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Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices.

ABSTRACT: Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and 'tonic' expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation.

SUBMITTER: Winter SM

PROVIDER: S-EPMC2691554 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices.

Winter Stefan M SM Fresemann Jens J Schnell Christian C Oku Yoshitaka Y Hirrlinger Johannes J Hülsmann Swen S

Pflugers Archiv : European journal of physiology 20090224 3

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and 'tonic' expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory b ...[more]

PMID: 19238427

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Project description:BackgroundEx vivo, whole-mount explant culture of the rodent retina has proved to be a valuable approach for studying retinal development. In a limited number of recent studies, this method has been coupled to live fluorescent microscopy with the goal of directly observing dynamic cellular events. However, retinal tissue thickness imposes significant technical limitations. To obtain 3-dimensional images with high quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as 2-photon, with a higher level of depth penetrance. Here, we report a retinal live imaging method that is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area.ResultsMouse retinal slice cultures were prepared and standard, inverted confocal microscopy was used to generate movies with high quality resolution of retinal cross-sections. To illustrate the ability of this method to capture discrete, physiologically relevant events during retinal development, we imaged the dynamics of the Fucci cell cycle reporter in both wild type and Cyclin D1 mutant retinal progenitor cells (RPCs) undergoing interkinetic nuclear migration (INM). Like previously reported for the zebrafish, mouse RPCs in G1 phase migrated stochastically and exhibited overall basal drift during development. In contrast, mouse RPCs in G2 phase displayed directed, apical migration toward the ventricular zone prior to mitosis. We also determined that Cyclin D1 knockout RPCs in G2 exhibited a slower apical velocity as compared to wild type. These data are consistent with previous IdU/BrdU window labeling experiments on Cyclin D1 knockout RPCs indicating an elongated cell cycle. Finally, to illustrate the ability to monitor retinal neuron differentiation, we imaged early postnatal horizontal cells (HCs). Time lapse movies uncovered specific HC neurite dynamics consistent with previously published data showing an instructive role for transient vertical neurites in HC mosaic formation.ConclusionsWe have detailed a straightforward method to image mouse retinal slice culture preparations that, due to its relative ease, extends live retinal imaging capabilities to a more diverse group of scientists. We have also shown that, by using a slice technique, we can achieve excellent lateral resolution, which is advantageous for capturing intracellular dynamics and overall cell movements during retinal development and differentiation.

Dataset Information

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices.

Publications

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices.

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