Effects of chronic exposure to 12‰ saltwater on the endocrine physiology of juvenile American alligator (Alligator mississippiensis).

American alligator (Alligator mississippiensis) habitats are prone to saltwater intrusion following major storms, hurricanes or droughts. Anthropogenic impacts affecting hydrology of freshwater systems may exacerbate saltwater intrusion into freshwater habitats. The endocrine system of alligators is susceptible to changes in the environment but it is currently not known how the crocodilian physiological system responds to environmental stressors such as salinity. Juvenile alligators were exposed to 12‰ saltwater for 5 weeks to determine the effects of chronic exposure to saline environments. Following 5 weeks, plasma levels of hormones [e.g. progesterone, testosterone, estradiol, corticosterone, aldosterone (ALDO), angiotensin II (ANG II)] were quantified using liquid chromatography and tandem mass spectrometry. Compared with freshwater-kept subjects, saltwater-exposed alligators had significantly elevated plasma levels of corticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone, testosterone, 17ß-estradiol, estrone and estriol whereas pregnenolone and ANG II were significantly depressed and ALDO levels were unchanged (slightly depressed). On the one hand, saltwater exposure did not affect gene expression of renal mineralocorticoid and glucorticoid and angiotensin type 1 (AT-1) receptors or morphology of lingual glands. On the other hand, saltwater exposure significantly reduced plasma glucose concentrations whereas parameters diagnostic of perturbed liver function (aspartate aminotransferase and alanine aminotransferase) and kidney function (creatinine and creatine kinase) were significantly elevated. Except for plasma potassium levels (K+), plasma ions Na+ and Cl- were significantly elevated in saltwater alligators. Overall, this study demonstrated significant endocrine and physiological effects in juvenile alligators chronically exposed to a saline environment. Results provide novel insights into the effects of a natural environmental stressor (salinity) on the renin-angiotensin-aldosterone system and steroidogenesis of alligators.

Temperature and species-specific effects on ß3-adrenergic receptor cardiac regulation in two freshwater teleosts: Channel catfish (Ictalurus punctatus) and common carp (Cyprinus carpio).

ß3-adrenergic receptors (AR) are important in teleost cardiovascular regulation. To date, it is unknown whether temperature acclimation changes ß3-AR functionality and consequently the involvement of this AR subtype in teleost cardiac regulation. Common carp (Cyprinus carpio) were acclimated at 12 °C or 23 °C (minimum 3 weeks) after which cardiovascular variables (cardiac output (Q), stroke volume (Sv) and heart rate (fH)) were measured upon injection of the ß3-AR agonist, BRL(37344), and antagonist, SR(59230A). In both 12 °C and 23 °C acclimated carp, BRL(37344) induced significant increases in fH and Q whereas Sv was significantly decreased. While temperature did not affect the change (increase vs. decrease) in cardiac variables, the magnitude and on-set of responses differed. For instance, fH, Sv and Q responded significantly faster to ß3-AR stimulation in 23 °C carp. In contrast, maximum responses of fH and Q were significantly higher in 23 °C carp whereas the maximum response of Sv was significantly greater in 12 °C carp. These findings suggest that temperature acclimation induced changes in ß3-AR receptor functionality (e.g. density and/or affinity). Stimulation of ß3-ARs in 23 °C acclimated channel catfish (Ictalurus punctatus) caused significant increases in fH, Sv and Q. The increase in Sv was opposite to the decrease observed in 23 °C acclimated common carp. SR(59230A) induced significant decreases in Sv and Q but had no effect in carp (23 °C). Results suggest species diversity in the density and affinity or structure of ß3-ARs which may explain the different cardiac responses to ß3-AR ligands.

Brainstem mechanisms controlling cardiovascular reflexes in channel catfish.

Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia.

Sensory receptors in the first gill arch of rainbow trout.

Afferent neural activity was recorded from sensory receptors innervated by the glossopharyngeal nerve (cranial nerve IX) in isolated, perfused first gill arch preparations from rainbow trout. The present study demonstrates the presence of every major type of peripheral cardio-respiratory receptor described in fish in this preparation. Oxygen-sensitive chemoreceptors responsive to internal and/or external hypoxia and cyanide were present. Qualitatively these receptors behaved in an identical fashion which was also similar to that described for mammalian carotid body chemoreceptors. About 5% of the sensory receptors examined were O2-sensitive. proprioceptors were the most numerous receptor type identified and were sensitive to mechanical stimulation of the arch, rakers or filaments. Finally, baroreceptors, the least numerous class of receptor identified, were also present with activity that was altered in response to changes in perfusion pressure. While the reflex responses elicited by the stimulation of these receptors were not addressed in this study, it is likely that these receptors contribute to the reflex cardio-respiratory responses to changes in gill perfusion, gill deflection and hypoxia (environmental or internal) described in fishes. These data thus support suggestions concerning homologies between the first gill arch of teleosts and the carotid bodies of mammals and the importance of the first gill arch in trout in cardio-respiratory control.

Cardio-ventilatory control in rainbow trout: II. Reflex effects of exogenous neurochemicals.

The effects of various neurochemicals were examined in intact, unanesthetized rainbow trout (Oncorhynchus mykiss) to assess the role of branchial O2-sensitive chemoreceptors in the cardio-ventilatory responses to exogenous neurochemicals. cyanide stimulated ventilation and elicited bradycardia when give externally but only stimulated ventilation when injected internally. Norepinephrine increased heart rate, blood pressure and ventilatory rate but opercular pressure was not affect. Dopamine had no effect on either heart or ventilatory rate but increased blood pressure and decreased opercular pressure. Serotonin stimulated heart rate and ventilation but decreased blood pressure. Acetylcholine and nicotine stimulated all cardio-ventilatory variables. Muscarine decreased heart rate and blood pressure and had a biphasic effect on ventilation. These results, combined with the results from the preceding study, suggest that the cardio-ventilatory effects of exogenously administered (1) cyanide are entirely mediated by gill O2 receptors, (2) serotonin, and cholinergic drugs could be partly mediated by O2 receptors and (3) catecholaminergic drugs are not mediated by O2 receptors.

Branchial chemoreceptors mediate ventilatory responses to hypercapnic acidosis in channel catfish.

The effects of hyperoxic hypercapnia on cardiovascular and ventilatory variables and blood gas and acid/base parameters were examined in conscious and anesthetized spontaneously breathing (ASB) channel catfish, Ictalurus punctatus. These separate experiments were designed to determine: (1) if channel catfish show a ventilatory response to hypercapnic acidosis when blood O(2) content is maintained in conscious animals; and (2) whether branchial receptors innervated by cranial nerves IX and X mediate this response. The combination of high O(2) and CO(2) tensions allowed the cardioventilatory effects of hypercapnic acidosis to be assessed independently of Root or Bohr mediated changes in blood O(2) content. In the absence of significant changes in dorsal or ventral aorta O(2) content, hyperoxic hypercapnia significantly stimulated ventilation, relative to hyperoxic exposure. Hypercapnic acidosis, however, had no significant effects on blood pressure or heart rate. Branchial denervation in ASB fish abolished the ventilatory response to hypercapnic acidosis. The results indicate that hypercapnic acidosis independently stimulates ventilation in channel catfish. This response is mediated by CO(2)/pH-sensitive branchial receptors innervated by cranial nerves IX and X.

Propranolol inhibits O2-sensitive chemoreceptor activity in trout gills.

The effects of propranolol on the activity of O2-sensitive chemoreceptors innervated by cranial nerve IX were studied using an isolated, perfused first gill arch preparation from rainbow trout (Onchyrhyncos mykiss). Perfusing the gill with hypoxic perfusate resulted in an increase in chemoreceptor activity. Propranolol (100-200 nmol) added to the perfusate inhibited O2-receptor discharge during both normoxia and hypoxia and attenuated the response to bolus injections of NaCN (25 micrograms). These results suggest that a beta-adrenergic mechanism is involved in O2 chemoreception. They further suggest that the inhibitory effects of propranolol on branchial, O2-sensitive chemoreceptors may contribute to the attenuated hypoxic ventilatory reflex observed in intact fish after propranolol injections.

Central nervous control of gill filament muscles in channel catfish.

The gills of fish are innervated by cranial nerves IX and X. There have been a number of studies on the characteristics of sensory activity carried by these nerves but remarkably little is known about motor control of the gills. Efferent, motor activity to the first gill arch was recorded from the glossopharyngeal nerve in spontaneously breathing channel catfish, Ictalurus punctatus. This study addressed two objectives. The first objective was to characterize efferent branchial nerve activity in spontaneously breathing fish. Nerve recordings show bursts of activity firing in synchrony with ventilation. These bursts occurred once during either abduction or adduction of the operculum with each breath. The observed patterns of neural activity indicate that it represents motor control of gill filament abductor and adductor muscles. The data show that rhythmic output from a central pattern generator controls filament musculature during the ventilatory cycle. The second objective was to use this efferent branchial nerve activity as an index of ventilation (fictive ventilation) in fish before and after paralysis to determine if feedback from phasic mechanoreceptors affects ventilatory timing. Breath-to-breath intervals measured before and after paralysis with gallamine were not significantly different, demonstrating that rhythmic feedback from phasic mechanoreceptors in the gills and/or ventilatory musculature is not involved in the breath-to-breath timing of the normal ventilatory cycle. During the course of these experiments many fish exhibited coughing. Coughs were characterized by a distinctive pattern of nerve activity that was not altered by paralysis. Overall, the data indicate that phasic mechanoreceptor feedback during normal breathing has no effect on the pattern of central motor control of gill filament muscles.

Cardio-ventilatory control in rainbow trout: I. Pharmacology of branchial, oxygen-sensitive chemoreceptors.

The effects of various neurochemicals on O2-sensitive chemoreceptor afferent discharge in the glossopharyngeal nerve (cranial nerve IX) were examined in an isolated, perfused first gill arch preparation from rainbow trout. Afferent neural activity from O2 receptors in the first gill arch increased in response to hypoxic perfusate and NaCN. Adrenergic agonists (epinephrine, norepinephrine and isoproterenol) had little effect on neural activity. Dopamine and 5-hydroxytryptamine (serotonin) caused a brief, small burst in chemoreceptor activity followed by a mild inhibition of receptor discharge. Acetylcholine and nicotine were potent neurochemical stimulants; muscarine had only a slight effect. While atropine completely blocked the effects of acetylcholine on receptor discharge, it only slightly inhibited responses to hypoxia and NaCN. Thus, although cholinergic mechanisms appear more likely than either adrenergic or serotonergic mechanisms to alter cardiovascular and ventilatory reflexes in fishes through their effects on O2-sensitive chemoreceptor activity, the transduction process involved in O2-chemoreception appears to be complex and not dependent on any single one of the neurochemicals tested.

Chemoreflexive responses to hypoxia and NaCN in longnose gar: evidence for two chemoreceptor loci.

Interactions between internal and external O2 stimulus levels were assessed by measuring the ventilatory and cardiovascular responses to varying water (PWO2) and air bladder (PabO2) O2 levels and intravascular NaCN in anesthetized spontaneously ventilating Lepisosteus osseus. As PWO2 fell, air-breathing frequency (fab) increased. Buccal pressure amplitude (Pb) also increased as PWO2 fell from hyperoxia to normoxia, but hypoxic water depressed Pb. The PO2 in the ventral aorta (VA) fell as PabO2 fell, which stimulated fab and Pb when the gar was in normoxic or hyperoxic water. Thus gill ventilation and air breathing were normally controlled by both internal and external O2 levels, but aquatic hypoxia uniformly depressed gill ventilation regardless of changes in PabO2 levels. Heart rate and blood pressure were unaffected by these changes. NaCN stimulated hypoxic reflexes and bradycardia more quickly when given into the VA or conus than when given into the dorsal aorta. The animals appear to possess internal chemoreceptors that set the level of hypoxic drive and external chemoreceptors that inhibit gill ventilation and shift the ventilatory emphasis from water to air breathing.

Branchial mechanoreceptor activity during spontaneous ventilation in channel catfish.

Extracellular afferent neural activity was recorded in vivo from cranial nerve IX (glossopharyngeal) from mechanoreceptors in the first gill arch of anesthetized, spontaneously breathing channel catfish (Ictalurus punctatus). Single unit and paucifiber recordings show that both phasic and tonic receptors were active during normal ventilation. Phasic receptors were characterized as having a burst of activity during some phase of the ventilatory cycle. Most of these occurred during peak adduction or peak abduction. Phasic receptors were not active during spontaneous apnic periods. Tonic receptors were always active, even during apneas, firing frequency was modulated by breathing movements with peak activity occurring during adduction. Flow-sensitive mechanoreceptors were identified in anesthetized, paralyzed catfish. These receptors decreased activity when the ventilatory water flow was stopped. Hypercapnia (5% CO(2) in air) stimulated ventilatory rate and amplitude but had no effect on mechanoreceptor activity. The discharge characteristics of branchial mechanoreceptors indicate that they could be involved in the timing and coordination of ventilatory movements and maintenance of the 'gill curtain' to minimize ventilatory dead space. Unlike ventilatory mechanoreceptors in the air breathing organs of gar and lungs of lungfish and tetrapods, branchial mechanoreceptors were insensitive to hypercapnia.