New insights on amygdala: Basomedial amygdala regulates the physiological response to social novelty.

The amygdala has been associated with a variety of functions linked to physiological, behavioral and endocrine responses during emotional situations. This brain region is comprised of multiple sub-nuclei. These sub-nuclei belong to the same structure, but may be involved in different functions, thereby making the study of each sub-nuclei important. Yet, the involvement of the basomedial amygdala (BMA) in the regulation of emotional states has yet to be defined. Therefore, the aim of our study was to investigate the regulatory role of the BMA on the responses evoked during a social novelty model and whether the regulatory role depended on an interaction with the dorsomedial hypothalamus (DMH). Our results showed that the chemical inhibition of the BMA by the microinjection of muscimol (γ-aminobutyric acid (GABAA) agonist) promoted increases in mean arterial pressure (MAP) and heart rate (HR), whereas the chemical inhibition of regions near the BMA did not induce such cardiovascular changes. In contrast, the BMA chemical activation by the bilateral microinjection of bicuculline methiodide (BMI; GABAA antagonist), blocked the increases in MAP and HR observed when an intruder rat was suddenly introduced into the cage of a resident rat, and confined to the small cage for 15min. Additionally, the increase in HR and MAP induced by BMA inhibition were eliminated by DMH chemical inhibition. Thus, our data reveal that the BMA is under continuous GABAergic influence, and that its hyperactivation can reduce the physiological response induced by a social novelty condition, possibly by inhibiting DMH neurons.

Increased α1-adrenoreceptor activity is required to sustain blood pressure in female rats under food restriction.

AIMS: We evaluated the effect of food restriction (FR) on the various reflexes involved in short term cardiovascular regulation; we also evaluated the contribution of the sympathetic nervous systemand of the plasmatic nitric oxide (NO) in the development of the counterregulatory cardiovascular changes triggered by FR. MAIN METHODS: Female rats were subjected to FR for 14 days, and after this period biochemical measurements of biochemical parameters were performed. For physiological tests, animals were anaesthetised, and a catheter was inserted into the femoral artery and vein for the acquisition of blood pressure and heart hate, and drug infusion, respectively.We then tested the Bezold­Jarisch reflex, the baroreflex and chemoreflex and the effect of the infusion of adrenergic receptor antagonists in control and food restricted animals. KEY FINDINGS: The rats subjected to severe FR presented biochemical changes characteristic of malnutrition with a great catabolic state. FR also led to hypotension and bradycardia besides reducing the plasmatic concentration of NO. Moreover, activation of the Bezold­Jarisch reflex induced a more pronounced hypotensive response in animals subjected to FR. Intravenous infusion of a α1-adrenoreceptor antagonist induced a greater hypotensive response and a more pronounced tachycardic response in animals under food restriction,while the infusion of ß-adrenoreceptor antagonist induced lower increases in blood pressure in these animals. SIGNIFICANCE: Our results suggest that an increased α1-adrenoreceptor activity in the resistance arteries coupled with a reduction of plasmatic NO contributes in a complementary manner to maintain the blood pressure levels in animals under FR.

The role of dorsomedial hypotalamus ionotropic glutamate receptors in the hypertensive and tachycardic responses evoked by Tityustoxin intracerebroventricular injection.

The scorpion envenoming syndrome is an important worldwide public health problem due to its high incidence and potential severity of symptoms. Some studies address the high sensitivity of the central nervous system to this toxin action. It is known that cardiorespiratory manifestations involve the activation of the autonomic nervous system. However, the origin of this modulation remains unclear. Considering the important participation of the dorsomedial hypotalamus (DMH) in the cardiovascular responses during emergencial situations, the aim of this work is to investigate the involvement of the DMH on cardiovascular responses induced by intracerebroventricular (icv) injection of Tityustoxin (TsTX, a α-type toxin extracted from the Tityus serrulatus scorpion venom). Urethane-anaesthetized male Wistar rats (n=30) were treated with PBS, muscimol or ionotropic glutamate receptor antagonists, bilaterally in DMH and later, with an icv injection of TsTX, or treated only with PBS in both regions. TsTX evoked a marked increase in mean arterial pressure and heart rate in all control rats. Interestingly, injection of muscimol, a GABAA receptor agonist, did not change the pressor and tachycardic responses evoked by TsTX. Remarkably, the injection ionotropic glutamate receptors antagonists in DMH abolished the pressor and the tachycardic response evoked by TsTX. Our data suggest that the central circuit recruited by TsTX, whose activation results in an array of physiological and behavioral alterations, depend on the activation of DMH ionotropic glutamate receptors. Moreover, our data provide new insights on the central mechanisms involved in the development of symptoms in the severe scorpion envenomation syndrome.

Malnutrition alters the cardiovascular responses induced by central injection of tityustoxin in Fischer rats.

Scorpion envenoming and malnutrition are considered two important public health problems in Brazil, involving mainly children. Both these conditions are more common among the economically stratified lower income portion of the population, thus suggesting that these factors should be analyzed concomitantly. It is known that cardiorespiratory manifestations, as cardiac arrhythmias, arterial hypertension and hypotension, pulmonary edema and circulatory failure are the main "causa mortis" of scorpion envenomation. Additionally, there are evidences in the literature that deficiencies in dietary intake endanger the CNS and modify the cardiovascular homeostasis. Then, the objective of this work is to evaluate the protein malnourished effect on cardiovascular responses induced by tityustoxin (TsTX, an α-type toxin extracted from the Tityus serrulatus scorpion venom). Fischer rats (n = 20) were injected i.c.v. with TsTX and divided in control and malnorished groups, which were, respectively, submitted to a control and a low-protein diet. Arterial pressure recordings were done until death of the animals. Although both groups presented an increased mean arterial pressure after TsTX injection, this increase was smaller and delayed in malnourished rats, when compared to control rats. In addition, heart rate increased only in rats from the control group. Finally, malnourished rats had an increase in survival time (9:9/13.5 vs. 15.5:10.5/18 min; p = 0.0009). In summary, our results suggest that the protein restriction attenuates the cardiovascular manifestations resulting from TsTX action on CNS.

Protein malnutrition modifies medullary neuronal recruitment in response to intermittent stimulation of the baroreflex.

Protein malnutrition after weaning changes the neurotransmission in neural pathways that organize cardiovascular reflexes in rats. The present study evaluates whether protein malnutrition alters the expression of c-fos protein (immediate-early gene expression) in central areas involved in the control of cardiovascular reflexes after intermittent stimulation of the baroreflex. The main nuclei we focused were paraventricular hypothalamus (PVH); nucleus tract solitarii (NTS); rostral ventromedial medulla (RVMM); rostral (RVLM) and caudal ventrolateral medulla (CVLM). Male Fisher rats at 28 days were submitted to two different isocaloric diets during the subsequent 35 days: control (CT) (15% protein) and malnourished (MN) (6% protein). thirtymin of intermittent (every 3 min) baroreflex stimulation was performed by infusing phenylephrine (Phe-0.25 mM) or, as control, 0.9% NaCl (Sal). Following ninety minutes, animals were anesthetized and perfused. The removed brains were sectioned (35 µm) and used for c-fos immunohistochemistry. Images were analyzed using the software Leica Q Win. Despite not altering the baseline MAP, malnutrition increased baseline HR and expression of c-fos in RVMM. Increases in c-fos expression after intermittent stimulation of baroreflex were evident in the PVH, medial NTS and CVLM in both dietary protocols. Current data further revealed a differential neuronal recruitment to stimulation of baroreflex in the caudal commissural and rostral NTS and RVLM of MN. We conclude that protein malnutrition modifies the cardiovascular control and the pattern of central response to baroreflex stimulation.

Cardiac responses of rats submitted to postnatal protein restriction.

Undernutrition during critical stages of development and childhood has important effects on cardiovascular homeostasis. The present study was undertaken to evaluate the in vivo and ex vivo cardiac function of rats submitted to postnatal protein restriction. Male Wistar rats (28 days old) were fed a regular (20%) or low-protein (6%) diet over 5 weeks. After this period, cardiac function was analyzed by echocardiography and isolated heart preparation. Furthermore, the density of cardiac noradrenergic fibers and hematological profile were evaluated. We found that malnourished rats exhibited elevated arterial blood pressure, increased fractional shortening (echocardiography), increased systolic tension, increased ±dT/dt (isolated heart technique), impaired diastolic function characterized by a slight increase in the left ventricular end-diastolic diameter (echocardiography) and decreased diastolic tension (isolated heart technique), and cardiac hypertrophy evidenced by augmentation of the posterior left ventricular wall and discrete hematological changes. In addition, malnourished rats exhibited increased noradrenergic fiber density in their hearts (0.08% ± 0.02% area in control rats vs. 0.17% ± 0.03% area in malnourished rats). Our current data demonstrate that postnatal protein restriction causes cardiac adaptation characterized by an early overworking heart. This is at least in part mediated by an increase in the efferent sympathetic fibers to the heart. These findings provide important information for efforts to prevent and manage the consequences of undernutrition in the human population.

Malnutrition during central nervous system growth and development impairs permanently the subcortical auditory pathway.

The brain that grows and develops under the continued influence of malnutrition presents permanent impairment on functioning and neurotransmitter release. The aim of this study was to investigate the chronic effects of neonatal food restriction on neurochemical and neurodynamical aspects within the primary auditory sensory pathway. Our working hypothesis is that neonatal malnutrition may affect the flow of primary sensory information both at a neurochemical and neurodynamical level. To test this hypothesis, three groups of rats were assigned, from birth to 370 days of life, to the following dietary scheme: a well-nourished (WN) group fed ad libitum lab chow diet; an undernourished (UN) group fed 60% of diet consumed by WN group; and a rehabilitated group, undergoing same dietary restriction as undernourished until 42 days of age and thereafter fed ad libitum until the end of the experiment. At 370 days of age, the animals were submitted to brainstem auditory-evoked potentials (BAEPs) recordings and sacrificed for neurochemical evaluation of glutamate release. Undernutrition decreased glutamate release in the cortex, hippocampus, midbrain and brainstem, and significantly increased the latency of BAEP wave V. In addition; the re-establishment of the dietary conditions was not sufficient to reverse the neurochemical and electrophysiological alterations observed in the UN group. Taken altogether, our results suggest that malnutrition imposed at a critical development period caused an irreversible effect within the auditory primary sensory pathway.

Malnutrition affects the pressor response to microinjection of L-glutamate into the RVLM of awake rats.

Post-weaning protein malnutrition is often related to the development of cardiovascular and metabolic diseases in humans, as well to changed content of neurotransmitters in the central nervous system under experimental conditions. The rostral ventrolateral medulla (RVLM) is a bulbar region that contains sympathetic premotor neurons; the excitatory amino acid L-glutamate seems to be the main neurotransmitter at this level. The aim of the present study was to evaluate the possible change in the L-glutamate sensitivity of the RVLM neurons of malnourished animals. Male Fischer rats were divided into two groups: control (n = 15) and malnourished (n = 19). Four days before the experiments, guide cannulas were implanted bilaterally in direction of the RVLM for microinjection of L-glutamate. Twenty-four hours before the experiments, the femoral artery was cannulated for cardiovascular recordings. The results showed that the baseline heart rate increased in malnourished compared to control animals (412.18 ± 16.03 bpm vs. 370.74 ± 9.59 bpm, respectively). Malnourished animals presented a dissimilar concentration-dependent pressor response curve to L-glutamate and an attenuated baroreflex gain. Our results suggest that post-weaning protein restriction affects glutamatergic neurotransmission of the baroreflex at the RVLM level.

Bezold-Jarisch reflex in sino-aortic denervated malnourished rats.

In this study we assessed the role of Bezold-Jarisch reflex (BJR) in the regulation of blood pressure (BP) of malnourished (MN) and control rats (CN) with sino-aortic denervation (SAD). Fischer rats were fed diets containing either 6% (MN) or 15% (CN) protein for 35 days after weaning. These rats underwent sham or SAD and catheterization of femoral artery and vein for BP measurements and drug injection. Phenylbiguanide (PBG 5 µg/kg, i.v.) for activation BJR, produced bradycardia (-317±22 bpm for CN vs. -372±16 bpm for MN) and hypotension (-57±4 mm Hg for CN vs. -54±6 mm Hg for MN. After SAD, MN rats had reduced hypotensive (-37±7 mm Hg for MN vs. -82±6 mm Hg for CN) and bradycardic (-124±17 for MN vs. -414±20 bpm CN) responses to BJR activation. To evaluate the contribution of the parasympathetic component due to BJR for the fall in BP, methyl atropine bromide, was given between two injections of PBG (5 µg/kg) separated by 10 min each other. Both bradycardic (-216±21 bpm before and -4±3 bpm after for CN -226±43 bpm before and -9±20 bpm after for MN) and hypotensive (-42±4 mm Hg before and -6±1 mm Hg after for CN -33±9 mm Hg before and -5±2 mm Hg after for MN) responses were abolished in CN and MN groups. These data indicate that dietary protein malnutrition changes the relation between baroreflex and BJR required for maintenance of the BP during malnourishment.

Sympathoinhibition to Bezold-Jarisch reflex is attenuated in protein malnourished rats.

Malnutrition affects cardiovascular reflexes, including chemoreflex and baroreflex. In this study we assessed the hypothesis that malnourishment changes the responses in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) evoked from Bezold-Jarisch reflex (BJR). Fischer rats were fed diets containing either (6% malnourished or 14% control) protein for 35 days after weaning. There were no differences in baseline MAP (102 ± 4 vs. 95 ± 3 mmHg) whereas higher baseline HR (478 ± 18 vs. 360 ± 11 bpm; P<0.05,) and reduced sympathoinhibition (ΔRSNA=-54 ± 9 vs. -84 ± 7%; P=0.0208) to BJR activation were found in malnourished rats. We conclude that malnutrition affects the sympathetic control of BJR.

Cardiovascular responses to hydrogen peroxide into the nucleus tractus solitarius.

The nucleus tractus solitarius (NTS), a major hindbrain area involved in cardiovascular regulation, receives primary afferent fibers from peripheral baroreceptors and chemoreceptors. Hydrogen peroxide (H(2)O(2)) is a relatively stable and diffusible reactive oxygen species (ROS), which acting centrally, may affect neural mechanisms. In the present study, we investigated effects of H(2)O(2) alone or combined with the glutamatergic antagonist kynurenate into the NTS on mean arterial pressure (MAP) and heart rate (HR). Conscious or anesthetized (urethane and alpha-chloralose) male Holtzman rats (280-320 g) were used. Injections of H(2)O(2) (125 to 1500 pmol/40 nl) into the intermediate NTS of anesthetized rats evoked dose-dependent and transient hypotension (-18 +/- 3 to -55 +/- 11 mmHg) and bradycardia (-16 +/- 5 to -116 +/- 40 bpm). Injection of the catalase inhibitor 3-amino-1,2,4-triazole (100 nmol/40 nl) into the NTS also produced hypotension and bradycardia. Previous injection of the ionotropic L-glutamate receptor antagonist kynurenate (7 nmol/40 nl) attenuated by 48% the bradycardic response, without changing the hypotension evoked by H(2)O(2) (500 pmol/40 nl) in anesthetized rats. The antioxidant L-ascorbate (600 pmol/80 nl) injected into the NTS attenuated the bradycardic (42%) and hypotensive (67%) responses to H(2)O(2) (500 pmol/40 nl) into the NTS. In conscious rats, injection of H(2)O(2) (50 nmol/100 nl) into the NTS also evoked intense bradycardia (-207 +/- 8 bpm) and hypotension (-54 +/- 6 mmHg) that were abolished by prior injection of kynurenate (7 nmol/100 nl). The results show that H(2)O(2) into the NTS induces hypotension and bradycardia probably due to activation of glutamatergic mechanisms.

Dose-dependent effect of carbamazepine on weanling rats submitted to subcutaneous injection of tityustoxin.

The scorpion envenoming syndrome is a serious public health matter in Brazil. The most severe cases occur during childhood and elderly. Previous results from our laboratory suggest that the effects of scorpion toxins on the central nervous system play a major role on the lethality induced by scorpion envenoming. The aim of this work is to evaluate the therapeutic potential of carbamazepine (CBZ) injected i.p. 90 min before s.c. tityustoxin (TsTX) injection in weanling rats. Rats were divided into six experimental groups according to s.c. injection (saline or TsTX) and i.p. treatment (vehicle or CBZ 12, 50 and 100 mg/kg): Sal/Veh group (n=4); Sal/CBZ100 (n=4); TsTX/CBZ12 (n=6); TsTX/CBZ50 (n=8); TsTX/CBZ100 (n=8) and, at last, TsTX/Veh (n=8). The dose of TsTX was the same for all groups: 6.0mg/kg, twice the DL50 for weanling rats. Video images were recorded until death or for a maximum period of 240 min. Lungs were excised and weighed to evaluate edema. The results showed that CBZ (12, 50 and 100mg/kg) was able to increase the survival rate and latency-to-death of the rats. Only the group treated with 100mg/kg of CBZ had a decrease in the pulmonary edema. The known effect of CBZ reducing neuronal excitability most likely protected the neural substrates targeted by TsTX. Although treatment was performed before TsTX inoculation, the results are promising regarding CBZ as a therapeutic coadjuvant in the treatment of scorpion poisoning. The pharmacokinetics of CBZ can be very much improved by either changing the form of administration or encapsulating the drug in order to enhance solubility.

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats.

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.