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

Winter SM, Fresemann J, Schnell C, Oku Y, Hirrlinger J, Hülsmann S - Pflugers Arch. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Abt Neuro- und Sinnesphysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.

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.

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Rhythmic calcium transients in glycinergic neurons. a Identification of GlyT2-EGFP neurons (arrowheads) by two-photon excitation at 900 nm and CCD camera detection with a CFP optimized filter (465–495 nm). b OGB-1-AM fluorescence is shown and measured by using 800 nm excitation wavelength and a 501–551-nm emission band pass filter. All GlyT2-EGFP cells (a) appear to be labeled by OGB-1-AM (b). c Intracellular free cytosolic calcium transients recorded from glycinergic neurons. The numbering of the traces corresponds with the numbering of the neurons in the images a and b. The lower trace (∫) shows the integrated population field potential from the preBötC. d Graphic summary of the experiments. The left bar (white) shows the percentage of inspiratory EGFP-expressing glycinergic neurons (over the total number of glycinergic neurons). The right bar (black) shows the percentage of inspiratory glycinergic neurons (over the total number of inspiratory neurons). Data are given as mean ± SEM based on 20 imaging planes from five slices
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Fig3: Rhythmic calcium transients in glycinergic neurons. a Identification of GlyT2-EGFP neurons (arrowheads) by two-photon excitation at 900 nm and CCD camera detection with a CFP optimized filter (465–495 nm). b OGB-1-AM fluorescence is shown and measured by using 800 nm excitation wavelength and a 501–551-nm emission band pass filter. All GlyT2-EGFP cells (a) appear to be labeled by OGB-1-AM (b). c Intracellular free cytosolic calcium transients recorded from glycinergic neurons. The numbering of the traces corresponds with the numbering of the neurons in the images a and b. The lower trace (∫) shows the integrated population field potential from the preBötC. d Graphic summary of the experiments. The left bar (white) shows the percentage of inspiratory EGFP-expressing glycinergic neurons (over the total number of glycinergic neurons). The right bar (black) shows the percentage of inspiratory glycinergic neurons (over the total number of inspiratory neurons). Data are given as mean ± SEM based on 20 imaging planes from five slices

Mentions: Two-photon excitation microscopy was used to test whether EGFP-positive glycinergic neurons are active during the inspiratory phase of the rhythm (Fig. 3a; see “Materials and Methods” and Supplementary Figs. 2 and 3 for details of separation of EGFP and OGB-1-AM fluorescence).Fig. 3


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

Winter SM, Fresemann J, Schnell C, Oku Y, Hirrlinger J, Hülsmann S - Pflugers Arch. (2009)

Rhythmic calcium transients in glycinergic neurons. a Identification of GlyT2-EGFP neurons (arrowheads) by two-photon excitation at 900 nm and CCD camera detection with a CFP optimized filter (465–495 nm). b OGB-1-AM fluorescence is shown and measured by using 800 nm excitation wavelength and a 501–551-nm emission band pass filter. All GlyT2-EGFP cells (a) appear to be labeled by OGB-1-AM (b). c Intracellular free cytosolic calcium transients recorded from glycinergic neurons. The numbering of the traces corresponds with the numbering of the neurons in the images a and b. The lower trace (∫) shows the integrated population field potential from the preBötC. d Graphic summary of the experiments. The left bar (white) shows the percentage of inspiratory EGFP-expressing glycinergic neurons (over the total number of glycinergic neurons). The right bar (black) shows the percentage of inspiratory glycinergic neurons (over the total number of inspiratory neurons). Data are given as mean ± SEM based on 20 imaging planes from five slices
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691554&req=5

Fig3: Rhythmic calcium transients in glycinergic neurons. a Identification of GlyT2-EGFP neurons (arrowheads) by two-photon excitation at 900 nm and CCD camera detection with a CFP optimized filter (465–495 nm). b OGB-1-AM fluorescence is shown and measured by using 800 nm excitation wavelength and a 501–551-nm emission band pass filter. All GlyT2-EGFP cells (a) appear to be labeled by OGB-1-AM (b). c Intracellular free cytosolic calcium transients recorded from glycinergic neurons. The numbering of the traces corresponds with the numbering of the neurons in the images a and b. The lower trace (∫) shows the integrated population field potential from the preBötC. d Graphic summary of the experiments. The left bar (white) shows the percentage of inspiratory EGFP-expressing glycinergic neurons (over the total number of glycinergic neurons). The right bar (black) shows the percentage of inspiratory glycinergic neurons (over the total number of inspiratory neurons). Data are given as mean ± SEM based on 20 imaging planes from five slices
Mentions: Two-photon excitation microscopy was used to test whether EGFP-positive glycinergic neurons are active during the inspiratory phase of the rhythm (Fig. 3a; see “Materials and Methods” and Supplementary Figs. 2 and 3 for details of separation of EGFP and OGB-1-AM fluorescence).Fig. 3

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Abt Neuro- und Sinnesphysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.

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.

Show MeSH
Related in: MedlinePlus