Intact Frogs Research Articles

In vivo confocal imaging of fast intrinsic optical signals correlated with frog retinal activation.

Using freshly isolated animal retinas, we have conducted a series of experiments to test fast intrinsic optical signals (IOSs) that have time courses comparable to electrophysiological kinetics. In this Letter, we demonstrate the feasibility of in vivo imaging of fast IOSs in intact frogs. A rapid line-scan confocal ophthalmoscope was constructed to achieve high-speed IOS recording. By rejecting out-of-focus background light, the line-scan confocal imager provided the resolution to differentiate individual photoreceptors in vivo. Rapid confocal imaging disclosed robust IOSs with time courses comparable to retinal electroretinogram kinetics. High-resolution IOS images revealed both positive (increasing) and negative (decreasing) light responses, with subcellular complexity.

Functional role of hypothalamic D1 and D2 receptors in organization of some forms of behavior of the common frog Rana temporaria

Dopamine is one of the most ancient, widely spread neurotransmitters. It performs a great number of neuromodulator effects in the vertebrates CNS. For the last few years there considerably increases an interest in study of functional role of this neurotransmitter in regulation of various forms of behavior of poikilothermal vertebrates. The present work deals with study of the role of the dopaminergic system, specifically of the hypothalamic dopaminergic system in providing some behavioral frog reactions. We studied behavior of the animals in the “open field” before and after administration to them of antagonists of D1 (SCH 23390) and D2 (haloperidol) receptors as well as of animals with destroyed anterior and posterior parts of hypothalamus. Administration of SCH 23390 to intact frogs caused a statistically significant decrease in the number of exploratory reactions and goal-oriented jumps, whereas haloperidol only moderately increased the number of the above reactions. Destruction of the posterior part of hypothalamus suppressed essentially all kinds of activity, while destruction of the anterior part inhibited them completely. Antagonist of D12 and D2 receptors of dopamine little changed the initial motor and emotional activity of the operated animals. The obtained data are discussed in terms of evolutionary origin of D1 and D2 receptors in vertebrates and allow concluding that D1 and D2 receptors of hypothalamic dopamine of the common frog are located predominantly in the anterior hypothalamic areas and that their effect on behavior can be mediated and is associated with other brain neurotransmitter systems in such brain structures as lateral hypothalamus, locus coereleus, and striatum that provide different aspects of wakefulness of amphibians.

Extracellular matrix molecules and their possible roles in the regeneration of frog nervous system

Recent biochemical and histochemical analyses explored different components of the extracellular matrix (ECM) in the nervous system, and either permissive or non-permissive roles in neuronal development and regeneration were suggested. The aim of this study was to detect the distribution pattern of a few of these molecules in the nervous system of intact frogs and during nerve regeneration. The hyaluronan (HA) and tenascin C reactions were negative in the peripheral nerves, but appeared in their entry zones. In the CNS, different populations of neurons were surrounded with HA and tenascin C-positive material, forming a perineuronal net (PN). The phosphacan reaction was weakly positive in the PNS, and a moderate intensity was detected in the entry zone and in the PN. Laminin and fibronectin immunoreactivity was strong in the PNS, but laminin could not be detected in the CNS. In animals with cut and regenerating vestibulocochlear nerve, the distribution of the ECM molecules in the CNS and PNS characteristically changed from that of the normal pattern. Our results showed a non-homogenous distribution of ECM components in the frog nervous system that could be associated with their different roles in physiological and pathological processes.

Morpho-Functional Characteristics of Neurosecretory Cells from Preoptic Nucleus of the Brown Frog Rana temporaria after Hypophysectomy and Vasotocin Administration

Vasotocin-positive cells from preoptic nucleus of frog Rana temporaria L. were studied morphometrically in intact and hypophysectomized individuals (in 10 days after adenohypophysectomy) in control and after intramuscular injections of arginine-vasotocin (VT) in the dose of 50 ng per 10 g weight. A statistically significant increase of nucleolar volume of vasotocin-positive cells in intact and particularly in hypophysectomized frogs was revealed after vasotocin administration. This was accompanied by a decrease of the number of vasotocin-poor cells and an increase of the number of cells storing vasotocin in intact frogs, while the inverse was true for hypophysectomized animals. The results obtained indicate that in the absence of adenopituitary hormones in frogs, a positive direct feedback is revealed between the blood level of vasotocin and intensity of its synthesis in and release from neurosecretory cells of preoptic nucleus.

Estrogen and Laryngeal Synaptic Strength in Xenopus laevis: Opposite Effects of Acute and Chronic Exposure

Synaptic transmission at the vocal synapse, the laryngeal neuromuscular junction, of Xenopus laevis has been shown to be regulated by long-term changes in circulating estrogen. In females, high levels of circulating estrogen also accompany gonadotropin-induced ovulation and oviposition and the switch from sexually unreceptive to receptive states, including changes in vocal behaviors (ticking to rapping). Here we examine the effects of gonadotropin injection on laryngeal synaptic strength and call type. Gonadotropin acutely reduced quantal content values of laryngeal synapses in intact, adult females; the lowest values were attained by 12 h post-injection. Estrogen and progesterone levels increased following human chorionic gonadotropin (hCG) injection; the time course was similar to, but negatively correlated with, changes in synaptic strength. In ovariectomized frogs, exogenous estrogen, but not progesterone or hCG, mimicked the acute effects of hCG in weakening laryngeal synapses of intact frogs. hCG injection suppressed ticking and sometimes induced rapping. Females could tick with either strong or weakened laryngeal synapses while rapping was only produced during the weakening action of hCG. The normally strong synapses of females may enable vocal production even when laryngeal synapses are weakened by hormones that induce ovulation. In contrast to the acute effect of estrogen on weakening laryngeal synapses, juveniles required more than 2 weeks of estrogen treatment to strengthen laryngeal synapses while at least 4 weeks postovariectomy were required to weaken synapses in adult females. We conclude that acute (hours) increases in circulating levels of estrogen weaken synapses while chronic (weeks) increases strengthen laryngeal synapses.

Effect of mesotocin on thyroid gland of the frogRana temporaria

The thyroid gland was studied in intact and hypophysectomized (10 and 60 days before the experiment), sexually mature male frogsRana temporaria after three injections of mesotocin at 30-min intervals (a dose of each injection was 5 ng/g body weight). As determined morphologically, height of the thyrocytes was statistically significantly decreased in intact frogs, while no statistically significant changed were revealed in hypophysectomized animals. The data obtained indicate that at stress responses in frogs, mesotocin can affect the thyroid gland via adenopituitary rather than via general circulation, i.e., by an indirect way.

Responses of Frog Skin,Rana pipiens, Calcium Ion Transport to Parathyroid Hormone, Calcitonin, and Vitamin D3

The responses of cutaneous Ca2+transport to parathyroid hormone, calcitonin, and vitamin D3were evaluated in intactRana pipiensand isolated skins from this species. Parathyroid hormone (12 pmol g−1) stimulated Ca2+influx in intact frogs but not in isolated skin (10–100 pmol ml−1) unless the frogs had been pretreated with vitamin D3injections (100–300 μg day−1) for 3 days prior to isolating the skins for flux measurements. Pretreatment of intact frogs with vitamin D3for 3 days (100 μg day−1) also resulted in greater responses to PTH (6–12 pmol g−1). Vitamin D3(most likely after conversion to 1,25(OH)2D3) had a direct stimulatory effect on Ca2+transport in isolated skins after three daily doses of 500 μg of D3. Calcitonin also stimulated Ca2+influx in both intact frogs (24 pmol g−1) and isolated skins (25–100 pmol ml−1). We conclude that parathyroid hormone, facilitated by vitamin D, stimulates Ca2+influx in the skin ofR. pipiens. We also conclude that calcitonin is stimulatory for cutaneous Ca2+influx in this species.

Immune response to "self" lens in Xenopus laevis enucleated during larval life.

We have reinvestigated an important issue in the amphibian immunology that has not been settled for years since the pioneer work of Triplett, concerning the necessity of being exposed to organ-specific antigens early in development. It was found that syngeneic lenses were rejected by frogs, Xenopus laevis, that had been enucleated (eye removed) during early larval life. This rejection did not occur in intact frogs or in those enucleated in later larval or adult life. Whereas the splenocytes from intact frogs did not proliferate in response to a co-cultured syngeneic lens, those from frogs that had been enucleated at any of the larval stages, or even after metamorphosis, proliferated intensely. Both of these responses were shown to be thymus- dependent. In conclusion, it was demonstrated that the frog immune system rejected even syngeheic lenses by enucleation in early larval life and that it began to recognize the syngeneic lenses by lymphoid proliferation after enucleation, even in later life.

Circadian Locomotor Activity Rhythms in the African Clawed Frog, Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.

Wiping reflex in the intact and spinal frogs

Studying the rostral wiping reflex, RWR, in intact grass frogs allowed us to identify two of its forms: flexor and extensor reflexes (FWR and EWR, respectively). Reflex fields of FWR and EWR considerably overlap; when the common zones are stimulated, both reflex forms can be observed. “Mixed” forms of the reflex are also possible. The probability of initiation of a certain RWR form depends on the stimulus position. Both FWR and EWR are preserved after spinalization, but their receptive fields are somewhat displaced. The hindlimb movement was divided into three phases: positioning (P), lowering, or orientation (L), and brushing aside (BA). In the intact frog, each phase is of approximately similar duration. The reflex phases and their temporal relations are preserved after spinalization. Total movement duration became longer in spinal animals, as compared with that in intact ones; in addition, this duration began to depend on the stimulus position. In the intact frogs, the position of a “working” limb shows the linear dependence on the stimulus localization; this dependence is preserved after spinalization. Our results allow us to conclude that the central generator of wiping reflex includes several subsystems determining the reflex form in a probabilistic manner.

CO2-sensitive olfactory and pulmonary receptor modulation of episodic breathing in bullfrogs.

Breathing was monitored during normocarbia, hypercarbia (6% CO2 in air), and the period immediately after the return to normocarbic conditions in intact, olfactory-denervated, and vagotomized bullfrogs. In intact frogs, ventilation increased during hypercarbia, but the breathing pattern remained episodic. Immediately upon return to air, there was a further paradoxical increase in breathing frequency, and breathing became continuous in most frogs. Results obtained from animals after olfactory receptor denervation indicate that tonic stimulation of olfactory receptors by airway CO2 inhibited breathing during hypercarbia. Measurements of the kinetics of changes in airway and arterial blood CO2 levels support the suggestion that the sudden release of this inhibition on the return to normocarbic conditions was responsible for the posthypercarbic hyperpnea. Vagotomy increased ventilation during normocarbia. Hypercarbia now caused a change in breathing pattern but had no net effect on total ventilation, suggesting that pulmonary vagal feedback inhibited ventilation during normocarbia but stimulated ventilation during hypercarbia. Although olfactory and pulmonary receptor feed-back shape the breathing pattern, they were not responsible for initiating or terminating the episodes of breathing.

Spinal plasticity after hemilabyrinthectomy and its relation to postural recovery in the frog

1. Brachial dorsal root-evoked ventral root responses were studied in the isolated brain/spinal cord preparation of frogs. One group of frogs (n = 20) had survived a hemilabyrinthectomy (HL) between 7 and 70 days. In another group of frogs (n = 30), a nerve branch to an individual labyrinthine organ was sectioned uni- or bilaterally 15 days before the recording session. In a third group of frogs (n = 5), a weight had been mounted eccentrically on the head for 15 days. A fourth group of intact frogs (n = 8) served as a control. 2. In chronic HL frogs (> or = 60 days postoperatively) the amplitudes of short- and long-latency ventral root potentials recorded on the operated side were consistently increased with respect to control values in response to all converging inputs tested. On the intact side most of these potentials were consistently increased as well, except for crossed long-latency responses after stimulation of the dorsal root on the operated side. 3. Practically identical responses were recorded in these preparations before and after the disconnection of the spinal cord from the brain stem at the level of the obex. Before this disconnection, ventral root potentials were recorded in response to electric stimulation of either one of the VIIIth nerves on the intact or on the operated side. Ventral root potentials recorded on the operated but not on the intact side were slightly increased in chronic HL frogs. 4. The time course of these changes was studied at intervals between 7 and 70 days after the lesion. The amplitudes of short-latency dorsal root-evoked ventral root potentials were increased relatively early (7-15 days) or relatively late (> or = 30 days) after HL. Ventral root potentials evoked by stimulation of either one of the N.VIII were significantly reduced in amplitude seven days after HL but normalized again or increased above control values after longer survival periods. These differences in the time courses suggest the presence of multiple, not singular mechanisms for intraspinal changes. 5. Changes in dorsal root-evoked ventral root potentials similar to those after HL were seen 15 days after a selective unilateral section of the utricular, but not after a unilateral section of the horizontal canal or saccular nerve branch. Therefore these changes were initiated either by asymmetric utricular afferent inputs or by asymmetric proprioceptive inputs resulting from lesion-induced postural deficits. 6. These two possibilities were investigated in two different sets of experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of gonadectomy and temperature on the N-acetyltransferase activity in the harderian gland of the green frog Rana esculenta

Since, in the Harderian gland (HG) of the hamster, the N-acetyltransferase (NAT), the specific enzyme involved in the biosynthesis of melatonin, exhibits a sexual dimorphism, in the present study, we investigated whether such a dimorphism is present also in the HG of the green frog Rana esculenta. In intact frogs, no significant differences emerged between males and females in the HG NAT activity under both cold (10°C) and warm (22°C) temperature conditions. In female frogs, the HG NAT activity was significantly decreased by both gonadectomy ( P < 0.001) and warm temperature ( P < 0.001), the two effects being not additive. In male animals, neither gonadectomy nor temperature alone significantly affected the activity of the NAT enzyme in the HG. However, gonadectomized male frogs exposed to warm temperature exhibited a significant drop in the HG NAT activity ( P < 0.005). These data show that, in Rana esculenta, although no sexual dimorphism exists in the HG NAT activity, a sex difference is evident in the modulation of the enzyme activity by gonads and temperature, the female frogs being more sensitive to the impairing effects of both gonadectomy and higher temperature.

On the existence of epithelial chloride channels in intact frogs and crayfish

Chloride channels in isolated amphibian skins have been shown to be open (high PCl) when the transepithelial potential (TEP) across the skin is > 30 mV, inside positive, and [Cl-] in the external medium is high. In the absence of either of these signals PCl is much lower. Using Cl- efflux as a criterion, it is shown here that when the same cues are presented to intact frogs, channels open as in the in vitro preparation. The augmented efflux is partially inhibited by high I- in both preparations. However, salt-depleted frogs do not show channel opening, differing in this regard from isolated skins. Elevating external [Cl-] resulted in an even larger efflux stimulation in crayfish than in frogs. The TEP in these animals is usually small and sometimes blood negative; it does not appear to play a role in augmenting the efflux. Gluconate and NO3- also caused Cl efflux to increase markedly. Salt-depletion reduced the stimulation by high [Cl-] but did not abolish it as in frogs. Iodide had no effect on chloride efflux in crayfish. The results may indicate that gated channels exist in these animals, but interpretation is complicated by the possibility that the augmented efflux is due to exchange diffusion.

Dopamine regulation of testicular activity in intact and hypophysectomized frogs, Rana esculenta.

In intact frogs, both GnRHA and L-dopa were able to increase testicular and plasma androgen levels and to induce spermiation. The dopamine antagonist pimozide inhibited both the effects of L-dopa but not those of GnRHa. Hypophysectomy reduced androgen levels, but spermiation was still induced by both GnRHa and L-dopa, suggesting that these agents can directly influence the testis through a route not involving the pars distalis. Again, pimozide antagonised spermiation induced by L-dopa but not that induced by GnRHa.

Bulk flow of the medium and cutaneous sodium uptake in frogs: potential significance of sodium and oxygen boundary layers.

To examine the potential impact of fluid dynamic boundary layers on cutaneous ion exchange, we investigated how bulk flow of dilute Na+ solutions (< or = 1.0 mmol l-1) over the skin of intact frogs (Rana catesbeiana and Rana pipiens) affects cutaneous Na+ uptake (JNa(in)) and transepithelial potential (TEP). Cessation of stirring resulted in a 14-35% decrease in TEP and a 14-65% decrease in JNa(in). Two weeks' acclimation to an unstirred bath increased JNa(in) to levels 70% greater than in frogs acclimated to a continuously stirred bath and to levels comparable to those of frogs acclimated to deionized water. These effects are consistent with depletion of Na+ in the boundary layer, but are also consistent with depletion of O2 in the boundary layer, which might limit generation of ATP consumed by ATPases responsible for cutaneous Na+ uptake. To investigate this latter possibility, we measured TEP and JNa(in) while manipulating the PO2 of well-stirred external media at constant [Na+]. Hyperoxia (PO2 > or = 97 kPa) increased JNa(in) by 28% and had little or no effect on TEP. Hypoxia (PO2 < or = 1.5 kPa) reduced JNa(in) by 48% and decreased TEP by 22%. These results suggest that ionic and gaseous boundary layers may interact to affect cutaneous ion transport.

Effects of cyproterone acetate on testicular and plasma androgen levels in the frog, Rana esculenta

The effects of an androgen antagonist, cyproterone acetate (CPA) on androgen production, was studied in intact and hypophysectomized (PDX) frogs,Rana esculenta. CPA, by competition with androgens for the same receptor sites, causes a decrease of androgen production both in plasma and in testis. These findings confirm the positive feedback exerted by testicular androgens either through the pituitary or at testicular level. Therefore in ranid species the regulation of testicular androgen production appears to be different from that of mammals.

Patterns of Metabolic Recovery from Exercise in Amphibians and Reptiles

ABSTRACT The physiological responses of amphibians and reptiles undergoing vigorous exercise are qualitatively similar to those of other vertebrates. Oxygen consumption increases rapidly to rates that are three-to 10-fold the rates at rest. The aerobic response to graded exercise in locomoting reptiles and amphibians is for the most part linear. Oxygen transport by the cardiovascular system during exercise is accomplished by factorial increases in heart rate and oxygen extraction from arterial blood in a fashion similar to that in mammals. Increments in stroke volume during exercise are small or in some cases negative. The influence of temperature or of intracardiac shunting on the cardiovascular function of active amphibians and reptiles is poorly understood. These aerobic responses to exercise are accompanied by robust anaerobic contributions to energy metabolism, resulting in significant lactate accumulation and glycogen depletion. The rate of lactate accumulation during exercise is generally greater in reptiles than in amphibians, but in all cases is so rapid that the only significant substrate source to support anaerobic energy production is muscle glycogen. Vigorous behavior in these animals is therefore limited to some degree by the maintenance and replenishment of muscle glycogen stores. Whereas data from rats and dogs suggest that most lactate is oxidized to CO2 following exercise, amphibians and reptiles appear to use lactate as a substrate for immediate muscle glycogen replenishment. Data from a variety of amphibians and lizards demonstrate that lactate removal following activity and glycogen replenishment are stoichiometrically and temporally related. Studies employing isotopically labelled compounds in intact frogs and lizards indicate that most lactate is resynthesized to glycogen during recovery. In vivo studies suggest skeletal muscle as the site for glycogenesis from lactate, and in vitro studies from many laboratories demonstrate a gluconeogenic capacity in skeletal muscle of lizards, frogs and salamanders. The liver appears to play no significant role in recovery metabolism in any of these classes. Data from lizard muscle suggest that oxidative fiber types have the most significant gluconeogenic capacity, and that the process may be stimulated by the hormonal milieu that exists following exercise. Whereas the recovery metabolism of many mammals seems to facilitate the rapid return of acid-base balance via lactate oxidation, the strategy of lactate removal employed by amphibians and reptiles provides for a mechanism of immediate muscle glycogen replenishment and consequently a reestablished capacity for subsequent activity.

The low density lipoprotein receptor in Xenopus laevis. II. Feedback repression mediated by conserved sterol regulatory element

The 5'-flanking regions of the two low density lipoprotein (LDL) receptor genes in Xenopus laevis contain three repeat sequences that are virtually identical to the repeats that mediate sterol-regulated transcription of the human LDL receptor gene. Like their human counterparts, Xenopus repeats 1 and 3, but not repeat 2, bind the transcription factor Sp1 and thus probably function as positive transcription elements. Xenopus repeat 2, like human repeat 2, contains all of the nucleotides that are required for sterol regulation. Administration of sterols repressed Xenopus LDL receptor mRNA in cultured A6 kidney cells and in the liver of intact frogs. In frogs this repression was associated with a 2-fold increase in plasma LDL levels. Xenopus LDL contains a protein corresponding in size to human apoB-100, a ligand for the LDL receptor. We found no evidence that frog plasma contains B-48, nor did we observe a clear-cut protein corresponding to apoE. We conclude that the structural gene for the LDL receptor has been under sterol-mediated regulation at least since the time of amphibian development more than 350 million years ago.

Reabsorption of water and electrolytes in the urinary bladder of intact frogs (genus Rana)

1. l. Water and electrolyte reabsorption of the urinary bladder epithelia has been studied in intact, fully hydrated frogs ( Rana temporaria R. lessonae R. ridibunda). 2. 2. The rates of water reabsorption were lower in frogs on wet soil than in those on dry soil and related to the degree of terrestrialism: R. temporaria > R. lessonae > R. ridibunda. 3. 3. Samples of urine stored up to 24 hr within the urinary bladder were analysed for osmolality and the concentration of urea, ammonia, sodium, potassium, magnesium and calcium. 4. 4. Selective reabsorption of sodium was detected in all species, that of calcium only in R. ridibunda. The efficiency of electrolyte reabsorption was also related to the degree of terrestrialism. 5. 5. In conclusion, in fully hydrated frogs reabsorption by the bladder epithelia contributes significantly to the water and electrolyte conservation.