Postmetamorphic Tadpoles Research Articles

Source of ventilatory complexity in the postmetamorphic tadpole brainstem, Pelophylax ridibundus: A pharmacological study

The neural respiratory output of isolated brainstems of post-metamorphic tadpoles displays two motor patterns, the buccal and the lung rhythms. Their global dynamics are complex and chaos-like. This study aimed at determining the source of this complexity. The neural respiratory output was recorded during exposure to increasing concentration of DAMGO or to reduced chloride concentration. Complexity was quantified with the noise limit (NL) and the largest Lyapunov exponent (LLE) values. DAMGO decreased lung frequency (p<0.0001), NL (p<0.0001) and LLE (p=0.0001) without changing buccal frequency (p=0.2392). Reduced concentration of chloride decreased buccal frequency (p=0.011) without changing lung frequency (p=0.2393) whereas NL (p=0.011) and LLE (p=0.027) increased significantly. When taking all the recordings into account, NL and LLE were correlated to lung frequency (r=0.661, p<0.0001 and r=0.3948, p=0.0012, respectively) but not to buccal frequency (r=0.1191, p=0.3487 and r=0.2083, p=0.0985, respectively). Therefore, the lung neural oscillator is both necessary and sufficient to the ventilatory complexity in the isolated brainstem of the post-metamorphic tadpole.

New insights in gill/buccal rhythm spiking activity and CO2 sensitivity in pre- and postmetamorphic tadpoles (Pelophylax ridibundus)

Central CO2 chemosensitivity is crucial for all air-breathing vertebrates and raises the question of its role in ventilatory rhythmogenesis. In this study, neurograms of ventilatory motor outputs recorded in facial nerve of premetamorphic and postmetamorphic tadpole isolated brainstems, under normo- and hypercapnia, are investigated using Continuous Wavelet Transform spectral analysis for buccal activity and computation of number and amplitude of spikes during buccal and lung activities. Buccal bursts exhibit fast oscillations (20–30Hz) that are prominent in premetamorphic tadpoles: they result from the presence in periodic time windows of high amplitude spikes. Hypercapnia systematically decreases the frequency of buccal rhythm in both pre- and postmetamorphic tadpoles, by a lengthening of the interburst duration. In postmetamorphic tadpoles, hypercapnia reduces buccal burst amplitude and unmasks small fast oscillations. Our results suggest a common effect of the hypercapnia on the buccal part of the Central Pattern Generator in all tadpoles and a possible effect at the level of the motoneuron recruitment in postmetamorphic tadpoles.

Effects of maturation and acidosis on the chaos-like complexity of the neural respiratory output in the isolated brainstem of the tadpole, Rana esculenta.

Human ventilation at rest exhibits mathematical chaos-like complexity that can be described as long-term unpredictability mediated (in whole or in part) by some low-dimensional nonlinear deterministic process. Although various physiological and pathological situations can affect respiratory complexity, the underlying mechanisms remain incompletely elucidated. If such chaos-like complexity is an intrinsic property of central respiratory generators, it should appear or increase when these structures mature or are stimulated. To test this hypothesis, we employed the isolated tadpole brainstem model [Rana (Pelophylax) esculenta] and recorded the neural respiratory output (buccal and lung rhythms) of pre- (n = 8) and postmetamorphic tadpoles (n = 8), at physiologic (7.8) and acidic pH (7.4). We analyzed the root mean square of the cranial nerve V or VII neurograms. Development and acidosis had no effect on buccal period. Lung frequency increased with development (P < 0.0001). It also increased with acidosis, but in postmetamorphic tadpoles only (P < 0.05). The noise-titration technique evidenced low-dimensional nonlinearities in all the postmetamorphic brainstems, at both pH. Chaos-like complexity, assessed through the noise limit, increased from pH 7.8 to pH 7.4 (P < 0.01). In contrast, linear models best fitted the ventilatory rhythm in all but one of the premetamorphic preparations at pH 7.8 (P < 0.005 vs. postmetamorphic) and in four at pH 7.4 (not significant vs. postmetamorphic). Therefore, in a lower vertebrate model, the brainstem respiratory central rhythm generator accounts for ventilatory chaos-like complexity, especially in the postmetamorphic stage and at low pH. According to the ventilatory generators homology theory, this may also be the case in mammals.

Do pacemakers contribute to respiratory rhythm generation in the developing bullfrog brainstem?

We tested the hypothesis that respiratory rhythm generation relies on pacemaker‐like activity in the bullfrog, Lithobates catesbeiana. We used in vitro brainstem preparations from pre‐metamorphic and post‐metamorphic tadpoles, superfused with oxygenated artificial CSF (20–22 °C), that generated spontaneous respiratory motor output. The potential role of pacemakers was tested with riluzole (RIL; 5–20 μM), a blocker of persistent Na+ currents, and flufenamic acid (FFA; 50 μM), a blocker of Ca2+‐activated cation currents. Both currents are hypothesized to be required for pacemaker activity in mammalian inspiratory neurons. RIL produced irreversible reductions in gill and lung respiratory frequency and burst amplitude in both pre‐ and post‐metamorphic brainstems, particularly at higher concentrations. FFA significantly reduced gill burst frequency and had no effect on lung burst frequency in pre‐metamorphic brainstems; in post‐metamorphic brainstems FFA had no effect on buccal burst frequency, but reversibly inhibited lung burst frequency. These data support the hypothesis that pacemaker‐like currents contribute to respiratory rhythm generation in the amphibian brainstem, although the specific role of these currents remains unclear. Supported by NIH SO6GM48135.

Glutamatergic regulation of respiratory rhythm generation during development in the bullfrog brainstem

This study examined the role of NMDA and non-NMDA glutamate receptors for regulating respiratory rhythm generation during development in the bullfrog, Rana catesbeiana. We used an in vitro brainstem preparation from pre-metamorphic tadpoles (T-K stages 8–16) and late-stage, post-metamorphic tadpoles (T-K stages 24–25). Respiratory-related neural activity corresponding with gill/lung activity in pre-metamorphic tadpole brainstems and buccal/lung activity in post-metamorphic brainstems were examined before and after blockade of ionotropic glutamate receptors. In both pre-metamorphic and post-metamorphic tadpoles, blockade of non-NMDA receptors with CNQX (0.1, 0.5, 1, 5 μM) significantly increased gill/buccal burst frequency (P<0.01), but reversibly abolished lung burst frequency at 5 μM (P<0.01). Blockade of NMDA receptors with AP-4 (0.1, 5, 10, 50, 100 mM) had no significant effect on gill/buccal or lung burst frequency at any stage of development, but did affect gill burst amplitude of pre-metamorphic tadpoles during the washout period. These data indicate that glutamate, acting at non-NMDA receptors, is inhibitory to gill burst activity but excitatory to lung burst activity and may be critical for activation of the lung rhythm generator. Glutamate acting at NMDA receptors, however, appears to have a role burst pattern formation, but not respiratory rhythm generation. These results indicate that glutamate differentially affects gill and lung rhythm generation with fast synaptic non-NMDA receptors having a major role on respiratory rhythm generation throughout development. Supported by NIH SO6 GM48135.

Nitric oxide changes its role as a modulator of respiratory motor activity during development in the bullfrog ( Rana catesbeiana)

Nitric oxide (NO) is a unique chemical messenger that has been shown to play a role in the modulation of breathing in amphibians and other vertebrates. In the post-metamorphic tadpole and adult amphibian brainstem, NO, acting via the neuronal isoform of nitric oxide synthase (nNOS), is excitatory to the generation of lung burst activity. In this study, we examine the modulation of breathing by NO during development of the amphibian brainstem. Isolated brainstem preparations from pre-metamorphic and late-stage post-metamorphic tadpoles ( Rana catesbeiana) were used to determine the role of NO in modulating central respiratory neural activity. Respiratory neural activity was monitored with suction electrodes recording extracellular activity of cranial nerve rootlets that innervate respiratory musculature. Brainstems were superfused with an artificial cerebrospinal fluid (aCSF) at 20–22 °C containing l-nitroarginine ( l-NA; 1–10 mM), a non-selective NOS inhibitor. In pre-metamorphic tadpoles, l-NA increased fictive gill ventilation frequency and amplitude, and increased lung burst frequency. By contrast, l-NA applied to the post-metamorphic tadpole brainstem had little effect on fictive buccal activity, but significantly decreased lung burst frequency and the frequency of lung burst episodes. These data indicate that early in development, NO provides a tonic inhibitory input to gill and lung burst activity, but as development progresses, NO provides an excitatory input to lung ventilation. This changing role for NO coincides with the shift in importance in the different respiratory modes during development in amphibians; that is, pre-metamorphic tadpoles rely predominantly on gill ventilation whereas post-metamorphic tadpoles have lost the gills and are obligate air-breathers primarily using lungs for gas exchange. We hypothesize that NO provides a tonic input to the respiratory CPG during development and this changing role reflects the modulatory influence of NO on inhibitory or excitatory modulators or neurotransmitters involved in the generation of respiratory rhythm.

Development of the respiratory response to hypoxia in the isolated brainstem of the bullfrogRana catesbeiana

The aim of this study was to examine the effects of cellular hypoxia, and the contribution of anaerobic metabolism, on respiratory activity in bullfrogs at different stages of development. Respiratory-related neural activity was recorded from cranial nerve rootlets in isolated brainstem preparations from pre-metamorphic (Taylor-Kollros (T-K) stages VIII-XVI) and postmetamorphic tadpoles (T-K stages XXIV-XXV) and adults. Changes in fictive gill/lung activity in brainstems from pre-metamorphic tadpoles and lung activity in postmetamorphic tadpoles and adults were examined during superfusion with control (98% O(2)/2% CO(2)) or hypoxic (98% N(2)/2% CO(2)) artificial cerebrospinal fluid (aCSF). Iodoacetate (IAA; 100 micromol l(-1)) was used in conjunction with hypoxic aCSF to inhibit glycolysis. Gill burst frequency in pre-metamorphic brainstems did not change over a 3 h exposure to hypoxia and fictive lung burst frequency slowed significantly, but only after 3 h hypoxia. Blockade of glycolysis with IAA during hypoxia significantly reduced the time respiratory activity could be maintained in pre-metamorphic, but not in adult, brainstems. In brainstems from post-metamorphic tadpoles and adults, lung burst frequency became significantly more episodic within 5-15 min hypoxic exposure, but respiratory neural activity was subsequently abolished in every preparation. The cessation of fictive breathing was restored to control levels upon reoxygenation. Neither tadpole nor adult brainstems exhibited changes in neural bursts resembling 'gasping' that is observed in mammalian brainstems exposed to severe hypoxia. There was also a significant increase in the frequency of 'non-respiratory' bursts in hypoxic postmetamorphic and adult brainstems, but not in pre-metamorphic brainstems. These results indicate that pre-metamorphic tadpoles are capable of maintaining respiratory activity for 3 h or more during severe hypoxia and rely to a great extent upon anaerobic metabolism to maintain respiratory motor output. Upon metamorphosis, however, hypoxia results in significant changes in respiratory frequency and pattern, including increased lung burst episodes, non-ventilatory bursts and a reversible cessation of respiratory activity. Adults have little or no ability to maintain respiratory activity through glycolysis but, instead, stop respiratory activity until oxygen is available. This 'switch' in the respiratory response to hypoxia coincides morphologically with the loss of gills and obligate air-breathing in the postmetamorphic frog. We hypothesize that the cessation of respiratory activity in post-metamorphic tadpoles and adults is an adaptive, energy-saving response to low oxygen.

Location of central respiratory chemoreceptors in the developing tadpole

The location of central respiratory chemoreceptors in amphibian larvae may change as the central chemoreceptive function shifts from driving gill to driving lung ventilation during metamorphosis. We examined this possibility in the in vitro brain stem of the pre- and postmetamorphic Rana catesbeiana tadpole by microinjecting hypercapnic artificial cerebrospinal fluid (aCSF) while recording fictive lung ventilation. The rostral and caudal brain stem were separately explored systematically using injections of 11 nl of aCSF equilibrated with 100% CO2 that transiently acidified a 500-microm region, producing a maximum reduction in pH of 0.23 +/- 0.06 at the site of injection. In postmetamorphic tadpoles, chemoreceptive sites were concentrated in the rostral compared with the caudal brain stem. No such segregation was observed in the premetamorphic tadpole. We conclude that, as in lung rhythmogenic function, respiratory chemosensitivity emerges rostrally in the amphibian brain stem during development.

Development of gill and lung breathing in amphibia

In the 25 morphological stages of larval bullfrog development there exists a gradual transition from gill to lung ventilation associated with a developmental decrease in the contribution of the skin in gas exchange. Bath application of GABA and/or glycine inhibited gill but not lung burst activities of cranial nerve (CN) VII in the premetamorphic (stages 16–19) in vitro tadpole brainstem preparation [1]. It was proposed that the neural basis of gill rhythmogenesis involved network inhibition, whereas lung rhythmicity was pacemaker driven [1]. Bath application of a bicuculline/strychnine solution abolished gill and enhanced lung bursting in stages 16–19 in vitro [1]. Bath application of the GABAB receptor antagonist 2-hydroxy-saclofen disinhibited the lung central pattern generator (CPG) resulting in precocious lung bursting patterns as early as developmental stage 6 [2]. We recorded efferent activity from CNVII and spinal nerve (SN)II in the in vitro tadpole brainstem preparation in three successive developmental groups (3–9; 10–15; 16–19) before and after bath application of a 10 μM bicuculline and 5 μM strychnine solution. We also exposed the brainstem to bath pH7.4, 7.8, and 8.2 before and after bath application of bicuculline/strychnine. Bicuculline/strychnine produced lung ventilatory bursts in all developmental stages tested, indicating the presence of the lung CPG as well as excitatory synapses to respiratory motor neurons as early as stage 3. We also designed an experiment to examine the importance of lung ventilation on the developmental shift from gill to lung bursting. Two groups of tadpoles were hatched from eggs. Control tadpoles had free access to the air-water interface throughout development, whereas tadpoles were denied access to the air-water interface via the placement of Plexiglas 2.5 cm below the surface of the water. CNVII and SNII efferent activities were recorded in vitro at bath pH7.4, 7.8, and 8.2 before and after bath application of 10 μM bicuculline and 5 μM strychnine. Postmetamorphic barrier tadpoles exhibited different motor patterns than stage-matched controls. Tadpoles reared in the barrier condition to stages 20–25 possessed fictive gill and lung ventilatory activities of premetamorphic tadpoles. All barrier tadpole preparations exhibited robust, spontaneous lung burst activity following bath application of bicuculline/strychnine. We propose that developmentally dependent GABA- and glycinergic mechanisms lead to disinhibition of the lung CPG such that early in development gill motor patterns are the dominant respiratory rhythm, whereas in late development the lung CPG is the dominant respiratory rhythm. Denying the tadpole the ability to practice lung breathing during metamorphosis produces a morphologically correct postmetamorphic tadpole with an immature, or premetamorphic respiratory rhythm. We propose that prevention of lung inflation in barrier tadpoles leads to premetamorphic levels of GABA- and glycinergic inhibition, and that this inhibition may be altered by pulmonary stretch receptor feedback in control tadpoles.

Baclofen eliminates cluster lung breathing of the tadpole brainstem, in vitro

Intermittent lung ventilation is a respiratory pattern wherein breaths occur in clusters. Intermittent lung ventilation is common in amphibians and can occur in mammals. Isolated brainstems from postmetamorphic tadpoles exhibiting episodic lung ventilatory bursts were superfused with baclofen, a potent γ-aminobutyric acid (GABA) B receptor agonist. At moderate concentrations (0.125 to 0.5 μM), the number of lung bursts per episode decreased but their overall frequency was unchanged. At 0.5 μM, only 1.22±0.24 lung bursts occurred per episode, indicating virtually no clustering. Only at higher concentrations was overall breathing frequency decreased. Therefore, at moderate concentrations of baclofen continuous ventilation replaced episodic ventilation, suggesting that a GABA B receptor-dependent pathway may regulate the clustering of lung breaths.

Fictive gill and lung ventilation in the pre- and postmetamorphic tadpole brain stem.

The pattern of efferent neural activity recorded from the isolated brain stem preparation of the tadpole Rana catesbeiana was examined to characterize fictive gill and lung ventilations during ontogeny. In vitro recordings from cranial nerve (CN) roots V, VII, and X and spinal nerve (SN) root II of premetamorphic tadpoles showed a coordinated sequence of rhythmic bursts occurring in one of two patterns, pattern1, high-frequency, low-amplitude bursts lacking corresponding activity in SN II and pattern 2, low-frequency, high-amplitude bursts with coincident bursts in SN II. These two patterns corresponded to gill and lung ventilatory burst patterns, respectively, recorded from nerve roots of decerebrate, spontaneously breathing tadpoles. Similar patterns were observed in brain stem preparations from postmetamorphic tadpoles except that they showed a greater frequency of lung bursts and they expressed fictive gill ventilation in SN II. The laryngeal branch of the vagus (Xl) displayed efferent bursts in phase with gill and lung activity, suggesting fictive glottal constriction during gill ventilation and glottal dilation during lung ventilation. The fictive gill ventilatory cycle of pre- and postmetamorphic tadpoles was characterized by a rostral to caudal sequence of CN bursts. The fictive lung ventilatory pattern in the premetamorphic animal was initiated by augmenting CN VII discharge followed by synchronous bursts in CN V, X, SN II, and Xl. By contrast, postmetamorphic patterns of fictive lung ventilation were characterized by lung burst activity in SN II that preceded burst onset in CN V and followed the lead burst in CN VII. We conclude that recruitment and timing of pattern 1 and pattern 2 rhythmic bursts recorded in vitro closely resemble that recorded during spontaneous respiratory behavior, indicating that the two patterns are the neural equivalent of gill and lung ventilation, respectively. Further, fictive gill and lung ventilatory patterns in postmetamorphic tadpoles differ in burst onset latency from premetamorphic tadpole patterns and resemble fictive oropharyngeal and pulmonary burst cycles in adult frogs.

Cloning, characterization and expression of two Xenopus bcl-2-like cell-survival genes

We describe two cloned cDNAs, termed xR1 and xR11, isolated from a Xenopus laevis stage 28–30 embryonic head cDNA library. Comparison of amino acid (aa) sequences derived from nucleotide (nt) sequences of xR1 and xR11 cDNAs revealed substantial homology with bcl-2-related genes, especially with bcl-x spl . In particular, there was a marked conservation of the BH1 and BH2 domains considered to be important for the anti-cell death and heterodimerisation properties of bcl-2. Constitutive expression of xR11 in cultured rat fibroblast (Rat-1) cells conferred a strong protection against cell death induced by the cytotoxic agents staurosporine and cycloheximide, by serum deprivation and specific deregulation of c-myc. Measurement of xR1 and xR11 mRNAs by RNase protection assay revealed similar widespread expression in Xenopus embryos and tadpoles. Except for an abrupt increase in the accumulation of xR1 and xR11 mRNAs in brains of mid-metamorphic and post-metamorphic tadpoles and adults, there was insignificant modulation of their expression in tissues undergoing total regression (tail) or morphogenesis (limb) during natural or thyroid hormone-induced metamorphosis. These findings raise the possibility of continuing expression of cell survival genes in tissues undergoing total regression during post-embryonic development.