Role of midbrain in the control of breathing in anuran amphibians

The present study was designed to explore systematically the midbrain of unanesthetized, decerebrate anuran amphibians (bullfrogs), using chemical and electrical stimulation and midbrain transections to identify sites capable of exciting and inhibiting breathing. Ventilation was measured as fictive motor output from the mandibular branch of the trigeminal nerve and the laryngeal branch of the vagus nerve. The results of our transection studies suggest that, under resting conditions, the net effect of inputs from sites within the rostral half of the midbrain is to increase fictive breathing frequency, whereas inputs from sites within the caudal half of the midbrain have no net effect on fictive breathing frequency but appear to act on the medullary central rhythm generator to produce episodic breathing. The results of our stimulation experiments indicate that the principal sites in the midbrain that are capable of exciting or inhibiting the fictive frequency of lung ventilation, and potentially clustering breaths into episodes, appear to be those primarily involved in visual and auditory integration, motor functions, and attentional state.

Locus coeruleus is a central chemoreceptive site in toads

The locus coeruleus (LC) has been suggested as a CO2 chemoreceptor site in mammals. This nucleus is a mesencephalic structure of the amphibian brain and is probably homologous to the LC in mammals. There are no data available for the role of LC in the central chemoreception of amphibians. Thus the present study was designed to investigate whether LC of toads (Bufo schneideri) is a CO2/H+ chemoreceptor site. Fos immunoreactivity was used to verify whether the nucleus is activated by hypercarbia (5% CO2 in air). In addition, we assessed the role of noradrenergic LC neurons on respiratory and cardiovascular responses to hypercarbia by using 6-hydroxydopamine lesion. To further explore the role of LC in central chemosensitivity, we examined the effects of microinjection of solutions with different pH values (7.2, 7.4, 7.6, 7.8, and 8.0) into the nucleus. Our main findings were that 1) a marked increase in c-fos-positive cells in the LC was induced after 3 h of breathing a hypercarbic gas mixture; 2) chemical lesions in the LC attenuated the increase of the ventilatory response to hypercarbia but did not affect ventilation under resting conditions; and 3) microinjection with acid solutions (pH = 7.2, 7.4, and 7.6) into the LC elicited an increased ventilation, indicating that the LC of toads participates in the central chemoreception.

Cardiovascular actions of rattlesnake bradykinin ([Val(1), Thr(6)] bradykinin) in the anesthetized South American rattlesnake Crotalus durissus terrificus

Incubation of heat-denatured plasma from the rattlesnake Crotalus atrox with trypsin generated a bradykinin (BK) that contained two amino acid substitutions (Arg(1) --> Val and Ser(6) --> Thr) compared with mammalian BK. Bolus intra-arterial injections of synthetic rattlesnake BK (0.01-10 nmol/kg) into the anesthetized rattlesnake, Crotalus durissus terrificus, produced a pronounced and concentration-dependent increase in systemic vascular conductance (Gsys). This caused a fall in systemic arterial blood pressure (Psys) and an increase in blood flow. Heart rate and stroke volume also increased. This primary response was followed by a significant rise in Psys and pronounced tachycardia (secondary response). Pretreatment with N-G-nitro-L-arginine methyl ester reduced the NK-induced systemic vasodilatation, indicating that the effect is mediated through increased NO synthesis. The tachycardia associated with the late primary and secondary response to BK was abolished with propranolol and the systemic vasodilatation produced in the primary phase was also significantly attenuated by pretreatment, indicating that the responses are caused, at least in part, by release of cathecholamines and subsequent stimulation of beta-adrenergic receptors. In contrast, the pulmonary circulation was relatively unresponsive to BK.

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.

Interaction of serotonin and cholecystokinin in the lateral parabrachial nucleus to control sodium intake

Serotonin [5-hydroxytryptamine (5-HT)] and CCK injected into the lateral parabrachial nucleus (LPBN) inhibit NaCl and water intake. In this study, we investigated interactions between 5-HT and CCK into the LPBN to control water and NaCl intake. Male Holtzman rats with cannulas implanted bilaterally in the LPBN were treated with furosemide + captopril to induce water and NaCl intake. Bilateral LPBN injections of high doses of the 5-HT antagonist methysergide (4 mug) or the CCK antagonist proglumide (50 mug), alone or combined, produced similar increases in water and 1.8% NaCl intake. Low doses of methysergide (0.5 mug) + proglumide (20 mug) produced greater increases in NaCl intake than when they were injected alone. The 5-HT2a/2c agonist 2,5-dimetoxy-4-iodoamphetamine hydrobromide (DOI; 5 mug) into the LPBN reduced water and NaCl intake. After proglumide (50 mug) + DOI treatment, the intake was not different from vehicle treatment. CCK-8 (1 mug) alone produced no effect. CCK-8 combined with methysergide (4 mug) reduced the effect of methysergide on NaCl intake. The data suggest that functional interactions between 5-HT and CCK in the LPBN may be important for exerting inhibitory control of NaCl intake.

Water deprivation-induced sodium appetite: humoral and cardiovascular mediators and immediate early genes

Adult rats deprived of water for 24-30 h were allowed to rehydrate by ingesting only water for 1-2 h. Rats were then given access to both water and 1.8% NaCl. This procedure induced a sodium appetite defined by the operational criteria of a significant increase in 1.8% NaCl intake (3.8 +/- 0.8 ml/2 h; n = 6). Expression of Fos (as assessed by immunohistochemistry) was increased in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), and supraoptic nucleus (SON) after water deprivation. After rehydration with water but before consumption of 1.8% NaCl, Fos expression in the SON disappeared and was partially reduced in the OVLT and MnPO. However, Fos expression did not change in the SFO. Water deprivation also 1) increased plasma renin activity (PRA), osmolality, and plasma Na+; 2) decreased blood volume; and 3) reduced total body Na+; but 4) did not alter arterial blood pressure. Rehydration with water alone caused only plasma osmolality and plasma Na+ concentration to revert to euhydrated levels. The changes in Fos expression and PRA are consistent with a proposed role for ANG II in the control of the sodium appetite produced by water deprivation followed by rehydration with only water.

Water deprivation-induced sodium appetite and differential expression of encephalic c-Fos immunoreactivity in the spontaneously hypertensive rat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of sodium appetite by lipopolysaccharide: involvement of alpha(2)-adrenoceptors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cardiovascular responses to hydrogen peroxide into the nucleus tractus solitarius

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antihypertensive effects of central ablations in spontaneously hypertensive rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitric oxide modulates the cardiovascular effects elicited by acetylcholine in the NTS of awake rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Energy substrate utilization during nightly vocal activity in three species of Scinax (Anura/Hylidae)

Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or locomotor activity. Finally, we found exceptionally high levels of carbohydrates and lipids in the liver of S. rizibilis, a trait perhaps related to a long and demanding breeding period.

Gluconeogenesis and P-enolpyruvate carboxykinase in liver and kidney of long-term fasted quails

The activity of cytoplasmic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) in kidney and liver, and in vivo gluconeogenic activity, were determined during different phases of prolonged fasting in quails. The fasting-induced changes in the activity of kidney cytoplasmic PEPCK were positively correlated with the changes in gluconeogenesis. Both activities increased at the initial phase (I) of fasting to levels 65% to 100% higher than fed values, and decreased during the protein-sparing period (phase II), although remaining higher than in fed birds. At the catabolic final phase (III) both kidney cytoplasmic PEPCK activity and gluconeogenesis increased markedly, attaining levels 115% to 150% higher than fed values. The activity of liver cytoplasmic PEPCK, present in appreciable amounts in quails, did not change during phases I and II of fasting, but increased to levels 60% higher than fed values at the final phase (III). Plasma glucose levels at phase III did not differ significantly from those at phases I and II. In both kidney and liver the activity of the mitochondrial PEPCK was not significantly affected by fasting. The data suggest that the kidney cytoplasmic PEPCK is the main enzyme responsible for gluconeogenesis adjustments during food deprivation in quails, and that this function is complemented at the final phase by enzyme present in liver cytosol.