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Background The explosion in the use of genetically modified mouse strains to investigate function in biology has an enormous potential to expand on pharmacological studies traditionally conducted in rats. A key limitation to date is the inability to record from multiple nerves in an anaesthetised mouse for long periods. New method Here we describe an in vivo preparation that maintains mice in a suitable physiological state, under anaesthesia, for at least 6hr and also enables multiple cardiorespiratory recordings over that time. Results Using the method described, blood pressure, heart rate, phrenic nerve activity, splanchnic nerve activity and heart rate were able to be recorded for hours in an anaesthetised, paralysed and mechanically ventilated mouse. Comparison with existing method Existing anaesthetised mouse preparations are limited by difficulties in maintaining mice under anaesthesia for long periods. This time constraint therefore limits the surgical time and number of cardiorespiratory variables recorded. It also limits the type of stimuli that can be administered and the length of recorded responses. The method described here optimises these variables to overcome these challenges. Conclusions In summary, we report an approach that enables physiological and pharmacological studies previously undertaken in larger animals or ‘reduced’ preparations, to be conducted in vivo in mice. We anticipate that the use of this preparation will enable a deeper understanding of genetic variation, and allow a much greater level of phenotypic characterisation in genetically modified mice.

New Findings What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal–maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal–maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal–maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has emerged as a principal and rate-limiting regulator of physiological stress responses in adult rodents and has been implicated in sudden infant death syndrome (SIDS). Recent studies show that PACAP plays a role in neonatal cardiorespiratory responses to hypoxia, hypercapnia, and hypothermia, but not hyperthermia, which is often associated with SIDS. Here we tested the hypothesis that, consistent with a role in SIDS, PACAP is involved in regulating the neonatal cardiorespiratory responses to severe heat. To address this, we used head-out plethysmography and surface ECG electrodes to study the cardiorespiratory physiology of conscious neonatal PACAP-null and wild-type mice at ambient temperatures of 32°C (baseline) and 40°C (heat stress). We also assessed body surface temperature as an indicator of cutaneous heat loss. Our results show that wild-type neonatal mice respond to heat stress by increasing ventilation ( P = 0.007) and associated expired CO2( P = 0.041), heart rate ( P < 0.001), and cutaneous heat loss ( P < 0.001). In PACAP-null neonates, this heat response is impaired, as indicated by a decrease in ventilation ( P = 0.04) and associated expired CO2( P = 0.006) and a blunted increase in heart rate ( P = 0.001) and cutaneous heat loss ( P = 0.0002). In addition, heart rate variability at baseline was lower in PACAP-null neonates than wild-type controls ( P < 0.01). These results suggest that, during heat stress, PACAP is important for neonatal cardiorespiratory responses that help regulate body temperature. Abnormal PACAP regulation could, therefore, contribute to neonatal disorders in which the autonomic response to stress is impaired, such as SIDS.

In unrestrained whole body plethysmography, tidal volume is commonly determined using the barometric method, which assumes that temperature and humidity changes (the 'barometric component') are solely responsible for breathing-related chamber pressure fluctuations. However, in small animals chamber pressure is also influenced by a 'mechanical component' dependent on airway resistance and airflow. We devised a novel 'mechanical lung' capable of simulating neonatal mouse breathing in the absence of temperature or humidity changes. Using this device, we confirm that the chamber pressure fluctuations produced by breathing of neonatal mice are dominated by the mechanical component, precluding direct quantitative assessment of tidal volume. Recognizing the importance of airway resistance to the chamber pressure signal and the ability of our device to simulate neonatal breathing at different frequencies and tidal volumes, we invented a novel in vivo, non-invasive method for conscious airway resistance and ventilation estimation (CARVE) in neonatal rodents. This technique will allow evaluation of developmental, pathological and pharmaceutical effects on airway resistance.

Aim Stress peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has been implicated in sudden infant death syndrome (SIDS). The aim of this exploratory study was to determine whether variants in the gene encoding the PACAP-specific receptor, PAC1, are associated with SIDS in Caucasian and African American infants. Methods Polymerase chain reaction and Sanger DNA sequencing was used to compare variants in the 5′-untranslated region, exons and intron–exon boundaries of the PAC1 gene in 96 SIDS cases and 96 race- and gender-matched controls. Results The intron 3 variant, A/G: rs758995 (variant ‘h’), and the intron 6 variant, C/T: rs10081254 (variant ‘n’), were significantly associated with SIDS in Caucasians and African Americans, respectively (p

Frog metamorphosis includes transition from water breathing to air breathing but the extent to which such a momentous change in behavior requires fundamental changes in the organization of the brainstem respiratory circuit is unknown. Here, we combine a vertically mounted isolated brainstem preparation, “the Sheep Dip,” with a search algorithm used in computer science, to identify essential rhombomeres for generation of ventilatory motor bursts in metamorphosing bullfrog tadpoles. Our data suggest that rhombomere 7, which in mammals hosts the PreBotC (PreBotzinger Complex; the likely inspiratory oscillator), is essential for gill and buccal bursts. Whereas rhombomere 5, in close proximity to a brainstem region associated with the mammalian expiratory oscillator, is essential for lung bursts at both stages. Therefore, we conclude there is no rhombomeric translocation of respiratory oscillators in bullfrogs as previously suggested. In premetamorphic tadpoles, functional ablation of rhombomere 7 caused ectopic expression of precocious lung bursts, suggesting the gill oscillator suppresses an otherwise functional lung oscillator in early development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 888–898, 2013

Pituitary adenylate cyclase-activating peptide (PACAP) is an important mediator of the stress response and is crucial in maintaining breathing in neonates. Here we investigate the role of exogenously applied PACAP in neonatal breathing using the neonatal rat in situ working heart-brainstem preparation. A 1-min bolus of 250 nM PACAP-38 caused an increased in respiratory frequency that was rapid and transient, but had no effect on neural tidal volume or neural minute ventilation. Denervation of the carotid body abolished this effect. PACAP had a persistent effect on breathing stability in both carotid body-intact and -denervated preparations, as shown by decreases in respiratory variability 5 min following application. These data suggest that PACAP released during stress acts via carotid body dependent and independent mechanisms to stimulate and stabilize breathing. These mechanisms may account for PACAP's critical role in defending neonatal breathing against environmental stress.

Central sleep apnoea is a condition characterized by oscillations between apnoea and hyperpnoea during sleep. Studies in sleeping dogs suggest that withdrawal of peripheral chemoreceptor (carotid body) activation following transient ventilatory overshoots plays an essential role in causing apnoea, raising the possibility that sustaining carotid body activity during ventilatory overshoots may prevent apnoea. To test whether sustained peripheral chemoreceptor activation is sufficient to drive breathing, even in the absence of central chemoreceptor stimulation and vagal feedback, we used a vagotomized, decerebrate dual-perfused in situ rat preparation in which the central and peripheral chemoreceptors are independently and artificially perfused with gas-equilibrated medium. At varying levels of carotid body stimulation (CB PO2/PCO2: 40/60, 100/40, 200/15, 500/15 Torr), we decreased the brainstem perfusate PCO2 in 5 Torr steps while recording phrenic nerve activity to determine the central apnoeic thresholds. The central apnoeic thresholds decreased with increased carotid body stimulation. When the carotid bodies were strongly stimulated (CB 40/60), the apnoeic threshold was 3.6 ± 1.4 Torr PCO2 (mean ± SEM, n = 7). Stimulating carotid body afferent activity with either hypercapnia (60 Torr PCO2) or the neuropeptide pituitary adenylate cyclase-activating peptide restored phrenic activity during central apnoea. We conclude that peripheral stimulation shifts the central apnoeic threshold to very hypocapnic levels that would likely increase the CO2 reserve and have a protective effect on breathing. These data demonstrate that peripheral respiratory chemoreceptors are sufficient to stave off central apnoeas when the brainstem is perfused with low to no CO2.

Febrile seizures (FS) are the most common seizure disorder to affect children. Although there is mounting evidence to support that FS occur when children have fever-induced hyperventilation leading to respiratory alkalosis, the underlying mechanisms of hyperthermia-induced hyperventilation and links to FS remain poorly understood. As transient receptor potential vanilloid-1 (TRPV1) receptors are heat-sensitive, play an important role in adult thermoregulation and modulate respiratory chemoreceptors, we hypothesize that TRPV1 activation is important for hyperthermia-induced hyperventilation leading to respiratory alkalosis and decreased FS thresholds, and consequently, TRPV1 KO mice will be relatively protected from hyperthermic seizures. To test our hypothesis we subjected postnatal (P) day 8-20 TRPV1 KO and C57BL/6 control mice to heated dry air. Seizure threshold temperature, latency and the rate of rise of body temperature during hyperthermia were assessed. At ages where differences in seizure thresholds were identified, head-out plethysmography was used to assess breathing and the rate of expired CO2 in response to hyperthermia, to determine if the changes in seizure thresholds were related to respiratory alkalosis. Paradoxically, we observed a pro-convulsant effect of TRPV1 deletion (∼4min decrease in seizure latency), and increased ventilation in response to hyperthermia in TRPV1 KO compared to control mice at P20. This pro-convulsant effect of TRPV1 absence was not associated with an increased rate of expired CO2, however, these mice had a more rapid rise in body temperature following exposure to hyperthermia than controls, and the expected linear relationship between body weight and seizure latency was absent. Based on these findings, we conclude that deletion of the TRPV1 receptor prevents reduction in hyperthermic seizure susceptibility in older mouse pups, via a mechanism that is independent of hyperthermia-induced respiratory alkalosis, but possibly involves impaired development of thermoregulatory mechanisms, although at present the mechanism remain unknown.

The shape of a neuron’s dendritic arbor is critical for its function as it determines the number of inputs the neuron can receive and how those inputs are processed. During development, a neuron initiates primary dendrites that branch to form a simple arbor. Subsequently, growth occurs by a process that combines the extension and retraction of existing dendrites, and the addition of new branches. The loss and addition of the fine terminal branches of retinal ganglion cells (RGCs) is dependent on afferent inputs from its synaptic partners, the amacrine and bipolar cells. It is unknown, however, whether neural activity regulates the initiation of primary dendrites and their initial branching. To investigate this, Xenopus laevis RGCs developing in vivo were made to express either a delayed rectifier type voltage-gated potassium (KV) channel, Xenopus Kv1.1, or a human inward rectifying channel, Kir2.1, shown previously to modulate the electrical activity of Xenopus spinal cord neurons. Misexpression of either potassium channel increased the number of branch points and the total length of all the branches. As a result, the total dendritic arbor was bigger than for control green fluorescent protein-expressing RGCs and those ectopically expressing a highly related mutant non-functional Kv1.1 channel. Our data indicate that membrane excitability regulates the earliest differentiation of RGC dendritic arbors.

Abnormal respiratory chemosensitivity is implicated in recurrent apnea syndromes, with the peripheral chemoreceptors, the carotid bodies, playing a particularly important role. Previous work suggests that supraphysiological concentrations of the endocannabinoid endovanilloid and TASK channel blocker anandamide (ANA) excite carotid bodies, but the mechanism(s) and physiological significance are unknown. Given that carotid body output is temperature-sensitive, we hypothesized that ANA stimulates carotid body chemosensory afferents via temperature-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, we used the dual-perfused in situ rat preparation to confirm that independent perfusion of carotid arteries with supraphysiological concentrations of ANA strongly excites carotid sinus nerve afferents and that this activity is sufficient to increase phrenic activity. Next, using ex vivo carotid body preparations, we demonstrate that these effects are mediated by TRPV1 receptors, not CB1 receptors or TASK channels: in CB1-null mouse preparations, ANA increased afferent activity across all levels of Po2, whereas in TRPV1-null mouse preparations, the stimulatory effect of ANA was absent. In rat ex vivo preparations, ANA's stimulatory effects were mimicked by olvanil, a nonpungent TRPV1 agonist, and suppressed by the TRPV1 antagonist AMG-9810. The specific CB1 agonist oleamide had no effect. Physiological levels of ANA had no effect alone but increased sensitivity to mild hyperthermia. AMG-9810 blocked ANA's effect on the temperature response. Immunolabeling and RT-PCR demonstrated that TRPV1 receptors are not expressed in carotid body glomus cells but reside in petrosal sensory afferents. Together, these results suggest that ANA plays a physiological role in augmenting afferent responses to mild hyperthermia by activating TRPV1 receptors on petrosal afferents.

Aims: Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) are prone to sudden death in the second post-natal week, having respiratory and metabolic disturbances reminiscent of the human Sudden Infant Death Syndrome (SIDS). Here we test the hypothesis that the human PACAP gene is a site of genetic variance associated with SIDS in a cohort of 92 victims and 92 matched controls. Methods: Using polymerase chain reaction and sequencing, we examined the PACAP gene in 92 SIDS cases (46 Caucasians and 46 African Americans) and 92 race- and gender-matched controls. Results: We found no significant associations between PACAP and SIDS in Caucasians. However, in the African Americans, a non-synonymous single nucleotide polymorphism (i.e. an aspartic acid/glycine coding variant, rs2856966) within exon 2 of PACAP was significantly associated with SIDS (p = 0.004), as were haplotypes containing this polymorphism (p < 0.0001). Glycine was three times more likely at this location in the African-American SIDS victims (17 cases) than African-American controls (5 cases). Conclusion: These data are the first to suggest an association between a variant within the coding region of the PACAP gene and SIDS. Based on these findings, further investigations are warranted into the functional importance of PACAP signaling in neonatal survival and the role of PACAP-signaling abnormalities in SIDS.

Interaction between central (brainstem) and peripheral (carotid body) respiratory chemosensitivity is vital to protect blood gases against potentially deleterious fluctuations, especially during sleep. Previously, using an in situ arterially perfused, vagotomized, decerebrate preparation in which brainstem and peripheral chemoreceptors are perfused separately (i.e. dual perfused preparation; DPP), we observed that the phrenic response to specific carotid body hypoxia was larger when the brainstem was held at 25 Torr P compared to 50 Torr P. This suggests a negative (i.e. hypo-additive) interaction between chemoreceptors. The current study was designed to (a) determine whether this observation could be generalized to all carotid body stimuli, and (b) exclude the possibility that the hypo-additive response was the simple consequence of ventilatory saturation at high brainstem P. Specifically, we tested how steady-state brainstem P modulates peripheral chemoreflex magnitude in response to carotid body P and P perturbations, both above and below eupnoeic levels. We found that the peripheral chemoreflex was more responsive the lower the brainstem P regardless of whether the peripheral chemoreceptors received stimuli which increased or decreased activation. These findings demonstrate a negative interaction between brainstem and peripheral chemosensitivity in the rat in the absence of ventilatory saturation. We suggest that a negative interaction in humans may contribute to increased controller gain associated with sleep-related breathing disorders and propose that the assumption of simple addition between chemoreceptor inputs used in current models of the respiratory control system be reconsidered.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an ancient neuropeptide that predates the evolution of vertebrates. While PACAP acts on multiple target organs and has multiple roles in development, neuronal network function and metabolic homeostasis, it also appears to play an important role in the control of breathing. Mice lacking PACAP die suddenly in the second week of life, a phenotype that is exaggerated by mild thermal stress and bares resemblance to human SIDS. Here we discuss several hypotheses as to why PACAP signaling is important for neonatal survival, focusing on data demonstrating an important role in the control of breathing. We review data suggesting that (a) breathing and respiratory chemosensitivity is blunted in PACAP-deficient mice; (b) PACAP plays an important role in protecting neonatal breathing during thermal stress; and (c) PACAP-signaling occurs in a number of loci important for respiratory control including the carotid bodies (the main peripheral respiratory chemoreceptors) and nuclei in the hypothalamus, pons and medulla, as well as pathways involved in setting sympathetic-parasympathetic tone. Whether PACAP gene abnormalities contribute to Sudden Infant Death Syndrome (SIDS) by reducing respiratory system efficacy during environmental stress remains unanswered.

Inputs from central (brainstem) and peripheral (carotid body) respiratory chemoreceptors are coordinated to protect blood gases against potentially deleterious fluctuations. However, the mathematics of the steady-state interaction between chemoreceptors has been difficult to ascertain. Further, how this interaction affects time-dependent phenomena (in which chemoresponses depend upon previous experience) is largely unknown. To determine how central PCO2 modulates the response to peripheral chemostimulation in the rat, we utilized an in situ arterially perfused, vagotomized, decerebrate preparation, in which central and peripheral chemoreceptors were perfused separately (i.e. dual perfused preparation (DPP)). We carried out two sets of experiments: in Experiment 1, we alternated steady-state brainstem PCO2 between 25 and 50 Torr in each preparation, and applied specific carotid body hypoxia (60 Torr PO2 and 40 Torr PCO2) under both conditions; in Experiment 2, we applied four 5 min bouts (separated by 5 min) of specific carotid body hypoxia (60 Torr PO2 and 40 Torr PCO2) while holding the brainstem at either 30 Torr or 50 Torr PCO2. We demonstrate that the level of brainstem PCO2 modulates (a) the magnitude of the phrenic responses to a single step of specific carotid body hypoxia and (b) the magnitude of time-dependent phenomena. We report that the interaction between chemoreceptors is negative (i.e. hypo-additive), whereby a lower brainstem PCO2 augments phrenic responses resulting from specific carotid body hypoxia. A negative interaction may underlie the pathophysiology of central sleep apnoea in populations that are chronically hypocapnic.

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted geneZac1.In humans, either loss or gain ofZAC1expression is associated with reduced growth rates and intellectual disability. To test whether increasedZac1expression directly perturbs neurodevelopment, we misexpressedZac1in murine neocortical progenitors. The effects were striking:Zac1delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons.Zac1misexpression also blocked neuronal migration, withZac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed onZac1knockdown, indicating thatZac1levels must be regulated precisely. Finally,Zac1controlled neuronal migration by regulatingPac1transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP).Pac1andZac1loss- and gain-of-function presented as phenocopies, and overexpression ofPac1rescued theZac1knockdown neuronal migration phenotype. Thus, dysregulatedZac1expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits.SIGNIFICANCE STATEMENTAltered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader–Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g.,Dlk1,Peg3,Ube3a,necdin, andGrb10). Herein we show that the maternally imprinted geneZac1is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevatedZAC1expression, which has been associated with neurocognitive defects.

Background and objectives Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) is a rare pediatric disease of unknown cause. Here, in response to a recent case report describing a ROHHAD patient who suffered from secondary narcolepsy confirmed by an absence of hypocretin-1 in the cerebrospinal fluid, we consider whether the ROHHAD phenotype is owing to one or more mutations in genes specific to hypocretin protein signalling. Methods DNA samples from 16 ROHHAD patients were analyzed using a combination of next-generation and Sanger sequencing to identify exonic sequence variations in three genes: HCRT , HCRTR1 , and HCRTR2 . Results No rare or novel mutations were identified in the exons of HCRT , HCRTR1 , or HCRTR2 genes in a set of 16 ROHHAD patients. Conclusions ROHHAD is highly unlikely to be caused by mutations in the exons of the genes for hypocretin and its two receptors.

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conserved transcription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarily conserved progenitor pool.

An essential role of the cardiorespiratory system is to ensure the brain is adequately oxygenated. This is achieved in part by responding to hypoxia with increases in peripheral vasoconstriction, h...

Time-dependent ventilatory responses to hypoxic and hypercapnic challenges, such as posthypoxic frequency decline (PHxFD) and posthypercapnic frequency decline (PHcFD), could profoundly affect breathing stability. However, little is known about the mechanisms that mediate these phenomena. To determine the contribution of specific carotid body chemostimuli to PHxFD and PHcFD, we developed a novel in situ arterially perfused, vagotomized, decerebrate rat preparation in which central and peripheral chemoreceptors are perfused separately (i.e., a nonanesthetized in situ dual perfused preparation). We confirmed that 1) the perfusion of central and peripheral chemoreceptor compartments was independent by applying specific carotid body hypoxia and hypercapnia before and after carotid sinus nerve transection, 2) the Pco2 chemoresponse of the dual perfused preparation was similar to other decerebrate preparations, and 3) the phrenic output was stable enough to allow investigation of time-dependent phenomena. We then applied four 5-min bouts (separated by 5 min) of specific carotid body hypoxia (40 Torr Po2 and 40 Torr Pco2) or hypercapnia (100 Torr Po2 and 60 Torr Pco2) while holding the brain stem Po2 and Pco2 constant. We report the novel finding that specific carotid body chemostimuli were sufficient to elicit several phrenic time-dependent phenomena in the rat. Hypoxic challenges elicited PHxFD that increased with bout, leading to progressive augmentation of the phrenic response. Conversely, hypercapnia elicited short-term depression and PHcFD, neither of which was bout dependent. These results, placed in the context of previous findings, suggest multiple physiological mechanisms are responsible for PHxFD and PHcFD, a redundancy that may illustrate that these phenomena have significant adaptive advantages.

The ventilatory response to several minutes of hypoxia consists of various time-dependent phenomena, some of which occur during hypoxia (e.g., short-term depression), whereas others appear on return to normoxia (e.g., posthypoxic frequency decline). Additional phenomena can be elicited by acute, intermittent hypoxia (e.g., progressive augmentation, long-term facilitation). Current data suggest that these phenomena originate centrally. We tested the hypothesis that carotid body afferent activity undergoes time-dependent modulation, consistent with a direct role in these ventilatory phenomena. Using an in vitro rat carotid body preparation, we found that 1) afferent activity declined during the first 5 min of severe (40 Torr Po2), moderate (60 Torr Po2), or mild (80 Torr Po2) hypoxia; 2) after return to normoxia (100 Torr Po2) and after several minutes of moderate or severe hypoxia, afferent activity was transiently reduced compared with prehypoxic levels; and 3) with successive 5-min bouts of mild, moderate, or severe hypoxia, afferent activity during bouts increased progressively. We call these phenomena sensory hypoxic decline, sensory posthypoxic decline, and sensory progressive augmentation, respectively. These phenomena were stimulus specific: similar phenomena were not seen with 5-min bouts of normoxic hypercapnia (100 Torr Po2 and 50–60 Torr Pco2) or hypoxic hypocapnia (60 Torr Po2 and 30 Torr Pco2). However, bouts of either normoxic hypercapnia or hypocapnic hypoxia resulted in sensory long-term facilitation. We suggest time-dependent carotid body activity acts in parallel with central mechanisms to shape the dynamics of ventilatory responses to respiratory chemostimuli.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice are more prone to sudden death during postnatal weeks 1–3 than wild-type littermates. Given that PACAP is localized in brainstem regions associated with respiratory chemosensitivity, we examined whether PACAP-null neonates have reduced respiratory responses to hypoxia and hypercapnia. Using unrestrained, whole-body, flow-through plethysmography we found that, by postnatal day 4, the PACAP-null neonates had significantly reduced ventilation during baseline breathing, and blunted responses to both hypoxia (10% O2–90% N2) and hypercapnia (8% CO2–92% air). To determine whether the respiratory phenotype of the PACAP-null mice may contribute to their greater neonatal mortality, we used ECG to examine respiration and cardiovascular function of littermates. We demonstrate that, under conditions that exacerbate mortality of knockout but not wild-type animals, PACAP-deficient mice experience prolonged apnoeas that precede atrio-ventricular block. Both apnoeas and atrio-ventricular block were absent in wild-type littermates. These data suggest that PACAP-deficiency results in higher neonatal mortality primarily as a result of respiratory control defects and raise the possibility that mutations in genes encoding components of the PACAP signalling pathways may contribute to neonatal breathing disorders in humans.

The neonatal mouse en bloc spinal cord-brainstem preparation used in combination with advances in mouse genomics provides a novel strategy for studying the spinal control of locomotion. How well the mouse en bloc prepara- tion is oxygenated however, is unknown. This is an important consideration given that (a) other superfused mammalian en bloc preparations have anoxic cores and (b) hypoxia can have profound effects on neuronal activity. Here we measure the level of tissue oxygenation in the mouse preparation and determine how neuronal activity within the spinal cord is influenced by poor superfusion and/or low oxygen. To measure tissue oxygenation, oxygen depth profiles were obtained (P0 -1 and P2-3; Swiss Webster mice). At P0 -1, spinal cords were oxygenated throughout under rest- ing conditions. When fictive locomotor activity was evoked (5-HT 10 M, dopamine 50 M, NMA 5 M), there was a substantial reduction in tissue PO2 starting within 5 min of drug application. Following washout, the PO2 slowly returned to control levels over a period of 30 min. The experiments described above were repeated using P2-3 preparations. In this older age group, the spinal cord preparations had a hypoxic/anoxic core that was exacerbated during metaboli- cally demanding tasks such as drug-evoked rhythmic activ- ity. To examine how an anoxic core affects neuronal activity within the spinal cord we either altered the flow-rate or ma- nipulated superfusate PO2. When the flow-rate was reduced a transient disruption in the rhythmicity of drug-induced loco- motion occurred during the first 15 min (P0 -1 preparations). However, the motor output adapted and stabilized. During prolonged superfusion with hypoxic artificial cerebrospinal fluid on the other hand, both the motor bursts in spinal nerves and the activity of most neurons near the center of the tissue were abolished. Overall, this study suggests that while oxygenation of P0 -P1 preparations is adequate for studies of locomotor function, oxygenation of older preparations is more problem- atic. Our data also show that neonatal spinal neurons require oxygen to maintain activity; and the spinal locomotor rhythm generator continues to function providing the peripheral tis- sue of the cord is oxygenated. Together, these results are consistent with the results of a previous study which suggest that the locomotor pattern generator is located close to the surface of the spinal cord. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

The occurrence of hiccoughs (hiccups) is very widespread and yet their neuronal origin and physiological significance are still unresolved. Several hypotheses have been proposed. Here we consider a phylogenetic perspective, starting from the concept that the ventilatory central pattern generator of lower vertebrates provides the base upon which central pattern generators of higher vertebrates develop. Hiccoughs are characterized by glottal closure during inspiration and by early development in relation to lung ventilation. They are inhibited when the concentration of inhaled CO(2) is increased and they can be abolished by the drug baclofen (an agonist of the GABA(B) receptor). These properties are shared by ventilatory motor patterns of lower vertebrates, leading to the hypothesis that hiccough is the expression of archaic motor patterns and particularly the motor pattern of gill ventilation in bimodal breathers such as most frogs. A circuit that can generate hiccoughs may persist in mammals because it has permitted the development of pattern generators for other useful functions of the pharynx and chest wall muscles, such as suckling or eupneic breathing.

Nitric oxide (NO) is a potent central neuromodulator of respiration, yet its scope and site of action are unclear. We used 7-nitroindazole (7-NI), a selective inhibitor of endogenous neuronal NO synthesis, to investigate the neurogenesis of respiration in larval bullfrog ( Rana catesbeiana) isolated brain stems. 7-NI treatment (0.0625–0.75 mM) increased the specific frequency of buccal ventilation (BV) events, indicating influence on BV central rhythm generators (CRGs). The drug reduced occurrence, altered burst shape, and disrupted clustering of lung ventilation (LV) events, without altering their specific frequency. LV burst occurrence and clustering also differed between pH conditions. We conclude that NO has diverse effects on respiratory rhythmogenesis, being necessary for the expression of respiratory rhythms, inhibiting the frequency of BV CRG, and affecting both shape and clustering of LV bursts through conditional modulation of LV CRG. We confirm central chemosensitivity in these preparations and demonstrate chemomodulation of LV burst clustering and occurrence but not specific frequency. Results support distinct oscillators underlying LV and BV CRGs.

Oxygen (O2) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O2 sensing and adaptive responses to low O2 (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O2 diffusion to the embryo. The O2 partial pressures (Po2) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po2 levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O2 sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O2 sensing. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 73–83, 2002

For most multiphasic motor patterns, rhythm and pattern are produced by the same circuit elements. For respiration, however, these functions have long been assumed to occur separately. In frogs, the ventilatory motor pattern produced by the isolated brainstem consists of buccal and biphasic lung bursts. Previously, two discrete necessary and sufficient sites for lung and buccal bursts were identified. Here we identify a third site, the Priming Area, important for and having neuronal activity correlated with the first phase of biphasic lung bursts. As each site is important for burst generation of a separate phase, we suggest each major phase of ventilation is produced by an anatomically distinct part of an extensive brainstem network. Embedding of discrete circuit elements producing major phases of respiration within an extensive rhythmogenic brainstem network may be a shared architectural characteristic of vertebrates.Ventilation in mammals consists of at least three distinct phases: inspiration, post-inspiration and late-expiration. While distinct brainstem rhythm generating and pattern forming networks have long been assumed, recent data suggest the mammalian brainstem contains two coupled neuronal oscillators: one for inspiration and the other for active expiration. However, whether additional burst generating ability is required for generating other phases of ventilation in mammals is controversial. To investigate brainstem circuit architectures capable of producing multiphasic ventilatory rhythms, we utilized the isolated frog brainstem. This preparation produces two types of ventilatory motor patterns, buccal and lung bursts. Lung bursts can be divided into two phases, priming and powerstroke. Previously we identified two putative oscillators, the Buccal and Lung Areas. The Lung Area produces the lung powerstroke and the Buccal Area produces buccal bursts and - we assumed - the priming phase of lung bursts. However, here we identify an additional brainstem region that generates the priming phase. This Priming Area extends rostral and caudal of the Lung Area and is distinct from the Buccal Area. Using AMPA microinjections and reversible synaptic blockade, we demonstrate selective excitation and ablation (respectively) of priming phase activity. We also demonstrate that the Priming Area contains neurons active selectively during the priming phase. Thus, we propose that three distinct neuronal components generate the multiphase respiratory motor pattern produced by the frog brainstem: the buccal, priming and powerstroke burst generators. This raises the possibility that a similar multi-burst generator architecture mediates the three distinct phases of ventilation in mammals.

We have previously shown that GABA and glycine modulate respiratory activity in the in vitro brainstem preparations of the lamprey and that blockade of GABAA and glycine receptors restores the respiratory rhythm during apnoea caused by blockade of ionotropic glutamate receptors. However, the neural substrates involved in these effects are unknown. To address this issue, the role of GABAA, GABAB and glycine receptors within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, and the vagal motoneuron region was investigated both during apnoea induced by blockade of glutamatergic transmission and under basal conditions through microinjections of specific antagonists. The removal of GABAergic, but not glycinergic transmission within the pTRG, causes the resumption of rhythmic respiratory activity during apnoea, and reveals the presence of a modulatory control of the pTRG under basal conditions. A blockade of GABAA and glycine receptors within the vagal region strongly increases the respiratory frequency through disinhibition of neurons projecting to the pTRG from the vagal region. These neurons were retrogradely labelled (neurobiotin) from the pTRG. Intense GABA immunoreactivity is observed both within the pTRG and the vagal area, which corroborates present findings. The results confirm the pTRG as a primary site of respiratory rhythm generation, and suggest that inhibition modulates the activity of rhythm-generating neurons, without any direct role in burst formation and termination mechanisms.

Evolution of central respiratory chemosensitivity has been linked traditionally to the need for carbon dioxide regulation that accompanied the evolution of air breathing in terresterial animals. We examined the validity of this linkage by investigating the possibility of central chemoreception in air breathing fish that diverged from the amphibian lineage long before the appearance of terrestriality. We showed that the isolated brainstem preparation of the long nose gar (Lepisosteus osseus) produces a putative motor pattern for lung ventilation, which is responsive to CO(2). These findings, together with more inferential evidence, suggest an association between air breathing and central chemosensitivity in aquatic animals that spans the major branches in vertebrate phylogeny. Furthermore, developmental observations in tadpoles suggest that the neural substrates for central chemoreception exist in proximity to that for rhythm generation. We postulate that a primitive ancestral CPG, sensitive to CO(2) is conserved and is evidenced in the intrinsic coupling of respiratory CPG and central chemoreception in modern tetrapods.

Spike frequency was recorded in the nerve of the isolated superfused first gill arch of the bullfrog larva, Rana catesbeiana and the response to different superfusate [Formula: see text] was evaluated. In the metamorphic tadpole, spike frequency increased significantly when the superfusate [Formula: see text] was decreased (mean ± SEM): 8.5 ± 1.6 Hz at 650 Torr, 11.7 ± 1.9 Hz at 140 Torr, 13.3 ± 1.8 Hz at 65 Torr, 14.8 ± 2.4 Hz at 0 Torr (ANOVA, p = 0.0002). The O2sensitive chemoreceptor stimulants NaCN and almitrine also increased the spike frequency. This study demonstrates the presence of O2sensitive chemoreceptors in the first gill arch of the tadpole.Key words: tadpole, Rana catesbeiana, gill, chemoreceptor, oxygen.

The rat working heart-brain stem preparation (WHBP) is an in situ preparation having many of the advantages associated with in vitro preparations while retaining cardiovascular response functionality and an eupnoeic respiratory motor pattern. The preparation is perfused arterially with an aqueous medium having a much lower oxygen-carrying capacity than blood. To evaluate the efficacy of the artificial perfusion in providing adequate gas exchange within the brain stem, we used polarographic Po2and pH microelectrodes to determine the tissue Po2and pH of the medulla oblongata at various depths. When the perfusate was equilibrated with 5% CO2and 95% O2, average tissue Po2was 294 Torr and no hypoxic areas were encountered. Tissue pH was remarkably uniform throughout the tissue, and on average was only 0.04 ± 0.02 pH units more acidic than that of the perfusate. Increasing the Pco2of the perfusate increased tissue Po2and decreased arterial resistance. Decreasing perfusate Pco2(while keeping pH constant) decreased tissue Po2and reduced the respiratory activity. These results suggest that arterial Pco2, independent of arterial pH, is an essential variable in determining both respiratory drive and cerebrovascular perfusion. We conclude that the medulla of the WHBP is oxygenated and within a physiological pH, which accounts for the eupneic pattern of respiratory motor activity it generates. Furthermore, this preparation may be a useful model for exploring mechanisms of central chemoreception as well as the dynamics of the cerebral vasculature responses following changes in blood gases.

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.

The ingestive behavior of the medicinal leech includes peristalsis-like movements of the midbody that are organized into both rostral-to-caudal and caudal-to-rostral waves (C. M. Lent, K. H. Fliegner, E. Freedman, & M. H. Dickenson, 1988). The neuronal control of this behavior is unknown. Using surgical manipulations and electromyograms, the authors show that (a) the head and tail ganglia are not necessary for this behavior; (b) the circuit is distributed, with components reiterated along the length of the leech; (c) excitatory signals transmitted from rostral segments via the nerve cord can initiate peristalsis in "empty" caudal segments; (d) inhibitory signals from caudal segments limit the frequency of peristalsis; and (e) stretch of the gut and/or body wall is sufficient to produce peristalsis in the absence of heat or chemical cues. These results are compared with peristalsis in the digestive tract of mammals. The leech may be a good model for studying peristalsis-like behaviors at the cellular level.

Development requires age-dependent changes in essential behaviors. While the mechanisms determining the developmental expression of such behavior in vertebrates remain largely unknown, a few studies have identified permissive mechanisms in which the appearance of promoting signals activates pre-established networks. Here we report a different developmental process. Specifically, we show that the neuronal substrate that produces putative lung breathing in tadpoles is formed early in development, but remains more or less inactive until metamorphosis because of suppression mediated by a GABA(B) receptor-dependent mechanism. Blocking this suppression using 2-hydroxy-saclofen, a GABA(B) receptor antagonist, results in the precocious production of the putative lung breathing motor pattern. This blocker failed to augment putative lung breaths after metamorphosis. Thus, our results suggest that loss of an inhibitory signal during development (i.e., developmental disinhibition) is responsible for the developmental expression of air breathing.

We have characterized the efficacy of a low volume (2 ml) two-compartment experimental chamber in which a gas inflow equilibrates and recirculates the bathing fluid. This type of chamber is suitable for experiments employing en bloc preparations that require the administration of expensive molecular probes. The fluid in the chamber is pumped from a compartment holding the preparation to an elevated reservoir compartment using gas bubbles. The fluid returns via gravity along a different path. The flow rate of superfusate in the chamber was 30 ml min −1 . To determine the effectiveness of the chamber in dissolving gas, we filled the chamber with bicarbonate-buffered physiological saline and measured pH and P O 2 with ion-selective and Clark-style microelectrodes. Steady state values of pH and P O 2 in the chamber were almost identical to those in an external tonometer bubbled vigorously with the same gas mixture. When CO 2 was increased from 2 to 4.4%, the chamber pH fell with a time constant of 56 s (about twice that of the tonometer). To determine the effectiveness of gas exchange between a brain preparation and the fluid in the chamber we measured pH and P O 2 depth profiles of the in vitro tadpole brainstem. We found virtually no unstirred layer owing to excellent mixing and the high flow created by the recirculating mechanism. We demonstrate that despite the high flow rates, preparations are mechanically stable allowing intracellular electrophysiological recordings.

Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca2+ mobilization, inhibition of various K+ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (∼41%), whereas the TASK-1-like K+ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (∼48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K+ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.

The Human immunodeficiency virus (HIV) and malaria are two of the world’s most formidable pathogens. Coinfection has been shown to amplify the effects of both diseases with HIV infection enhancing the severity of malaria. Previous work in our laboratory has shown that individuals infected with malaria and HIV who are taking anti-retrovirals have the Plasmodium parasite in their bloodstream suggesting that the lack of anti-malarials in their drug regimen resulted in Plasmodium infection. In this study, we set out to determine the status of Plasmodium infection in a cohort of patients taking both anti-malarial and anti-retroviral drugs. Blood samples were collected from patients of the Edo district of Nigeria in Benin City co-infected with Plasmodium and HIV. We have found that 31 out of the 317 (9.78%) HIV patients on HAART and ACT had Plasmodium in their blood based on microscopic counts. Surprisingly, using the polymerase chain reaction (PCR), the prevalence was at 25.6% for Plasmodium. In addition, we have identified by PCR that Plasmodium falciparum is the only species infecting these patients. Furthermore, no significant relationship was found to exist between CD4+ T-cell counts and malarial infections (CD4 count

A mathematical model of the hydrostatic skeleton of the leech has been developed to predict the shape of and internal pressure within the animal in response to a given pattern of motor neuron activity in different behaviors. The model incorporates experimental data on: the dimensions of the animal at behavioral extremes, the passive properties of the tissues, the active length-tension behavior of the muscles in response to neural activation, the relations between firing frequencies and forces developed by the muscles. The model is based on three general assumptions: (i) the cross-sectional geometry of each segment is elliptical, (ii) the volume of each segment remains constant during movement, (iii) the shape of the animal reflects dimensions that minimize the total potential energy. Presently the model is implemented to simulate the vermiform elongation of the leech, predicting the shape and the pressure changes during behavior. The results are in good agreement with the experimental measurements. The pattern of motor neuronal activity was determined by the known intersegmental travel time and estimated delay time between relaxation of the longitudinal muscles and the activation of the circular muscles. The anesthetized state of the leech was taken as the reference state for the model in which the active and passive stresses are zero.

Several behaviour patterns have been studied in the leech at both the kinematic and neuronal levels. However, very little is known about how patterns of motor neurone activity map to actual movements. Internal pressure is an essential biomechanical property in this process, being responsible for producing the rigidity and posture that allow the directed delivery of forces produced by muscle contraction. To obtain a better understanding of the biomechanical processes involved in movement of the leech, we have measured the internal pressure of the animal by placing catheters through the body wall and into the gut of intact animals showing normal patterns of behaviour. Each type of behaviour had a characteristic pressure waveform. The elongation phase of crawling produced a rapid increase in pressure that peaked when midbody segments were maximally elongated. The pressure produced during the contraction phase of crawling depended on the type of crawl, only inchworm crawling producing a second peak. Whole-body shortening in response to a head poke also produced a pressure peak, but it had a faster rise time. Swimming produced the largest pressure, which was marked by a large sustained increase that fluctuated phasically with undulations of the body. Dual pressure recordings using two catheters demonstrated that pressure was not uniform along the length of the leech, indicating that the body cavity is functionally compartmentalised. Injecting fluid into the gut via a recording catheter allowed us to determine the effects of increasing internal volume on pressure. In line with previous predictions made using an abstract biomechanical model of the leech hydroskeleton, we found that an increase in the volume caused a reduction in the pressure. We are in the process of constructing a more realistic biomechanical model of the leech, based on actual data reported elsewhere. The results in this paper will provide key tests for refining these models.

Frog metamorphosis includes transition from water breathing to air breathing but the extent to which such a momentous change in behavior requires fundamental changes in the organization of the brainstem respiratory circuit is unknown. Here, we combine a vertically mounted isolated brainstem preparation, "the Sheep Dip," with a search algorithm used in computer science, to identify essential rhombomeres for generation of ventilatory motor bursts in metamorphosing bullfrog tadpoles. Our data suggest that rhombomere 7, which in mammals hosts the PreBötC (PreBötzinger Complex; the likely inspiratory oscillator), is essential for gill and buccal bursts. Whereas rhombomere 5, in close proximity to a brainstem region associated with the mammalian expiratory oscillator, is essential for lung bursts at both stages. Therefore, we conclude there is no rhombomeric translocation of respiratory oscillators in bullfrogs as previously suggested. In premetamorphic tadpoles, functional ablation of rhombomere 7 caused ectopic expression of precocious lung bursts, suggesting the gill oscillator suppresses an otherwise functional lung oscillator in early development.

Spatially distinct, interacting oscillators in the bullfrog medulla generate and coordinated buccal and lung ventilatory rhythms, but how these rhythms are transmitted onto trigeminal and hypoglossal motor neurons is unknown. Using a vertically-mounted isolated brainstem preparation, the Sheep Dip, we identified the regions of the brainstem containing motor nuclei using a solution capable of blocking synaptic release and, following washout, locally exposed these regions to 5 μM NBQX and/or 50 μM AP5. Local application of NBQX significantly reduced the amplitude of buccal and lung bursts on the trigeminal nerve, and lung bursts on the hypoglossal nerve. Local AP5 caused a significant reduction in lung burst amplitude on both nerves, but for buccal bursts, hypoglossal amplitude increased and trigeminal amplitude was unchanged. Local co-application of NBQX and AP5 eliminated fictive respiratory motor output completely in both nerves. These results are consistent with mammalian data, suggesting a critical role for glutamate in transmission of respiratory activity from oscillators to motor neurons.

We describe fabrication methods and the characterisation and use of extracellular microelectrode arrays for the detection of action potentials from neurons in culture. The 100 microns2 platinised gold microelectrodes in the 64 electrode array detect the external current which flows during an action potential with S:N ratios of up to 500:1, giving a maximum recorded signal of several millivolts. The performance of these electrodes is enhanced if good sealing of the cells over the electrodes is obtained and further enhanced if the electrodes and the cells lie in a deep groove in the substratum. The electrodes can be used for both recording and stimulation of activity in cultured neurons and for recording from multiple sites on a single cell. The use of such electrodes to obtain recordings from invertebrate neurons is described. The particular advantages of these electrodes, their long term stability, non-invasive nature, high packing density, and utility in stimulation, are demonstrated.

Sudden infant death syndrome (SIDS) is one of the most common causes of postneonatal infant mortality in the developed world. An insufficient cardiorespiratory response to multiple environmental stressors (such as prone sleeping positioning, overwrapping, and infection), during a critical period of development in a vulnerable infant, may result in SIDS. However, the effect of multiple risk factors on cardiorespiratory responses has rarely been tested experimentally. Therefore, this study aimed to quantify the independent and possible interactive effects of infection, hyperthermia, and hypoxia on cardiorespiratory control in rats during the neonatal period. We hypothesized that lipopolysaccharide (LPS) administration will negatively impact cardiorespiratory responses to increased ambient temperature and hypoxia in neonatal rats. Sprague–Dawley neonatal rat pups were studied at postnatal day 6–8. Rats were examined at an ambient temperature of 33°C or 38°C. Within each group, rats were allocated to control, saline, or LPS (200 μg/kg) treatments. Cardiorespiratory and thermal responses were recorded and analyzed before, during, and after a hypoxic exposure (10% O2). Serum samples were taken at the end of each experiment to measure cytokine concentrations. LPS significantly increased cytokine concentrations (such as TNF α, IL‐1β, MCP‐1, and IL‐10) compared to control. Our results do not support a three‐way interaction between experimental factors on cardiorespiratory control. However, independently, heat stress decreased minute ventilation during normoxia and increased the hypoxic ventilatory response. Furthermore, LPS decreased hypoxia‐induced tachycardia. Herein, we provide an extensive serum cytokine profile under various experimental conditions and new evidence that neonatal cardiorespiratory responses are adversely affected by dual interactions of environmental stress factors.