Neuron populations use variable combinations of short-term feedback mechanisms to stabilize firing rate.

Neurons tightly regulate firing rate and a failure to do so leads to multiple neurological disorders. Therefore, a fundamental question in neuroscience is how neurons produce reliable activity patterns for decades to generate behavior. Neurons have built-in feedback mechanisms that allow them to monitor their output and rapidly stabilize firing rate. Most work emphasizes the role of a dominant feedback system within a neuronal population for the control of moment-to-moment firing. In contrast, we find that respiratory motoneurons use 2 activity-dependent controllers in unique combinations across cells, dynamic activation of an Na+ pump subtype, and rapid potentiation of Kv7 channels. Both systems constrain firing rate by reducing excitability for up to a minute after a burst of action potentials but are recruited by different cellular signals associated with activity, increased intracellular Na+ (the Na+ pump), and membrane depolarization (Kv7 channels). Individual neurons do not simply contain equal amounts of each system. Rather, neurons under strong control of the Na+ pump are weakly regulated by Kv7 enhancement and vice versa along a continuum. Thus, each motoneuron maintains its characteristic firing rate through a unique combination of the Na+ pump and Kv7 channels, which are dynamically regulated by distinct feedback signals. These results reveal a new organizing strategy for stable circuit output involving multiple fast activity sensors scaled inversely across a neuronal population.

Plasticity in the Functional Properties of NMDA Receptors Improves Network Stability during Severe Energy Stress.

Brain energy stress leads to neuronal hyperexcitability followed by a rapid loss of function and cell death. In contrast, the frog brainstem switches into a state of extreme metabolic resilience that allows them to maintain motor function during hypoxia as they emerge from hibernation. NMDA receptors (NMDARs) are Ca2+-permeable glutamate receptors that contribute to the loss of homeostasis during hypoxia. Therefore, we hypothesized that hibernation leads to plasticity that reduces the role of NMDARs within neural networks to improve function during hypoxia. To test this, we assessed a circuit with a large involvement of NMDAR synapses, the brainstem respiratory network of female bullfrogs, Lithobates catesbeianus Contrary to our expectations, hibernation did not alter the role of NMDARs in generating network output, nor did it affect the amplitude, kinetics, and hypoxia sensitivity of NMDAR currents. Instead, hibernation strongly reduced NMDAR Ca2+ permeability and enhanced desensitization during repetitive stimulation. Under severe hypoxia, the normal NMDAR profile caused network hyperexcitability within minutes, which was mitigated by blocking NMDARs. After hibernation, the modified complement of NMDARs protected against hyperexcitability, as disordered output did not occur for at least one hour in hypoxia. These findings uncover state-dependence in the plasticity of NMDARs, whereby multiple changes to receptor function improve neural performance during metabolic stress without interfering with their normal role during healthy conditions.

Neural processing without O2 and glucose delivery: from the pond to the clinic.

Neuronal activity requires a large amount of ATP, leading to a rapid collapse of brain function when aerobic respiration fails. Here, we summarize how rhythmic motor circuits in the brainstem of adult frogs, which normally have high metabolic demands, transform to produce proper output during severe hypoxia associated with emergence from hibernation. We suggest that general principles underlying plasticity in brain bioenergetics may be uncovered by studying non-mammalian models that face extreme environments, yielding new insights to combat neurological disorders involving dysfunctional energy metabolism.

Synaptic modifications transform neural networks to function without oxygen.

BACKGROUND: Neural circuit function is highly sensitive to energetic limitations. Much like mammals, brain activity in American bullfrogs quickly fails in hypoxia. However, after emergence from overwintering, circuits transform to function for approximately 30-fold longer without oxygen using only anaerobic glycolysis for fuel, a unique trait among vertebrates considering the high cost of network activity. Here, we assessed neuronal functions that normally limit network output and identified components that undergo energetic plasticity to increase robustness in hypoxia. RESULTS: In control animals, oxygen deprivation depressed excitatory synaptic drive within native circuits, which decreased postsynaptic firing to cause network failure within minutes. Assessments of evoked and spontaneous synaptic transmission showed that hypoxia impairs synaptic communication at pre- and postsynaptic loci. However, control neurons maintained membrane potentials and a capacity for firing during hypoxia, indicating that those processes do not limit network activity. After overwintering, synaptic transmission persisted in hypoxia to sustain motor function for at least 2 h. CONCLUSIONS: Alterations that allow anaerobic metabolism to fuel synapses are critical for transforming a circuit to function without oxygen. Data from many vertebrate species indicate that anaerobic glycolysis cannot fuel active synapses due to the low ATP yield of this pathway. Thus, our results point to a unique strategy whereby synapses switch from oxidative to exclusively anaerobic glycolytic metabolism to preserve circuit function during prolonged energy limitations.

Series resistance errors in whole cell voltage clamp measured directly with dual patch-clamp recordings: not as bad as you think.

Whole cell patch clamp has provided much insight into the function of voltage-gated ion channels in central neurons. However, voltage errors caused by the resistance of the recording electrode [series resistance (Rs)] limit its application to relatively small ionic currents. Ohm's law is often applied to estimate and correct the membrane potential for these voltage errors. We tested this assumption in brainstem motoneurons of adult frogs with dual patch-clamp recordings, one performing whole cell voltage clamp of K+ currents and the other directly recording the membrane potential. We hypothesized that Ohm's law-based correction would approximate the measured voltage error. We found that voltage errors averaged <5 mV for currents considered to be large for patch clamp (∼7-13 nA) and <10 mV for massive currents thought to be experimentally intractable (25-30 nA), each error falling within commonly accepted inclusion limits. In most cases Ohm's law-based correction overpredicted these measured voltage errors by roughly 2.5-fold. Consequently, the use of Ohm's law to correct for voltage errors led to erroneous current-voltage (I-V) relationships, showing the greatest distortion for inactivating currents. Finally, recordings with low electrode Rs compensated moderately by the amplifier circuitry appeared to have smaller voltage errors than those with larger Rs and high compensation despite the same "effective Rs" and current magnitude. Therefore, when Rs is low, large currents may be studied with better-than-expected voltage control. These results suggest that patch-clamp may be used to study ionic currents often interpreted to be off limits because of size.NEW & NOTEWORTHY Voltage errors occur in whole cell voltage clamp. We make, to our knowledge, the first direct measurements of these errors and find that voltage errors are far smaller than standard calculations would predict. Since voltage errors were often minimal during the measurement of large ion channel currents, this technique may be applied to large neurons of adults to gain insight into ion channel function across the life span and progression of disease.

Activation of respiratory-related bursting in an isolated medullary section from adult bullfrogs.

Breathing is generated by a rhythmic neural circuit in the brainstem, which contains conserved elements across vertebrate groups. In adult frogs, the 'lung area' located in the reticularis parvocellularis is thought to represent the core rhythm generator for breathing. Although this region is necessary for breathing-related motor output, whether it functions as an endogenous oscillator when isolated from other brainstem centers is not clear. Therefore, we generated thick brainstem sections that encompass the lung area to determine whether it can generate breathing-related motor output in a highly reduced preparation. Brainstem sections did not produce activity. However, subsaturating block of glycine receptors reliably led to the emergence of rhythmic motor output that was further enhanced by blockade of GABAA receptors. Output occurred in singlets and multi-burst episodes resembling the intact network. However, burst frequency was slower and individual bursts had longer durations than those produced by the intact preparation. In addition, burst frequency was reduced by noradrenaline and µ-opioids, and increased by serotonin, as observed in the intact network and in vivo. These results suggest that the lung area can be activated to produce rhythmic respiratory-related motor output in a reduced brainstem section and provide new insights into respiratory rhythm generation in adult amphibians. First, clustering breaths into episodes can occur within the rhythm-generating network without long-range input from structures such as the pons. Second, local inhibition near, or within, the rhythmogenic center may need to be overridden to express the respiratory rhythm.

Molecular profiling of CO2/pH-sensitive neurons in the locus coeruleus of bullfrogs reveals overlapping noradrenergic and glutamatergic cell identity.

Locus coeruleus (LC) neurons regulate breathing by sensing CO2/pH. Neurons within the vertebrate LC are the main source of norepinephrine within the brain. However, they also use glutamate and GABA for fast neurotransmission. Although the amphibian LC is recognized as a site involved in central chemoreception for the control of breathing, the neurotransmitter phenotype of these neurons is unknown. To address this question, we combined electrophysiology and single-cell quantitative PCR to detect mRNA transcripts that define norepinephrinergic, glutamatergic, and GABAergic phenotypes in LC neurons activated by hypercapnic acidosis (HA) in American bullfrogs. Most LC neurons activated by HA had overlapping expression of noradrenergic and glutamatergic markers but did not show strong support for GABAergic transmission. Genes that encode the pH-sensitive K+ channel, TASK2, and acid-sensing cation channel, ASIC2, were most abundant, while Kir5.1 was present in 1/3 of LC neurons. The abundance of transcripts related to norepinephrine biosynthesis linearly correlated with those involved in pH sensing. These results suggest that noradrenergic neurons in the amphibian LC also use glutamate as a neurotransmitter and that CO2/pH sensitivity may be linkedto the noradrenergic cell identity.

Transformation to ischaemia tolerance of frog brain function corresponds to dynamic changes in mRNA co-expression across metabolic pathways.

Neural activity is costly and requires continuous ATP from aerobic metabolism. Brainstem motor function of American bullfrogs normally collapses after minutes of ischaemia, but following hibernation, it becomes ischaemia-tolerant, generating output for up to 2 h without oxygen or glucose delivery. Transforming the brainstem to function during ischaemia involves a switch to anaerobic glycolysis and brain glycogen. We hypothesized that improving neural performance during ischaemia involves a transcriptional program for glycogen and glucose metabolism. Here we measured mRNA copy number of genes along the path from glycogen metabolism to lactate production using real-time quantitative PCR. The expression of individual genes did not reflect enhanced glucose metabolism. However, the number of co-expressed gene pairs increased early into hibernation, and by the end, most genes involved in glycogen metabolism, glucose transport and glycolysis exhibited striking linear co-expression. By contrast, co-expression of genes in the Krebs cycle and electron transport chain decreased throughout hibernation. Our results uncover reorganization of the metabolic transcriptional network associated with a shift to ischaemia tolerance in brain function. We conclude that modifying gene co-expression may be a critical step in synchronizing storage and use of glucose to achieve ischaemia tolerance in active neural circuits.

A brainstem preparation allowing simultaneous access to respiratory motor output and cellular properties of motoneurons in American bullfrogs.

Breathing is generated by a complex neural circuit, and the ability to monitor the activity of multiple network components simultaneously is required to uncover the cellular basis of breathing. In neonatal rodents, a single brainstem slice can be obtained to record respiratory-related motor nerve discharge along with individual rhythm-generating cells or motoneurons because of the close proximity of these neurons in the brainstem. However, most ex vivo preparations in other vertebrates can only capture respiratory motor outflow or electrophysiological properties of putative respiratory neurons in slices without relevant synaptic inputs. Here, we detail a method to horizontally slice away the dorsal portion of the brainstem to expose fluorescently labeled motoneurons for patch-clamp recordings in American bullfrogs. This 'semi-intact' preparation allows tandem recordings of motor output and single motoneurons during respiratory-related synaptic inputs. The rhythmic motor patterns are comparable to those from intact preparations and operate at physiological temperature and [K+]. Thus, this preparation provides the ability to record network and cellular outputs simultaneously and may lead to new mechanistic insights into breathing control across vertebrates.

Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis.

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling-RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.

Lactate ions induce synaptic plasticity to enhance output from the central respiratory network.

Lactate ion sensing has emerged as a process that regulates ventilation during metabolic challenges. Most work has focused on peripheral sensing of lactate for the control of breathing. However, lactate also rises in the central nervous system (CNS) during disturbances to blood gas homeostasis and exercise. Using an amphibian model, we recently showed that lactate ions, independently of pH and pyruvate metabolism, act directly in the brainstem to increase respiratory-related motor outflow. This response had a long washout time and corresponded with potentiated excitatory synaptic strength of respiratory motoneurons. Thus, we tested the hypothesis that lactate ions enhance respiratory output using cellular mechanisms associated with long-term synaptic plasticity within motoneurons. In this study, we confirm that 2 mM sodium lactate, but not sodium pyruvate, increases respiratory motor output in brainstem-spinal cord preparations, persisting for 2 h upon the removal of lactate. Lactate also led to prolonged increases in the amplitude of AMPA-glutamate receptor (AMPAR) currents in individual motoneurons from brainstem slices. Both motor facilitation and AMPAR potentiation by lactate required classic effectors of synaptic plasticity, L-type Ca2+ channels and NMDA receptors, as part of the transduction process but did not correspond with increased expression of immediate-early genes often associated with activity-dependent neuronal plasticity. Altogether these results show that lactate ions enhance respiratory motor output by inducing conserved mechanisms of synaptic plasticity and suggest a new mechanism that may contribute to coupling ventilation to metabolic demands in vertebrates. KEY POINTS: Lactate ions, independently of pH and metabolism, induce long-term increases in respiratory-related motor outflow in American bullfrogs. Lactate triggers a persistent increase in strength of AMPA-glutamatergic synapses onto respiratory motor neurons. Long-term plasticity of motor output and synaptic strength by lactate involves L-type Ca2+ channels and NMDA-receptors as part of the transduction process. Enhanced AMPA receptor function in response to lactate in the intact network is causal for motor plasticity. In sum, well-conserved synaptic plasticity mechanisms couple the brainstem lactate ion concentration to respiratory motor drive in vertebrates.

Neuromodulation or energy failure? Metabolic limitations silence network output in the hypoxic amphibian brainstem.

Hypoxia tolerance in the vertebrate brain often involves chemical modulators that arrest neuronal activity to conserve energy. However, in intact networks, it can be difficult to determine whether hypoxia triggers modulators to stop activity in a protective manner or whether activity stops because rates of ATP synthesis are insufficient to support network function. Here, we assessed the extent to which neuromodulation or metabolic limitations arrest activity in the respiratory network of bullfrogs-a circuit that survives moderate periods of oxygen deprivation, presumably, by activating an inhibitory noradrenergic pathway. We confirmed that hypoxia and norepinephrine (NE) reduce network output, consistent with the view that hypoxia may cause the release of NE to inhibit activity. However, these responses differed qualitatively; hypoxia, but not NE, elicited a large motor burst and silenced the network. The stereotyped response to hypoxia persisted in the presence of both NE and an adrenergic receptor blocker that eliminates sensitivity to NE, indicating that noradrenergic signaling does not cause the arrest. Pharmacological inhibition of glycolysis and mitochondrial respiration recapitulated all features of hypoxia on network activity, implying that reduced ATP synthesis underlies the effects of hypoxia. Finally, activating modulatory mechanisms that dampen neuronal excitability when ATP levels fall, KATP channels and AMP-dependent protein kinase, did not resemble the hypoxic response. These results suggest that energy failure-rather than inhibitory modulation-silences the respiratory network during hypoxia and emphasize the need to account for metabolic limitations before concluding that modulators arrest activity as an adaptation for energy conservation in the nervous system.

A direct excitatory action of lactate ions in the central respiratory network of bullfrogs, Lithobates catesbeianus.

Chemoreceptors that detect O2 and CO2/pH regulate ventilation. However, recent work shows that lactate ions activate arterial chemoreceptors independent of pH to stimulate breathing. Although lactate rises in the central nervous system (CNS) during metabolic challenges, the ability of lactate ions to enhance ventilation by directly targeting the central respiratory network remains unclear. To address this possibility, we isolated the amphibian brainstem-spinal cord and found that small increases in CNS lactate stimulate motor output that causes breathing. In addition, lactate potentiated the excitatory postsynaptic strength of respiratory motor neurons, thereby coupling central lactate to the excitatory drive of neurons that trigger muscle contraction. Lactate did not affect motor output through pH or pyruvate metabolism, arguing for sensitivity to lactate anions per se. In sum, these results introduce a mechanism whereby lactate ions in the CNS match respiratory motor output to metabolic demands.

The differential contribution of pacemaker neurons to synaptic transmission in the pyloric network of the Jonah crab, Cancer borealis.

Many neurons receive synchronous input from heterogeneous presynaptic neurons with distinct properties. An instructive example is the crustacean stomatogastric pyloric circuit pacemaker group, consisting of the anterior burster (AB) and pyloric dilator (PD) neurons, which are active synchronously and exert a combined synaptic action on most pyloric follower neurons. Previous studies in lobster have indicated that AB is glutamatergic, whereas PD is cholinergic. However, although the stomatogastric system of the crab Cancer borealis has become a preferred system for exploration of cellular and synaptic basis of circuit dynamics, the pacemaker synaptic output has not been carefully analyzed in this species. We examined the synaptic properties of these neurons using a combination of single-cell mRNA analysis, electrophysiology, and pharmacology. The crab PD neuron expresses high levels of choline acetyltransferase and the vesicular acetylcholine transporter mRNAs, hallmarks of cholinergic neurons. In contrast, the AB neuron expresses neither cholinergic marker but expresses high levels of vesicular glutamate transporter mRNA, consistent with a glutamatergic phenotype. Notably, in the combined synapses to follower neurons, 70-75% of the total current was blocked by putative glutamatergic blockers, but short-term synaptic plasticity remained unchanged, and although the total pacemaker current in two follower neuron types was different, this difference did not contribute to the phasing of the follower neurons. These findings provide a guide for similar explorations of heterogeneous synaptic connections in other systems and a baseline in this system for the exploration of the differential influence of neuromodulators.NEW & NOTEWORTHY The pacemaker-driven pyloric circuit of the Jonah crab stomatogastric nervous system is a well-studied model system for exploring circuit dynamics and neuromodulation, yet the understanding of the synaptic properties of the two pacemaker neuron types is based on older analyses in other species. We use single-cell PCR and electrophysiology to explore the neurotransmitters used by the pacemaker neurons and their distinct contribution to the combined synaptic potentials.

Chronic femoral artery ligation exaggerates the pressor and sympathetic nerve responses during dynamic skeletal muscle stretch in decerebrate rats.

Mechanical and metabolic signals arising during skeletal muscle contraction reflexly increase sympathetic nerve activity and blood pressure (i.e., the exercise pressor reflex). In a rat model of simulated peripheral artery disease in which a femoral artery is chronically (~72 h) ligated, the mechanically sensitive component of the exercise pressor reflex during 1-Hz dynamic contraction is exaggerated compared with that found in normal rats. Whether this is due to an enhanced acute sensitization of mechanoreceptors by metabolites produced during contraction or involves a chronic sensitization of mechanoreceptors is unknown. To investigate this issue, in decerebrate, unanesthetized rats, we tested the hypothesis that the increases in mean arterial blood pressure and renal sympathetic nerve activity during 1-Hz dynamic stretch are larger when evoked from a previously "ligated" hindlimb compared with those evoked from the contralateral "freely perfused" hindlimb. Dynamic stretch provided a mechanical stimulus in the absence of contraction-induced metabolite production that closely replicated the pattern of the mechanical stimulus present during dynamic contraction. We found that the increases in mean arterial blood pressure (freely perfused: 14 ± 1 and ligated: 23 ± 3 mmHg, P = 0.02) and renal sympathetic nerve activity were significantly greater during dynamic stretch of the ligated hindlimb compared with the increases during dynamic stretch of the freely perfused hindlimb. These findings suggest that the exaggerated mechanically sensitive component of the exercise pressor reflex found during dynamic muscle contraction in this rat model of simulated peripheral artery disease involves a chronic sensitizing effect of ligation on muscle mechanoreceptors and cannot be attributed solely to acute contraction-induced metabolite sensitization. NEW & NOTEWORTHY We found that the pressor and sympathetic nerve responses during dynamic stretch were exaggerated in rats with a ligated femoral artery (a model of peripheral artery disease). Our findings provide mechanistic insights into the exaggerated exercise pressor reflex in this model and may have important implications for peripheral artery disease patients.

High Fat Feeding in Rats Alters Respiratory Parameters by a Mechanism That Is Unlikely to Be Mediated by Carotid Body Type I Cells.

The carotid bodies (CB) respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin augments the hypoxic sensory response of the intact in-vitro CB (Pye RL, Roy A, Wilson RJ, Wyatt CN. FASEB J 30(1 Supplement):983.1, 2016) and isolated CB type I cell (Pye RL, Dunn EJ, Ricker EM, Jurcsisn JG, Barr BL, Wyatt CN. Arterial chemoreceptors in physiology and pathophysiology. Advances in experimental medicine and biology. Springer, Cham, 2015). This study's aim was to examine, in-vivo, if elevated leptin modulated CB function and breathing.Rats were fed high fat or control chow for 16-weeks. High fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals and had significantly higher serum leptin levels compared to CF. Utilizing whole-body plethysmography, HFF animals demonstrated significantly depressed breathing compared to CF at rest and during hypoxia. However, amplitudes in the change in breathing from rest to hypoxia were not significantly different between groups. CB type I cells were isolated and intracellular calcium levels recorded. Averaged and peak cellular hypoxic responses were not significantly different.Despite a small but significant rise in leptin, differences in breathing caused by high fat feeding are unlikely caused by an effect of leptin on CB type I cells. However, the possibility remains that leptin may have in-vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated.

How important is the CO2 chemoreflex for the control of breathing? Environmental and evolutionary considerations.

Haldane and Priestley (1905) discovered that the ventilatory control system is highly sensitive to CO2. This "CO2 chemoreflex" has been interpreted to dominate control of resting arterial PCO2/pH (PaCO2/pHa) by monitoring PaCO2/pHa and altering ventilation through negative feedback. However, PaCO2/pHa varies little in mammals as ventilation tightly couples to metabolic demands, which may minimize chemoreflex control of PaCO2. The purpose of this synthesis is to (1) interpret data from experimental models with meager CO2 chemoreflexes to infer their role in ventilatory control of steady-state PaCO2, and (2) identify physiological causes of respiratory acidosis occurring normally across vertebrate classes. Interestingly, multiple rodent and amphibian models with minimal/absent CO2 chemoreflexes exhibit normal ventilation, gas exchange, and PaCO2/pHa. The chemoreflex, therefore, plays at most a minor role in ventilatory control at rest; however, the chemoreflex may be critical for recovering PaCO2 following acute respiratory acidosis induced by breath-holding and activity in many ectothermic vertebrates. An apparently small role for CO2 feedback in the genesis of normal breathing contradicts the prevailing view that central CO2/pH chemoreceptors increased in importance throughout vertebrate evolution. Since the CO2 chemoreflex contributes minimally to resting ventilation, these CO2 chemoreceptors may have instead decreased importance throughout tetrapod evolution, particularly with the onset and refinement of neural innovations that improved the matching of ventilation to tissue metabolic demands. This distinct and elusive "metabolic ventilatory drive" likely underlies steady-state PaCO2 in air-breathers. Uncovering the mechanisms and evolution of the metabolic ventilatory drive presents a challenge to clinically-oriented and comparative respiratory physiologists alike.

Activation of respiratory muscles does not occur during cold-submergence in bullfrogs, Lithobates catesbeianus.

Semiaquatic frogs may not breathe air for several months because they overwinter in ice-covered ponds. In contrast to many vertebrates that experience decreased motor performance after inactivity, bullfrogs, Lithobates catesbeianus, retain functional respiratory motor processes following cold-submergence. Unlike mammalian hibernators with unloaded limb muscles and inactive locomotor systems, respiratory mechanics of frogs counterintuitively allow for ventilatory maneuvers when submerged. Thus, we hypothesized that bullfrogs generate respiratory motor patterns during cold-submergence to avoid disuse and preserve motor performance. Accordingly, we measured activity of respiratory muscles (buccal floor compressor and glottal dilator) via electromyography in freely behaving bullfrogs at 20 and 2°C. Although we confirm that ventilation cycles occur underwater at 20°C, bullfrogs did not activate either respiratory muscle when submerged acutely or chronically at 2°C. We conclude that cold-submerged bullfrogs endure respiratory motor inactivity, implying that other mechanisms, excluding underwater muscle activation, maintain a functional respiratory motor system throughout overwintering.

Environmentally induced return to juvenile-like chemosensitivity in the respiratory control system of adult bullfrog, Lithobates catesbeianus.

KEY POINTS: The degree to which developmental programmes or environmental signals determine physiological phenotypes remains a major question in physiology. Vertebrates change environments during development, confounding interpretation of the degree to which development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) produces phenotypes. Tadpoles mainly breathe water for gas exchange and frogs may breathe water or air depending on their environment and are, therefore, exemplary models to differentiate the degree to which life-stage vs. environmental context drives developmental phenotypes associated with neural control of lung breathing. Using isolated brainstem preparations and patch clamp electrophysiology, we demonstrate that adult bullfrogs acclimatized to water-breathing conditions do not exhibit CO2 and O2 chemosensitivity of lung breathing, similar to water-breathing tadpoles. Our results establish that phenotypes associated with developmental stage may arise from plasticity per se and suggest that a developmental trajectory coinciding with environmental change obscures origins of stage-dependent physiological phenotypes by masking plasticity. ABSTRACT: An unanswered question in developmental physiology is to what extent does the environment vs. a genetic programme produce phenotypes? Developing animals inhabit different environments and switch from one to another. Thus a developmental time course overlapping with environmental change confounds interpretations as to whether development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) generates phenotypes. Tadpoles of the American bullfrog, Lithobates catesbeianus, breathe water at early life-stages and minimally use lungs for gas exchange. As adults, bullfrogs rely on lungs for gas exchange, but spend months per year in ice-covered ponds without lung breathing. Aquatic submergence, therefore, removes environmental pressures requiring lung breathing and enables separation of adulthood from environmental factors associated with adulthood that necessitate control of lung ventilation. To test the hypothesis that postmetamorphic respiratory control phenotypes arise through permanent developmental changes vs. reversible environmental signals, we measured respiratory-related nerve discharge in isolated brainstem preparations and action potential firing from CO2 -sensitive neurons in bullfrogs acclimatized to semi-terrestrial (air-breathing) and aquatic-overwintering (no air-breathing) habitats. We found that aquatic overwintering significantly reduced neuroventilatory responses to CO2 and O2 involved in lung breathing. Strikingly, this gas sensitivity profile reflects that of water-breathing tadpoles. We further demonstrated that aquatic overwintering reduced CO2 -induced firing responses of chemosensitive neurons. In contrast, respiratory rhythm generating processes remained adult-like after submergence. Our results establish that phenotypes associated with life-stage can arise from phenotypic plasticity per se. This provides evidence that developmental time courses coinciding with environmental changes obscure interpretations regarding origins of stage-dependent physiological phenotypes by masking plasticity.

Control of lung ventilation following overwintering conditions in bullfrogs, Lithobates catesbeianus.

Ranid frogs in northern latitudes survive winter at cold temperatures in aquatic habitats often completely covered by ice. Cold-submerged frogs survive aerobically for several months relying exclusively on cutaneous gas exchange while maintaining temperature-specific acid-base balance. Depending on the overwintering hibernaculum, frogs in northern latitudes could spend several months without access to air, the need to breathe or the chemosensory drive to use neuromuscular processes that regulate and enable pulmonary ventilation. Therefore, we performed experiments to determine whether aspects of the respiratory control system of bullfrogs, Lithobates catesbeianus, are maintained or suppressed following minimal use of air breathing in overwintering environments. Based on the necessity for control of lung ventilation in early spring, we hypothesized that critical components of the respiratory control system of bullfrogs would be functional following simulated overwintering. We found that bullfrogs recently removed from simulated overwintering environments exhibited similar resting ventilation when assessed at 24°C compared with warm-acclimated control bullfrogs. Additionally, ventilation met resting metabolic and, presumably, acid-base regulation requirements, indicating preservation of basal respiratory function despite prolonged disuse in the cold. Recently emerged bullfrogs underwent similar increases in ventilation during acute oxygen lack (aerial hypoxia) compared with warm-acclimated frogs; however, CO2-related hyperventilation was significantly blunted following overwintering. Overcoming challenges to gas exchange during overwintering have garnered attention in ectothermic vertebrates, but this study uncovers robust and labile aspects of the respiratory control system at a time point correlating with early spring following minimal to no use of lung breathing in cold-aquatic overwintering habitats.