Complex interaction of dietary fat and Alaskan bog blueberry supplementation influences manganese mediated neurotoxicity and behavioral impairments.

Dietary fat modulates neuronal health and contributes to age-related nervous system disorders. However, the complex interaction between dietary fat and supplementation and its consequences on neurotoxic pathophysiology has been sparsely explored. The indigenous Alaskan bog blueberry (BB), Vaccinum uliginosum, is known to have anti-inflammatory properties, mostly attributed to its rich polyphenolic content. Here, we evaluate the interplay between dietary fat and BB supplementation on sub-chronic manganese (Mn) exposure that inflicts neurotoxicity and behavioral impairments. In both low-fat and normal-fat diets, BB supplementation attenuated the behavioral and the molecular hallmarks of Mn-induced neurotoxicity. On the contrary, a high-fat diet was found to exacerbate these Mn-induced pathological features. Furthermore, BB supplementation failed to recover the behavioral deficits in mice subjected to a high fat diet in Mn-treated mice. Overall, our results demonstrate the importance of including a dietary regimen comprised of polyphenolic rich supplements with low-fat content in combating age-related neurodegenerative disorders.

Genetic Silencing of Fatty Acid Desaturases Modulates α-Synuclein Toxicity and Neuronal Loss in Parkinson-Like Models of C. elegans.

The molecular basis of Parkinson's disease (PD) is currently unknown. There is increasing evidence that fat metabolism is at the crossroad of key molecular pathways associated with the pathophysiology of PD. Fatty acid desaturases catalyze synthesis of saturated fatty acids from monounsaturated fatty acids thereby mediating several cellular mechanisms that are associated with diseases including cancer and metabolic disorders. The role of desaturases in modulating age-related neurodegenerative manifestations such as PD is poorly understood. Here, we investigated the effect of silencing Δ9 desaturase enzyme encoding fat-5 and fat-7 genes which are known to reduce fat content, on α-synuclein expression, neuronal morphology and dopamine-related behaviors in transgenic PD-like models of Caenorhabditis elegans (C. elegans). The silencing of the fat-5 and fat-7 genes rescued both degeneration of dopamine neurons and deficits in dopamine-dependent behaviors, including basal slowing and ethanol avoidance in worm models of PD. Similarly, silencing of these genes also decreased the formation of protein aggregates in a nematode model of PD expressing α-synuclein in the body wall muscles and rescued deficits in resistance to heat and osmotic stress. On the contrary, silencing of nhr-49 and tub-1 genes that are known to increase total fat content did not alter behavioral and pathological endpoints in the PD worm strains. Interestingly, the genetic manipulation of all four selected genes resulted in differential fat levels in the PD models without having significant effect on the lifespan, further indicating a complex fat homeostasis unique to neurodegenerative pathophysiology. Overall, we provide a comprehensive understanding of how Δ9 desaturase can alter PD-like pathology due to environmental exposures and proteotoxic stress, suggesting new avenues in deciphering the disease etiology and possible therapeutic targets.

Development of central respiratory control in anurans: The role of neurochemicals in the emergence of air-breathing and the hypoxic response.

Physiological and environmental factors impacting respiratory homeostasis vary throughout the course of an animal's lifespan from embryo to adult and can shape respiratory development. The developmental emergence of complex neural networks for aerial breathing dates back to ancestral vertebrates, and represents the most important process for respiratory development in extant taxa ranging from fish to mammals. While substantial progress has been made towards elucidating the anatomical and physiological underpinnings of functional respiratory control networks for air-breathing, much less is known about the mechanisms establishing these networks during early neurodevelopment. This is especially true of the complex neurochemical ensembles key to the development of air-breathing. One approach to this issue has been to utilize comparative models such as anuran amphibians, which offer a unique perspective into early neurodevelopment. Here, we review the developmental emergence of respiratory behaviours in anuran amphibians with emphasis on contributions of neurochemicals to this process and highlight opportunities for future research.

Buccal rhythmogenesis and CO2 sensitivity in Lithobates catesbeianus tadpole brainstems across metamorphosis.

Bullfrog tadpoles ventilate both the buccal cavity and lung. In isolated brainstems, the midbrain/pons influences CO2 responsiveness and timing of lung ventilatory bursting, depending on larval development. However, little is known about midbrain/pons influences on buccal burst patterns. As such, we investigated how removal of this region affects buccal burst shape and CO2 responsiveness across development. We measured facial nerve activity in brainstems isolated from tadpoles during early and late developmental stages, under normal and elevated levels of CO2. Brainstems were either left intact or transected by removing the midbrain/pons. In late stage preparations, buccal burst pattern differed between intact and reduced preparations, and bursts were responsive to elevated CO2 in these reduced preparations. These results suggest the midbrain/pons affects tadpole buccal burst pattern and CO2 responsiveness, perhaps similar to its influences on lung ventilation.

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms.

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode research community, there is a need for an affordable and effective way to maintain large, age-matched populations of C. elegans. Here, we present a methodology for mechanically sorting and cleaning C. elegans. Our aim is to provide a cost-effective, efficient, fast, and simple process to obtain animals of uniform sizes and life stages for their use in experiments. This tool, the Caenorhabditis Sieve, uses a custom-built lid system that threads onto common conical lab tubes and sorts C. elegans based on body size. We also demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to another allowing for a rapid sorting, synchronizing, and cleaning without impacting markers of health, including motility and stress-inducible gene reporters. This accessible and innovative tool is a fast, efficient, and non-stressful option for maintaining C. elegans populations.

Sir-2.1 mediated attenuation of α-synuclein expression by Alaskan bog blueberry polyphenols in a transgenic model of Caenorhabditis elegans.

Misfolding and accumulation of cellular protein aggregates are pathological hallmarks of aging and neurodegeneration. One such protein is α-synuclein, which when misfolded, forms aggregates and disrupts normal cellular functions of the neurons causing Parkinson's disease. Nutritional interventions abundant in pharmacologically potent polyphenols have demonstrated a therapeutic role for combating protein aggregation associated with neurodegeneration. The current study hypothesized that Alaskan bog blueberry (Vaccinum uliginosum), which is high in polyphenolic content, will reduce α-synuclein expression in a model of Caenorhabditis elegans (C. elegans). We observed that blueberry extracts attenuated α-synuclein protein expression, improved healthspan in the form of motility and restored lipid content in the transgenic strain of C. elegans expressing human α-synuclein. We also found reduced gene expression levels of sir-2.1 (ortholog of mammalian Sirtuin 1) in blueberry treated transgenic animals indicating that the beneficial effects of blueberries could be mediated through partial reduction of sirtuin activity. This therapeutic effect of the blueberries was attributed to its xenohormetic properties. The current results highlight the role of Alaskan blueberries in mediating inhibition of sir-2.1 as a novel therapeutic approach to improving pathologies of protein misfolding diseases. Finally, our study warrants further investigation of the structure, and specificity of such small molecules from indigenous natural compounds and its role as sirtuin regulators.

The rostral medulla of bullfrog tadpoles contains critical lung rhythmogenic and chemosensitive regions across metamorphosis.

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO2 responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis.

Behavioral Phenotyping and Pathological Indicators of Parkinson's Disease in C. elegans Models.

Parkinson's disease (PD) is a neurodegenerative disorder with symptoms that progressively worsen with age. Pathologically, PD is characterized by the aggregation of α-synuclein in cells of the substantia nigra in the brain and loss of dopaminergic neurons. This pathology is associated with impaired movement and reduced cognitive function. The etiology of PD can be attributed to a combination of environmental and genetic factors. A popular animal model, the nematode roundworm Caenorhabditis elegans, has been frequently used to study the role of genetic and environmental factors in the molecular pathology and behavioral phenotypes associated with PD. The current review summarizes cellular markers and behavioral phenotypes in transgenic and toxin-induced PD models of C. elegans.

BUILDing BLaST: promoting rural students' biomedical research careers using a culturally responsive, one health approach.

BACKGROUND AND PURPOSE: Most postsecondary institutions in the state of Alaska (USA) have a broad mission to serve diverse students, many of whom come from schools in rural villages that are accessible only by plane, boat, or snowmobile. The major research university, the University of Alaska in Fairbanks (UAF), serves a population whereby 40% are from groups recognized as underrepresented in the biomedical workforce. The purpose of this article is to describe the Building Infrastructure Leading to Diversity (BUILD)-supported program in the state of Alaska that seeks to engage students from rural areas with a culturally relevant approach that is centered on the One Health paradigm, integrating human, animal, and environmental health. PROGRAM AND KEY HIGHLIGHTS: The Biomedical Learning and Student Training (BLaST) program distinguished by broad themes that address recruitment, retention, and success of students in biomedical programs, especially for students from rural backgrounds. Targeted rural outreach emphasizes that biomedical research includes research on the integration of human, animal, and environmental health. This One Health perspective gives personal relevance and connection to biomedical research. This outreach is expected to benefit student recruitment, as well as foster family and community support for pursuit of college degrees. BLaST promotes integration of research into undergraduate curricula through curriculum development, and by creating a new class of instructors, laboratory research and teaching technicians, who provide research mentorship, course instruction, and comprehensive advising. Finally, BLaST facilitates early and sustained undergraduate research experiences in collaborations with graduate students and faculty. IMPLICATIONS: BLaST's approach is highly adapted to the Alaskan educational and physical environment, but components and concepts could be adapted to other rural areas as a means to engage students from rural backgrounds, who often have a closer relationship with the natural environment than urban students.

Mechanosensory Neuron Aging: Differential Trajectories with Lifespan-Extending Alaskan Berry and Fungal Treatments in Caenorhabditis elegans.

Many nutritional interventions that increase lifespan are also proposed to postpone age-related declines in motor and cognitive function. Potential sources of anti-aging compounds are the plants and fungi that have adapted to extreme environments. We studied the effects of four commonly consumed and culturally relevant Interior Alaska berry and fungus species (bog blueberry, lowbush cranberry, crowberry, and chaga) on the decline in overall health and neuron function and changes in touch receptor neuron morphology associated with aging. We observed increased wild-type Caenorhabditis elegans lifespan and improved markers of healthspan upon treatment with Alaskan blueberry, lowbush cranberry, and chaga extracts. Interestingly, although all three treatments increased lifespan, they differentially affected the development of aberrant morphologies in touch receptor neurons. Blueberry treatments decreased anterior mechanosensory neuron (ALM) aberrations (i.e., extended outgrowths and abnormal cell bodies) while lowbush cranberry treatment increased posterior mechanosensory neuron (PLM) aberrations, namely process branching. Chaga treatment both decreased ALM aberrations (i.e., extended outgrowths) and increased PLM aberrations (i.e., process branching and loops). These results support the large body of knowledge positing that there are multiple cellular strategies and mechanisms for promoting health with age. Importantly, these results also demonstrate that although an accumulation of abnormal neuron morphologies is associated with aging and decreased health, not all of these morphologies are detrimental to neuronal and organismal health.

Morphological remodeling of C. elegans neurons during aging is modified by compromised protein homeostasis.

Understanding cellular outcomes, such as neuronal remodeling, that are common to both healthy and diseased aging brains is essential to the development of successful brain aging strategies. Here, we used Caenorhabdits elegans to investigate how the expression of proteotoxic triggers, such as polyglutamine (polyQ)-expanded huntingtin and silencing of proteostasis regulators, such as the ubiquitin-proteasome system (UPS) and protein clearance components, may impact the morphological remodeling of individual neurons as animals age. We examined the effects of disrupted proteostasis on the integrity of neuronal cytoarchitecture by imaging a transgenic C. elegans strain in which touch receptor neurons express the first 57 amino acids of the human huntingtin (Htt) gene with expanded polyQs (128Q) and by using neuron-targeted RNA interference in adult wild-type neurons to knockdown genes encoding proteins involved in proteostasis. We found that proteostatic challenges conferred by polyQ-expanded Htt and knockdown of specific genes involved in protein homeostasis can lead to morphological changes that are restricted to specific domains of specific neurons. The age-associated branching of PLM neurons is suppressed by N-ter polyQ-expanded Htt expression, whereas ALM neurons with polyQ-expanded Htt accumulate extended outgrowths and other soma abnormalities. Furthermore, knockdown of genes important for ubiquitin-mediated degradation, lysosomal function, and autophagy modulated these age-related morphological changes in otherwise normal neurons. Our results show that the expression of misfolded proteins in neurodegenerative disease such as Huntington's disease modifies the morphological remodeling that is normally associated with neuronal aging. Our results also show that morphological remodeling of healthy neurons during aging can be regulated by the UPS and other proteostasis pathways. Collectively, our data highlight a model in which morphological remodeling during neuronal aging is strongly affected by disrupted proteostasis and expression of disease-associated, misfolded proteins such as human polyQ-Htt species.

CO2-inhibited neurons in the medullary raphé are GABAergic.

Previous studies have reported subsets of medullary raphé neurons that are either stimulated or inhibited by CO2/pH in vitro, in situ, and in vivo. We tested the hypothesis that medullary raphé CO2-inhibited neurons are GABAergic. Extracellular recordings in unanesthetized juvenile in situ rat preparations showed reversible hypercapnia-induced suppression of 19% (63/323) of medullary raphé neurons, and this suppression persisted after antagonism of NMDA, AMPA/kainate, and GABAA receptors. We stained a subset of CO2-inhibited cells and found that most (11/12) had glutamic acid decarboxylase 67 immunoreactivity (GAD67-ir). These data indicate that the majority of acidosis-inhibited medullary raphé neurons are GABAergic, and that their chemosensitivity is independent of major fast synaptic inputs. Thus, CO2-sensitive GABAergic neurons may play a role in central CO2/pH chemoreception.

Insulin signaling in the aging of healthy and proteotoxically stressed mechanosensory neurons.

Insulin signaling is central to cellular metabolism and organismal aging. However, the role of insulin signaling in natural and proteotoxically stressed aging neurons has yet to be fully described. We studied aging of Caenorbaditis elegans mechanosensory neurons expressing a neurotoxic expanded polyglutamine transgene (polyQ128), or lacking this proteotoxicity stressor (polyQ0), under conditions in which the insulin signaling pathway was disrupted by RNA interference (RNAi). We describe specific changes in lifespan, mechanosensory neuronal morphologies, and mechansensory function following RNAi treatment targeting the insulin signaling pathway. Overall, we confirmed that transcription factor DAF-16 is neuroprotective in the proteotoxically stressed model, though not strikingly in the naturally aging model. Decreased insulin signaling through daf-2 RNAi improved mechanosensory function in both models and decreased protein aggregation load in polyQ128, yet showed opposing effects on accumulation of neuronal aberrations in both strains. Decreased daf-2 signaling slightly enhanced mechanosensation while greatly enhancing branching of the mechanosensory neuron axons and dendrites in polyQ0 animals, suggesting that branching is an adaptive response in natural aging. These effects in polyQ0 did not appear to involve DAF-16, suggesting the existence of a non-canonical DAF-2 pathway for the modulation of morphological adaptation. However, in polyQ128 animals, decreased daf-2 signaling significantly enhanced mechanosensation while decreasing neuronal aberrations. Unlike other interventions that reduce the strength of insulin signaling, daf-2 RNAi dramatically redistributed large polyQ128 aggregates to the cell body, away from neuronal processes. Our results suggest that insulin signaling strength can differentially affect specific neurons aging naturally or under proteotoxic stress.

Intermittent hypercapnia enhances CO2 responsiveness and overcomes serotonergic dysfunction.

Serotonergic dysfunction compromises ventilatory chemosensitivity and may enhance vulnerability to pathologies such as the Sudden Infant Death Syndrome (SIDS). We have shown raphé contributions to central chemosensitivity involving serotonin (5-HT)-and γ-aminobutyric acid (GABA)-mediated mechanisms. We tested the hypothesis that mild intermittent hypercapnia (IHc) induces respiratory plasticity, due in part to strengthening of GABA mechanisms. Rat pups were IHc-pretreated (eight consecutive cycles; 5 min 5% CO2 - air, 10 min air) or constant normocapnia-pretreated as a control, each day for 5 consecutive days beginning at P12. We subsequently assessed CO2 responsiveness using the in situ perfused brainstem preparation. Hypercapnic responses were determined with and without pharmacological manipulation. Results show IHc-pretreatment induces plasticity sufficient for responsiveness despite removal of otherwise critical ketanserin-sensitive mechanisms. Responsiveness following IHc-pretreatment was absent if ketanserin was combined with GABAergic antagonism, indicating that plasticity depends on GABAergic mechanisms. We propose that IHc-induced plasticity could reduce the severity of reflex dysfunctions underlying pathologies such as SIDS.

Chronic nicotine and ethanol exposure both disrupt central ventilatory responses to hypoxia in bullfrog tadpoles.

The central hypoxic ventilatory response (HVR) comprises a reduction in ventilatory activity that follows a peripherally mediated ventilatory augmentation. Chronic early developmental exposure to nicotine or ethanol are both known to impair the peripherally mediated HVR, and nicotine impairs the central HVR, but the effect of ethanol on the central HVR has not been investigated. Additionally, chronic nicotine and ethanol exposure are known to impair ventilatory responses to hypercapnia in bullfrog tadpoles but HVRs have not been tested. Here early and late metamorphic tadpoles were exposed to either 30 µg/L nicotine or 0.15-0.05 g/dL ethanol for 10 wk. Tadpole brainstems were then isolated and the neurocorrelates of ventilation were monitored in vitro over 180 min of hypoxia (PO2=5.05±1.04 kPa). Both nicotine and ethanol exposure disrupted central HVRs. Nicotine impairments were dependent on development. Central HVRs were impaired only in early metamorphic nicotine-exposed tadpoles. Both early and late metamorphic ethanol-exposed tadpoles failed to exhibit central HVRs. Thus, central HVRs are impaired following both nicotine and ethanol exposure. Such failure to decrease ventilatory activity during hypoxia indicates that central hypoxic ventilatory depression is an active suppression of neural activity in response to hypoxia rather than a metabolic consequence of O2 limitation, and that exposure to ethanol (across development) or nicotine (during early development) disrupts mechanisms that normally induce active ventilatory depression.

Chronic, but not acute, ethanol exposure impairs central hypercapnic ventilatory drive in bullfrog tadpoles.

Chronic ethanol exposure early in development is deleterious to neural development and may impair responses to ventilatory stimuli (ventilatory drive) that maintain homeostasis. Central hypercapnic ventilatory drive (CHVD) increases ventilation to ensure pH homeostasis and accommodate the metabolic production of CO(2). We tested the hypothesis that chronic ethanol exposure impairs CHVD in bullfrog tadpoles. Early and late metamorphic tadpoles were exposed in vivo to 0.12-0.06 g/dL ethanol for either 3- or 10-wk durations. Brainstems from these animals were isolated and the neural correlates of ventilation were recorded in vitro during superfusion with normocapnic (1.5% CO(2):98.5% O(2)) and hypercapnic (5.0% CO(2):95.0% O(2)) artificial cerebral spinal fluid. Normocapnic neuroventilation was unaffected by chronic ethanol exposure. The typical response to hypercapnia, an increase in lung burst frequency, was lost following 10 but not 3 wk of ethanol exposure in both early and late metamorphic tadpoles. The neuroventilatory effects of chronic ethanol exposure were distinguishable from those of acute central ethanol (0.08 g/dL) exposure, which attenuated early metamorphic tadpole normocapnic neuroventilation, but had no effect on tadpole CHVD. Thus, 10 wk of ethanol exposure both early and late in metamorphosis impairs CHVD in bullfrog tadpoles.

Neuroplasticity of the central hypercapnic ventilatory response: teratogen-induced impairment and subsequent recovery during development.

Neuroventilation is highly plastic and exposure to either of two distinct teratogens, nicotine or ethanol, during development results in a similar loss of the neuroventilatory response to hypercapnia in bullfrog tadpoles. Whether this functional deficit is permanent or transient following nicotine or ethanol exposure was unknown. Here, we tested the persistence of hypercapnic neuroventilatory response impairments in tadpoles exposed to either 30 microg/L nicotine or 0.12-0.06 g/dL ethanol for 10 weeks. Brainstem breathing-related neural activity was assessed in tadpoles allowed to develop teratogen-free after either nicotine or ethanol exposure. Nicotine-exposed animals responded normally to hypercapnia after a 3-week teratogen-free period but the hypercapnic response in ethanol-exposed tadpoles remained impaired. Tadpoles allowed to develop for only 1 week nicotine free after chronic exposure were unable to respond to hypercapnia. The hypercapnic response of ethanol-exposed tadpoles returned by 6 weeks following chronic ethanol exposure. These findings suggest that some nicotine- and ethanol-induced impairments can be resolved during early development. Understanding both the disruptive effects of nicotine and ethanol exposure and how impaired responses return when teratogen exposure stops may offer insight on the function and plasticity of respiratory control.

Cholinergic sensitivity of the developing bullfrog (Rana catesbeiana) does not explain vulnerability to chronic nicotine exposure.

Juvenile bullfrogs previously identified as highly sensitive to acute nicotine, demonstrated normal neuroventilation following 3 wk of chronic nicotine exposure. Acute bath application of 1 microM galantamine, an acetylcholinesterase inhibitor, significantly attenuated both bullfrog normocapnic neuroventilation and response to hypercapnia in a fashion similar to that of acute nicotine. This would suggest that the developmental increase in nicotine sensitivity does not enhance vulnerability to chronic exposure, and that acute nicotine acts via endogenous acetylcholine pathways to depress neuroventilation and hypercapnic drive.

Nicotine elicits a developmentally dependent depression in bullfrog neuroventilatory response to CO(2).

Nicotine exposure is associated with numerous neurodevelopmental aberrations, including impairment of the neuroventilatory response to hypercapnia in bullfrog tadpoles and mouse neonates following prolonged developmental exposure. It is unclear how acute nicotine exposure affects neuroventilation and the neuroventilatory response to hypercapnia, or how these effects might differ from those of chronic exposure. In this study the neural correlates of ventilation were recorded from in vitro brainstem preparations derived from early and late metamorphic tadpoles and juvenile bullfrogs. Lung and gill/buccal breath parameters were compared during control (=0), 18, 50, 100, and 200microg/L nicotine conditions, applied during normocapnia (1.5% CO(2)) and hypercapnia (5.0% CO(2)). All preparations demonstrated a reduction in normocapnic lung burst frequency and an attenuated hypercapnic response during acute nicotine treatment. The concentrations necessary to elicit both of these responses decreased from 200microg/L nicotine in early metamorphic tadpole brainstems to 18microg/L nicotine in juvenile bullfrog brainstems, which suggests a developmental increase in acute nicotine sensitivity that is distinguishable from the developmental changes in vulnerability to chronic nicotine exposure. In summary, acute nicotine exposure impaired central CO(2) response, attenuated rather than enhanced respiratory drive, and had more pronounced effects at progressively lower concentrations as development proceeded through metamorphosis.

Timing and duration of developmental nicotine exposure contribute to attenuation of the tadpole hypercapnic neuroventilatory response.

The ability for air-breathing vertebrates to adjust ventilation in response to increased CO(2) (hypercapnia) is fundamental to maintaining pH homeostasis. Developmental nicotine exposure has been shown to impair tadpole neuroventilatory responses to hypercapnia following 8-12 weeks of exposure. It is not clear, however, to what extent the timing of exposure during development and/or the duration over which the exposure takes place contribute to this impairment. Here, tadpoles were exposed to 30 microg/L of nicotine for 3- or 10-week durations, either early or late in tadpole development. Correlates of tadpole lung neuroventilation were monitored during normocapnic (1.5% CO(2)) and hypercapnic (5% CO(2)) conditions of isolated brainstems. Preparations derived from early metamorphic tadpoles failed to increase lung neuroventilation in response to hypercapnia whether they had been exposed to nicotine for 3 or 10 weeks. Preparations derived from late metamorphic tadpoles failed to respond to hypercapnia after being exposed to nicotine for 10 weeks. These results suggest that both the developmental timing and duration of exposure are important when considering nicotine's effect on the hypercapnic neuroventilatory response.