9 of gestation until birth. Sixty day old offspring from MMI-treated dams (OMTD, n = 13) or water-treated dams (OWTD, n = 13) had femoral arteries surgically assessed for the measurements of heart rate (HR), mean (MAP), systolic (SAP) and diastolic arterial pressure (DAP), and spontaneous baroreflex sensitivity (BRS). To investigate the balance of ANS, we established the high (HF) and low frequency (LF) bands of pulse interval (PI) and LF band of SAP spectrum. OMTD had increased MAP (130.2 +/- 2.0 vs 108.8 +/- 3.0 mm Hg, p<0.001), SAP (157.3 +/- 2.9 vs 135.7 +/- 4.5 mm Hg, p<0.001) and DAP (109.7 +/- 1.9 vs 88.4 +/- 2.6 mm Hg, p<0.001) when compared to OWED, and had lower HR (355.1 +/- 8.9 vs 386.8 +/- 9.2 bpm, p<0.05). After spectral analysis of PI and SAP, only LF band of SAP spectrum was higher (7.2 +/- 0.8 vs 4.0 +/- 0.6 mm Hg-2, p<0.01) in OMTD under spontaneous condition. Despite bradycardia, EGH promotes spontaneous hypertension in 60 day old offspring, probably due to increased sympathetic modulation of vessels, which is suggested by the higher LF of SAP. These findings suggest a critical role of maternal THs in the development of fetal cardiovascular and autonomic nervous systems. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

alpha 1 and alpha 2-adrenoceptors in the medial amygdaloid nucleus modulate differently the cardiovascular responses to restraint stress in rats

Medial amygdaloid nucleus (MeA) neurotransmission has an inhibitory influence on cardiovascular responses in rats submitted to restraint, which are characterized by both elevated blood pressure (BP) and intense heart rate (HR) increase. In the present study, we investigated the involvement of MeA adrenoceptors in the modulation of cardiovascular responses that are observed during an acute restraint. Male Wistar rats received bilateral microinjections of the selective alpha 1-adrenoceptor antagonist WB4101 (10, 15, and 20 nmol/100 nL) or the selective alpha 2-adrenoceptor antagonist RX821002 (10, 15, and 20 nmol/nL) into the MeA, before the exposure to acute restraint. The injection of WB4101 reduced the restraint-evoked tachycardia. In contrast, the injection of RX821002 increased the tachycardia. Both drugs had no influence on BP increases observed during the acute restraint. Our findings indicate that alpha 1 and alpha 2-adrenoceptors in the MeA play different roles in the modulation of the HR increase evoked by restraint stress in rats. Results suggest that alpha 1-adrenoceptors and alpha 2-adrenoceptors mediate the MeA-related facilitatory and inhibitory influences on restraint-related HR responses, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Glutamatergic neurotransmission in the hypothalamus PVN on heart rate variability in exercise trained rats

The paraventricular nucleus of hypothalamus (PVN) is a well known site of integration for autonomic and cardiovascular responses, and the glutamate neurotransmitter plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after ionotropic glutamate receptor inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After exercise training protocol, adult male Wistar rats, instrumented with guide cannulae to PVN and artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, physical training induced a resting bradycardia (S: 379 +/- 3, ST: 349 +/- 2 bpm, P<0.05) and promoted adaptations in HRV characterized by an increase of HF in normalized values and a decrease of LF in absolute and normalized units compared with the sedentary group. Microinjection of kynurenic acid (KYNA) in the PVN of sedentary and trained rats promoted decreases in MAP and HR, but the decrease in HR was smaller in the trained animals (Delta HRS: -48 +/- 7, ST: -28 +/- 4 bpm, P<0.05). Furthermore, the differences in baseline parameters of pulse interval, found between sedentary and trained animals, disappeared after KYNA microinjection in the PVN. Our data suggest that the cardiovascular and autonomic adaptations to the heart induced by exercise training may involve glutamatergic mechanisms in the PVN. (C) 2012 Elsevier B.V. All rights reserved.

Cannabidiol injected into the bed nucleus of the stria terminalis reduces the expression of contextual fear conditioning via 5-HT1A receptors

Systemic administration of cannabidiol (CBD) attenuates cardiovascular and behavioral changes induced by re-exposure to a context that had been previously paired with footshocks. Previous results from our group using cFos immunohistochemistry suggested that the bed nucleus of the stria terminalis (BNST) is involved in this effect. The mechanisms of CBD effects are still poorly understood, but could involve 5-HT1A receptor activation. Thus, the present work investigated if CBD administration into the BNST would attenuate the expression of contextual fear conditioning and if this effect would involve the activation of 5-HT1A receptors. Male Wistar rats with cannulae bilaterally implanted into the BNST were submitted to a 10 min conditioning session (six footshocks, 1.5 mA/3 s). Twenty-four hours later freezing and cardiovascular responses (mean arterial pressure and heart rate) to the conditioning box were measured for 10 min. CBD (15, 30 or 60 nmol) or vehicle was administered 10 min before the re-exposure to the aversive context. The second experiment was similar to the first one except that animals received microinjections of the 5-HT1A receptor antagonist WAY100635 (0.37 nmol) 5 min before CBD (30 nmol) treatment. The results showed that CBD (30 and 60 nmol) treatment significantly reduced the freezing and attenuated the cardiovascular responses induced by re-exposure to the aversive context. Moreover, WAY100635 by itself did not change the cardiovascular and behavioral response to context, but blocked the CBD effects. These results suggest that CBD can act in the BNST to attenuate aversive conditioning responses and this effect seems to involve 5-HT1A receptor-mediated neurotransmission.

Effect of caloric restriction on myenteric neuroplasticity in the rat duodenum during aging

The objective of this study was to evaluate the effects of caloric restriction (CR) on myenteric neurons in the duodenum of Wistar rats during aging. Thirty rats were divided into three groups: the C group (six-month-old animals that were fed a normal diet from weaning until six months of age), the SR group (18-month-old animals that were fed a normal diet from weaning until 18 months of age) and the CR group (18-month-old animals that were fed a 30% CR diet after six months of age). After 12 months, the animals were euthanized. Whole-mount preparations of the duodenums were either stained with Giemsa or underwent NADPH-diaphorase histochemistry to determine the general myenteric neuron population and the nitrergic neuron subpopulation (NADPH-d +), respectively. The NADPH-d-negative (NADPH-d -) neuron population was estimated based on the difference between the Giemsa-stained and NADPH-d + neurons. The neurons were counted, and the cell body areas were measured. Aging was associated with neuronal loss in the SR group, which was minimized by caloric restriction in the CR group. The density (mm(2)) of the Giemsa-stained neurons was higher in the SR group (79.09 +/- 6.25) than in the CR (92.37 +/- 11.6) and C (111.68 +/- 15.26) groups. The density of the NADPH-d + neurons was higher in the SR group (44.90 +/- 5.88) than in the C (35.75 +/- 1.6) and RC (39.14 +/- 7.02) groups. The density of NADPH-d - neurons was higher in the CR (49.73 +/- 12.08) and C (75.64 +/- 17.05) groups than in the SR group (33.82 +/- 4.5). In the C group, 32% and 68% of the Giemsa-stained myenteric neurons were NADPH-d + or NADPH-d -, respectively. With aging (SR group), the percentage of nitrergic neurons (56.77%) increased, whereas the percentage of NADPH-d - neurons (43.22%) decreased. In the CR group, the change in the percentage of nitrergic (42.37%) and NADPH-d - (57.62%) neurons was lower. As NADPH-d - neurons will be mostly cholinergic neurons, CR appears to reduce the loss of cholinergic neurons during aging. The cell body dimensions (mu m(2)) were not altered by aging or CR. Thus. CR had a protective effect on myenteric neurons during aging. (C) 2012 Elsevier B.V. All rights reserved.

Cardiometabolic benefits of exercise training in an experimental model of metabolic syndrome and menopause

Objective: The aim of this study was to investigate the cardiometabolic effects of exercise training in ovariectomized hypertensive rats both submitted and not submitted to fructose overload. Methods: Spontaneously hypertensive ovariectomized rats were divided into sedentary and trained (THO) groups submitted to normal chow and sedentary and trained groups submitted to fructose overload (100 g/L in drinking water for 19 wk). Exercise training was performed on a treadmill (8 wk). Arterial pressure (AP) was directly recorded. Cardiovascular autonomic control was evaluated through pharmacological blockade (atropine and propranolol) and in the time and frequency domains by spectral analysis. Results: The THO group presented reduced AP (approximately 16 mm Hg) and enhanced cardiac vagal tonus (approximately 49%) and baroreflex sensitivity (approximately 43%) compared with the sedentary hypertensive ovariectomized group. Exercise training attenuated metabolic impairment, resting tachycardia, cardiac and vascular sympathetic increases, and baroreflex sensitivity decrease induced by fructose overload in hypertensive rats. However, the trained hypertensive ovariectomized group submitted to fructose overload presented higher AP (approximately 32 mm Hg), associated with baroreflex sensitivity (approximately 69%) and parasympathetic dysfunctions compared with the THO group. Conclusions: These data suggest that the metabolic disorders in hypertensive rats after ovarian hormone deprivation could blunt and/or attenuate some exercise training benefits.

Aging reduces complexity of heart rate variability assessed by conditional entropy and symbolic analysis

Increasing age is associated with a reduction in overall heart rate variability as well as changes in complexity of physiologic dynamics. The aim of this study was to verify if the alterations in autonomic modulation of heart rate caused by the aging process could be detected by Shannon entropy (SE), conditional entropy (CE) and symbolic analysis (SA). Complexity analysis was carried out in 44 healthy subjects divided into two groups: old (n = 23, 63 +/- A 3 years) and young group (n = 21, 23 +/- A 2). It was analyzed SE, CE [complexity index (CI) and normalized CI (NCI)] and SA (0V, 1V, 2LV and 2ULV patterns) during short heart period series (200 cardiac beats) derived from ECG recordings during 15 min of rest in a supine position. The sequences characterized by three heart periods with no significant variations (0V), and that with two significant unlike variations (2ULV) reflect changes in sympathetic and vagal modulation, respectively. The unpaired t test (or Mann-Whitney rank sum test when appropriate) was used in the statistical analysis. In the aging process, the distributions of patterns (SE) remain similar to young subjects. However, the regularity is significantly different; the patterns are more repetitive in the old group (a decrease of CI and NCI). The amounts of pattern types are different: 0V is increased and 2LV and 2ULV are reduced in the old group. These differences indicate marked change of autonomic regulation. The CE and SA are feasible techniques to detect alteration in autonomic control of heart rate in the old group.

Strength training with and without a vibration platform is capable to modulate heart rate variability?

The current research compared resting heart rate variability (VFC) before and after 10 weeks of strength training in groups that used and did not use a vibration platform. Seventeen healthy men were divided into conventional strength training (TF) or strength training using a vibration platform with a frequency of 30 Hz (TF+V30) training groups. One repetition maximum load (1-RM) on half squat exercise and VFC measurements were determined pre- and post-training program. Both groups had improved 1-RM load after the program (15.1% in TF group and 16.4% in TF+V30 group), although this increase was changed in the same extent for the two groups and there was no difference in 1-RM load between groups pre- and post-training program. No significant difference was observed in resting VFC measurements between groups pre and post-training program, however the magnitude of the effect size was moderated (ES = 0.50-0.80) for some variables (R-R interval, standard deviation of all R-R interval - SDNN, RMSSD, log-transformed of low frequency - InLF, and log-transformed of high frequency - InHF) in TF+V30 group. It was concluded that 10 weeks of strength training program with or without the vibration platform provided similar increase in 1-RM load in both groups, and although some evidences in this study indicate that vibration can increase vagal activity analyzed by ES, in neither groups the strength training was able to change VFC significantly.

Bed nucleus of the stria terminalis and the cardiovascular responses to chemoreflex activation

Several studies from our group have indicated that the BNST play an important role in baroreflex modulation. However, the involvement of the BNST in the chemoreflex activity is unknown. Thus, in the present study, we investigated the effect of the local bed nucleus of stria terminalis (BNST) neurotransmission inhibition by bilateral microinjections of the non-selective synaptic blocker cobalt chloride (CoCl2) on the cardiovascular responses to chemoreflex activation in rats. For this purpose, chemoreflex was activated with KCN (i.v.) before and after microinjections of CoCl2 into the BNST. Reversible BNST inactivation produced no significant changes in the magnitude and durations of both pressor and bradycardic responses to intravenous KCN infusion. These findings suggesting that BNST neurotransmission have not influence on both sympathoexcitatory and parasympathoexcitatory components of the peripheral chemoreflex activation. (C) 2012 Elsevier B.V. All rights reserved.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG + projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. (C) 2012 Elsevier Inc. All rights reserved.

Induction of chronic non-inflammatory widespread pain increases cardiac sympathetic modulation in rats

Fibromyalgia (FM) is characterized by chronic non-inflammatory widespread pain (CWP) and changes in sympathetic function. In attempt to elucidate the pathophysiological mechanisms of FM we used a well-established CWP animal model. We aimed to evaluate changes in cardiac autonomic balance and baroreflex function in response to CWP induction in rats. CWP was induced by two injections of acidic saline (pH 4.0, n = 8) five days apart into the left gastrocnemius muscle. Control animals were injected twice with normal saline (pH 7.2, n = 6). One day after the second injection of acidic saline or normal saline, the animals had pulse interval (PI) and systolic arterial pressure (SAP) variability, and spontaneous baroreflex sensitivity (BRS) evaluated. After induction of CWP, there was an increase of power in the low frequency (LF) band of PI spectrum (12.75 +/- 1.04 nu), a decrease in the high frequency (HF) band (87.25 +/- 1.04 nu) and an increase of LF/HF ratio (0.16 +/- 0.01), when compared to control animals (7.83 +/- 1.13 nu LF; 92.16 +/- 1.13 nu HF; 0.08 +/- 0.01 LF/HF). In addition, there was an increase of power in the LF band of SAP spectrum (7.93 +/- 1.39 mmHg(2)) when compared to control animals (2.97 +/- 0.61 mmHg(2)). BRS was lower in acidic saline injected rats (0.59 +/- 0.06 ms/mmHg) when compared to control animals (0.71 +/- 0.03 ms/mmHg). Our results showed that induction of CWP in rats shifts cardiac sympathovagal balance towards sympathetic predominance and decreases BRS. These data corroborate findings in humans with FM. (C) 2011 Elsevier B.V. All rights reserved.

Both the dorsal hippocampus and the dorsolateral striatum are needed for rat navigation in the Morris water maze

The multiple memory systems theory proposes that the hippocampus and the dorsolateral striatum are the core structures of the spatial/relational and stimulus-response (S-R) memory systems, respectively. This theory is supported by double dissociation studies showing that the spatial and cue (S-R) versions of the Morris water maze are impaired by lesions in the dorsal hippocarnpus and dorsal striatum, respectively. In the present study we further investigated whether adult male Wistar rats bearing double and bilateral electrolytic lesions in the dorsal hippocampus and dorsolateral striatum were as impaired as rats bearing single lesions in just one of these structures in learning both versions of the water maze. Such a prediction, based on the multiple memory systems theory, was not confirmed. Compared to the controls, the animals with double lesions exhibited no improvement at all in the spatial version and learned the cued version very slowly. These results suggest that, instead of independent systems competing for holding control over navigational behaviour, the hippocampus and dorsal striatum both play critical roles in navigation based on spatial or cue-based strategies. (C) 2011 Elsevier B.V. All rights reserved.

Alpha(2)-adrenergic receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats during development

The nucleus tractus solitarii (NTS), located in the brainstem, is one of the main nuclei responsible for integrating different signals in order to originate a specific and orchestrated autonomic response. Antihypertensive drugs are well known to stimulate alpha(2)-adrenoceptor (alpha(2R)) in brainstem cardiovascular regions to induce reduction in blood pressure. Because alpha(2R) impairment is present in several models of hypertension, the aim of the present study was to investigate the distribution and density of alpha(2R) binding within the NTS of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats during development (1,15,30 and 90 day-old) by an in vitro autoradiographical study. The NTS shows heterogeneous distribution of alpha(2R) in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. Alpha(2R) increased from rostral to caudal dorsomedial/dorsolateral subnuclei in 30 and 90 day-old SHR but not in WKY. Alpha(2R) decreased from rostral to caudal subpostremal subnucleus in 15, 30 and 90 day-old SHR but not in WKY. Medial/intermediate subnuclei did not show any changes in alpha(2R) according to NTS levels. Furthermore, alpha(2R) are decreased in SHR as compared with WKY in all NTS subnuclei and in different ages. Surprisingly, alpha(2R) impairment was also found in pre-hypertensive stages, specifically in subpostremal subnucleus of 15 day-old rats. Finally, alpha(2R) decrease from 1 to 90 day-old rats in all subnuclei analyzed. This decrease is different between strains in rostral dorsomedial/dorsolateral and caudal subpostremal subnuclei within the NTS. In summary, our results highlight the importance of alpha(2R) distribution within the NTS regarding the neural control of blood pressure and the development of hypertension. (C) 2011 Elsevier B.V. All rights reserved.

RGMa and RGMb expression pattern during chicken development suggest unexpected roles for these repulsive guidance molecules in notochord formation, somitogenesis, and myogenesis

Background: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues. Results: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo. Conclusions: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development. Developmental Dynamics, 2012. (C) 2012 Wiley Periodicals, Inc.