Structural and functional identification of two distinct inspiratory neuronal populations at the level of the phrenic nucleus in the rat cervical spinal cord

Abstract

The diaphragm is driven by phrenic motoneurons that are located in the cervical spinal cord. Although the anatomical location of the phrenic nucleus and the function of phrenic motoneurons at a single cellular level have been extensively analyzed, the spatiotemporal dynamics of phrenic motoneuron group activity have not been fully elucidated. In the present study, we analyzed the functional and structural characteristics of respiratory neuron population in the cervical spinal cord at the level of the phrenic nucleus by voltage imaging, together with histological analysis of neuronal and astrocytic distribution in the cervical spinal cord. We found spatially distinct two cellular populations that exhibited synchronized inspiratory activity on the transversely cut plane at C4-C5 levels and on the ventral surface of the mid cervical spinal cord in the isolated brainstem-spinal cord preparation of the neonatal rat. Inspiratory activity of one group emerged in the central portion of the ventral horn that corresponded to the central motor column, and the other appeared in the medial portion of the ventral horn that corresponded to the medial motor column. We identified by retrogradely labeling study that the anatomical distributions of phrenic and scalene motoneurons coincided with optically detected central and medial motor regions, respectively. Furthermore, we anatomically demonstrated closely located features of putative motoneurons, interneurons and astrocytes in these regions. Collectively, we report that phrenic and scalene motoneuron populations show synchronized inspiratory activities with distinct anatomical locations in the mid cervical spinal cord.

Keywords: Astrocyte; Cervical spinal cord; Interneuron; Phrenic motoneuron; Phrenic nucleus; Respiratory control; Scalene motoneuron; Voltage imaging.

Fig. 1

Inspiratory-related activity imaged on the transverse cut plane at the C4/C5 spinal cord of brainstem–spinal cord preparation superfused with normocapnic CSF. a Time course of optical inspiratory-related activity from − 10 to 800 ms. Traces below the optical images are integrated C4 inspiratory activity, where the peak of C4 inspiratory activity was defined as 0 ms, and red broken lines on these traces indicate the timing when the images shown above the traces were captured. b Integrated C4 activity. c Depolarizing optical signal in the central region. d Depolarizing optical signal in the medial region. Central and medial regions are indicated with red and blue circles, respectively, b–d were recorded simultaneously

Fig. 2

Representative depolarizing inspiratory optical signals obtained in the experimental hypocapnic (2% CO₂) and hypercapnic (8% CO₂) conditions. Changes in CO₂ did not affect depolarizing inspiratory optical signals in either central or medial motor region. a, b Depolarizing optical signals on the transverse cut plane at C4/C5 level. Central and medial regions are indicated with red and blue circles, respectively. c, d Integrated C4 activity. e, f Depolarizing optical signals in the central region. g, h Depolarizing optical signals in the medial region, a, c, e, g correspond to hypocapnia, b, d, f, h correspond to hypercapnia

Fig. 3

Comparison of depolarizing inspiratory optical signals obtained in hypocapnic (2% CO₂) and hypercapnic (8% CO₂) conditions (n = 13). Changes in CO₂ did not appreciably affect the peak amplitude or area under the depolarizing wave curve between 0.84 and 2.5 s after the onset of the recording in either central or medial motor region. Each ordinate, arbitrary unit (au)

Fig. 4

Inspiratory-related optical signals recorded from the ventral surface of the C3–C5 spinal cord superfused with normocapnic CSF, with C4 integrated activity and its time course from − 40 to 320 ms, where the peak of C4 inspiratory activity was defined as 0 ms. Two longitudinal columnar depolarizing regions, corresponding to central and medial regions, were observed

Fig. 5

Representative optical images of inspiratory-related activity on the ventral surface of the cervical spinal cord in hypocapnic (2% CO₂) and hypercapnic (8% CO₂) conditions. In either central or medial motor region, changes in CO₂ did not appreciably affect depolarizing inspiratory signals. a, b Images showing depolarizing optical signals on the ventral surface of the cervical spinal cord at C3–C5 level. Two longitudinal columnar depolarizing regions, corresponding to central and medial regions, were observed. Representative areas of central and medial regions for calculation of optical signal wave forms in e–h are indicated with red and blue circles, respectively. c, d Integrated C4 activity. e, f Depolarizing optical signals in the central region. g, h Depolarizing optical signals in the medial region. Each ordinate, arbitrary unit, a, c, e, g correspond to hypocapnia, b, d, f, h correspond to hypercapnia

Fig. 6

Photomicrographs and confocal images showing the distribution of DiI-labeled neurons in the spinal cord after DiI application to the phrenic nerve. a Ventral view of the brainstem–spinal cord. Location of DiI-labeled neurons is marked in red in the photomicrograph. b–e DiI-labeled neurons in horizontal section of the spinal cord (b–e ventral to dorsal). Red broken line in f indicates the midline of the spinal cord. g, h DiI-labeled neurons in transverse section of the spinal cord. The area enclosed with a rectangle in g is shown at higher magnification in h. Phrenic motoneurons are located in the central motor region between the C3 and C5 levels. Scale bars, a–e 1 mm; f 100 µm; g 200 µm; h 50 µm

Fig. 7

Photomicrographs and confocal images showing the distribution of DiI-labeled neurons in the spinal cord after DiI injection into the scalene muscle. a Ventral view of the brainstem–spinal cord. Location of DiI-labeled neurons is marked in red in the photomicrograph. b–e DiI-labeled neurons in horizontal section of the spinal cord (b–e ventral to dorsal). Red broken line in f indicates the midline of the spinal cord. g, h DiI-labeled neurons in transverse section of the spinal cord. The area enclosed with rectangles in g is shown at higher magnification in h. Distribution of scalene motoneurons is longer (between the C3 and C5 levels) and more medial as compared to that of phrenic motoneurons as shown in Fig. 8. Scale bars, a–e 1 mm; f 100 µm; g 200 µm; h 50 µm

Fig. 8

Photomicrographs taken at C4 level of the cervical spinal cord. a Nissl-staining. b Choline acetyltransferase (ChAT)-immunoreactive neurons. c ChAT-immunoreactive neurons in the horizontal section of the ventral horn of C3–C5 spinal cord. Red broken line indicates the midline of the spinal cord. d–f Immunostaining of neuronal nuclear antigen (NeuN), glial fibrillary acidic protein (GFAP) and S-100-protein β-subunit (S100), respectively. g–i Enlarged images of the d–f, respectively. Arrowheads, double-arrowheads, and triple-arrowheads indicate the medial, central, and lateral motor columns, respectively. Scale bars: a–f 500 µm; g–i 200 µm

Fig. 9

Confocal images showing distribution of immunoreactivity of various cell markers at C4 level of the cervical spinal cord (a, i). The areas enclosed with rectangles in a and i are shown at higher magnifications in b and j, respectively. The central, medial, and lateral motor columns enclosed with rectangles in b, j are shown at higher magnification in c, d, k, l and e, f, m, n and g, h, o, p, respectively. Immunoreactivities for ChAT, S100, GFAP, and NeuN are indicated in green, magenta, white and yellow, respectively, and DAPI-positive cell nuclei are indicated in cyan. c, e, g, k, m, and o are same area as d, f, h, l, n, and p, respectively. Putative phrenic and scalene motoneurons (ChAT-ir large neurons in the central and medial motor regions, respectively) as well as motoneurons in the lateral motor region are surrounded by interneurons (ChAT-negative and NeuN-positive cells) and astrocytes (GFAP-ir or S100-ir cells). Scale bars: a, i 300 µm; b, j 200 µm; c–f, k–n 50 µm; g, h, o, p 100 µm