This work was funded by the National Science Foundation IOS-1 022442

Reed, Mitchell D.

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MethodsWhole-Nerve Recording• The tadpole brainstem was isolated in arecording chamber and bathed in artificialcerebrospinal fluid (aCSF). Cranial nerve 5 or 7was drawn into a suction electrode, whichrecorded whole-nerve respiratory activity.Extracellular Recording• A micro-electrode filled with 5%biotinamide was vertically inserted into thebrainstem proximal to CN 5 or 10 or in themedullary raphe. Single-neuron activity wasrecorded and correlated with whole-nerveactivity to determine its respiratory relatedness.Treatments• The brainstem is bathed in normocapnia(1.5% CO2) or hypercapnia (5% CO2) aCSF.ConclusionsSimultaneous recordings of singleneurons and whole nerves, withsubsequent labeling of the recordedcell provides a platform to askquestions about the functionalrelationships of respiratory-relatedneurons in central chemosensitivity.Future Work• Immunohistochemistry will beconducted to identify theneurotransmitter phenotype ofrespiratory-related, CO2-sensitiveneurons.• Blockade of fast-synaptictransmission to identify neurons withintrinsic CO2 chemosensitivity.• Resolve temporal versus spatialrecruitment of neurons contributingto chemoreceptor, motoneuron, andoscilattor neuron pools.IntroductionTadpoles exhibit bimodal breathing; acombination of buccal and lung breaths.Breathing is controled by a central neuralnetwork that is sensitive to changes incentral CO2. The respiratory neuralnetwork can be isolated as the en blocbrainstem preparation and remains activeunder physiologically relevant conditionsfor over 48h.Thus, theisolatedtadpolebrainstempreparationis a powerfultool forinvestigatingneuralcontrol ofbreathing invertebrates.AimsTo simultaneously recordrespiratory-related activity fromcranial nerves and single neuronsTo label the recorded neuronTo characterize and quantifycentral chemosensitivity in thebrainstemChemosensitive Sites• Single-neuonrecordings were made inrostral (A & B) and caudalchemosensitive sites andthe raphe (C)• Firing of respiratory-related neurons (A & B)exhibits synchronicity tolung and/or buccal bursts• CO2-sensitive neurons,respiratory-related andunrelated, were found inall three chemosensitivesitesCO2 Sensitivity•CO2-sensitive neuronschanged firingfrequency and patternin response tohypercapniaCO2-induced firingchanges includedincreases and decreases(see right) in frequencyCO2-induced changesin pattern includedincreases (see right) anddecreases insynchronicitySimultaneous Recordings• Single-neuron and whole-nerve recordings are made simultaneously in vitro• Neuron firing that is coincident with whole-nerve activity indicates respriratory-relatedness of the cellJuxtacellular LabelingNeurons are entrained with juxtacellularn application of current pulses that induces electroporation of the cellmembrane, and facilitates uptake of neurobiotin ejected from the microelectrode during the current pulse.Tissue is fixed in 4% paraformaldehyde and then sliced into 60μm sections. Sections are stained with astreptavidin dye which binds to biotin, and imaged to visualize the recorded neuron.AbstractThe isolated frog brainstem is an established preparation to quantify respiratory drive andthe role of central CO2 sensitivity in control of breathing. Here, we establish a techniqueto quantify neuronal response to high CO2 (hypercapnia) and compare it to theconcomitant response by the intact respiratory network. Recorded neurons weresubsequently juxtacellularly labeled, facilitating identification of location and phenotype ofrespiratory CO2-sensitive neurons. Neurograms representing buccal and lung breathingwere obtained with suction electrodes recording wholenerve activity in cranial nerves 5and 7. Simultaneously, extracellular electrodes recorded single neurons in establishedCO2-sensitive sites of the frog brainstem. Neural activity was recorded while brainstemswere exposed to normocapnia and hypercapnia (1.5 and 5.0% CO2, balance O2). Neuronswere then entrained to an injected current and juxtacellularly labeled with biotinamide.After electrophysiology experiments, brainstem slices were stained with streptavidin-AlexaFluor and the recorded cells were imaged. Our results validate this technique, whichis the first reported capacity to simultaneously assess CO2 sensitivity of the centralnetwork that controls breathing and a single neuron embedded in the network.We expectto use this technique to elucidate neural mechanisms responsible for respiratory drive.NSF IOS-1022442Simultaneous assessment of CO2 sensitivity in the respiratorynetwork and its neuronsNormocapniaHypercapniaRecovery NormocapniaMitchell D Reed, Kimberly E Iceman, Barbara E TaylorUniversity of Alaska Fairbanks, Institute of Arctic Biology, Fairbanks, AKD657 894.1 4ReferencesDavies BL, Brundage CM, Taylor BE. 2009. Lung respiratory rhythm and pattern generation inthe frog: role of neurokinin--opioid receptors. J Comp Physiol B (Epub ahead of print)Pinault D. 1996. A novel single-cell staining procedure performed in vivo underelectrophysiological control: morpho-functional features of juxtacellularly labeled thalamiccells and other central neurons with biocytin or Neurobiotin. J Neurosci Methods 65(2):113-36.Stuesse SL, Adli DS, Cruce WL. 2001. Immunohistochemical Distribution of Enkephalin,Substance P, and Somatostatin in the Brainstem of the Leopard Frog, Rana pipiens. Microscopyand Research Techniques 54:229–245.AcknowledgementsThis work was funded by the National Science Foundation IOS-1 022442.The novel techniqueof simultaneouslyrecording cranialnerves and singleneurons provides newinsight into centralCO2 sensitivity.Juxtacellular Recording Juxtacellular Entrainment Juxtacellular LabelingABCDavies et al.Stuesse et al.

Simultaneous assessment of CO2 sensitivity in the respiratory network and its neurons

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Simultaneous assessment of CO2 sensitivity in the respiratory network and its neurons

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