Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat

Abstract

Although monosynaptic bulbospinal projections to phrenic motoneurons have been extensively described, little is known about the organization of phrenic premotor neurons in the adult rat spinal cord. Because interneurons may play an important role in normal breathing and recovery following spinal cord injury, the present study has used anterograde and transneuronal retrograde tracing to study their distribution and synaptic relations. Exclusive unilateral, first-order labeling of the phrenic motoneuron pool with pseudorabies virus demonstrated a substantial number of second-order, bilaterally distributed cervical interneurons predominantly in the dorsal horn and around the central canal. Combined transneuronal and anterograde tracing revealed ventral respiratory column projections to prephrenic interneurons, suggesting that some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual-labeling studies with pseudorabies virus recombinants also showed prephrenic interneurons integrated with either contralateral phrenic or intercostal motoneuron pools. The stability of interneuronal pseudorabies virus labeling patterns following lateral cervical hemisection was then addressed. Except for fewer infected contralateral interneurons at the level of the central canal, the number and distribution of phrenic-associated interneurons was not significantly altered 2 weeks posthemisection (i.e., the point at which the earliest postinjury recovery of phrenic activity has been reported). These results demonstrate a heterogeneous population of phrenic-related interneurons. Their connectivity and relative stability after cervical hemisection raise speculation for potentially diverse roles in modulating phrenic function normally and postinjury.

Figure 1

A transverse spinal cord section is shown that was obtained from approximately the C3 spinal level ∼58 hours following PRV labeling of the left hemidiaphragm. In addition to infected ipsilateral phrenic motoneurons (* in A and B), some PRV-positive cervical interneurons (**) were also observed (A and C). No labeling was detected in the brainstem at this time point. This suggests that the cervical interneurons are second-order labeled. CC = central canal. Scale bar is 500 (A), and 100 (B & C) micrometers.

Figure 2

Transverse semi-thin (2μm) plastic sections labeled with antibodies to PRV (brown) and counterstained with Toluidine blue, 64 hours following delivery of PRV to the left hemidiaphragm. Labeled cells were visible in the dorsal horn (arrowheads in A, B) and at the level of the central canal (arrowheads in C). B is a higher magnification image from A (*). At this time post-PRV there is an accumulation of mononuclear cells (**) around PRV-labeled PhMNs (D). This most likely reflects imminent lysis of the earliest infected cells and thus provides an indirect, but useful, index of second-order vs. primary infection. Scale bar is 100 (A), 50 (C) and 25 (B, D) micrometers.

Figure 3

Three representative longitudinal sections are shown that were obtained from a serial section series of cervical spinal cord stained with PRV-specific antibody. Schematic diagrams of transverse spinal cord sections indicate the approximate horizontal level that each image was obtained. At 64 hours post-diaphragm PRV delivery, immunopositive cells were observed bilaterally in the dorsal horn (A) and more extensively in around the central canal (B). Cells within the dorsal horn are more sparsely distributed throughout the cervical cord (arrowheads in A) than those cells at the level of the central canal (B). The majority of labeled cells were observed ipsilateral to the labeled phrenic motoneuron pool (C). Cells within the dorsal horn and at the level of the central canal are shown at higher power in Fig. 4. Definitive second-order labeling of cells medial and lateral to the phrenic motoneuron pool was not determined in this study (see text). Emphasis in this study is thus restricted to PRV-immunopositive cells dorsal the level of the phrenic motoneuron pool. Rostro-caudal orientation is from right to left. Scale bar is 1 millimeter. NB. A high resolution version of this image is available as a Supplementary Figure (Supp. Fig. 1).

Figure 4

High magnification images of putative pre-phrenic interneurons seen in longitudinal sections through the dorsal horn (A) and at the level of the central canal (B and C). Labeled interneurons were distributed bilaterally in all cervical segments. The dendritic processes from the majority of labeled cells lateral to the central canal were orientated in a rostro-caudal direction. However, some cells located more medially (over the central canal) had bilaterally directed processes some of which crossed the spinal midline (asterisk in B and C). Rostro-caudal orientation is from right to left. Scale bar is 200 (A, B) and 100 micrometers (C).

Figure 5

To determine the pattern of first- and second-order labeling, dual retrograde tracing studies with PRV (transynaptically transported) and cholera toxin B-subunit (monosynaptically transported) were used. Sixty-four hours following delivery of these tracers to the hemidiaphragm, both PhMNs (A, transverse section) and interneurons (B, longitudinal section) were PRV-labeled (green); however, only motoneurons were labeled with the cholera toxin (red). Dual-labeled cells thus appear yellow. Rostro-caudal orientation in B is from left to right. Scale bar is 50 (A) and 100 (B) micrometers. NB. A magenta-green version of this image is available as a Supplementary Figure (Supp. Fig. 2).

Figure 6

The number of PRV-positive cells counted in the dorsal horn (A) and in laminae VII and X (B) are presented as a proportion of the number of PRV-positive PhMNs (±SEM). Forty eight hours following delivery of PRV to the left diaphragm (n=4), no cells other than the PhMNs were labeled. Sixty-four (n=4) to seventy-two hours (n=4) post-PRV delivery, a significant number of cells were observed bilaterally in the dorsal horn and at the level of the central canal. The number of cells within each area relative to labeled PhMNs remained constant between 64 and 72 hours. However, the number of cells labeled in both the ipsilateral dorsal horn and in laminae VII and X was significantly (P<0.05) greater than seen contralaterally (*). Two weeks following injury (n=4), there is a reduction in the number of cells observed in all topographical regions. The cell number around the central canal post-injury, contralateral to the labeled hemidiaphragm (●), was statistically less than seen in uninjured animals 64 hours post-PRV delivery (P=0.03)

Figure 7

Shown are longitudinal sections of the cervical spinal cord 64 hours following co-administration of PRV152 (green) and PRV614 (red) to the left and right halves of the diaphragm, respectively. Bilaterally-labeled interneurons were observed (A) as shown previously. In addition, there was a sub-population of bilaterally distributed interneurons at the level of the central canal that were labeled with both PRV variants (arrowheads). Rostro-caudal orientation is from top to bottom. Scale bar is 25 (A) and 100 micrometers (B). NB. A magenta-green version of this image is available as a Supplementary Figure (Supp. Fig. 3).

Figure 8

Neurophysiologically-guided anterograde tracing of axons originating within the VRC (a.k.a., rVRG see text) is illustrated. Sections were labeled with Vectastain ABC to detect Miniruby (conjugated with biotin, brown) and counter-stained with cresyl violet. (A) Discrete stereotaxic localization of inspiratory medullary neurons was facilitated by using a Carbostar-3 electrode (B, image courtesy of Kation Scientific). Miniruby (10,000kDa BDA conjugated with rhodamine) was then iontophoretically delivered (see Methods). In a subset of animals (n=4) the rVRG recording was matched with diaphragm EMG recordings (C) to ensure correct placement of the electrode in the medulla. Note that the heartbeat was also detected in left-side diaphragm EMG traces. Anterogradely-labeled axons were observed in horizontal sections of the cervical spinal cord at the level of the phrenic motoneurons (D) and bilaterally around the central canal. Boutons were observed on the soma of motoneurons (asterisk in D) and between cells, presumably on dendrites (black arrowheads in D). Boutons were also found around the soma of neurons at the central canal (E). Labeled axons were seen crossing the spinal midline (white arrowhead in F). Rostro-caudal orientation in D-F is from top to bottom. Scale bars are 1mm (A), and 10 (B) 25 (D, E) and 50 microns (F). SEM = scanning electron microscope. cc = central canal.

Figure 9

Longitudinal sections through the cervical spinal cord. Miniruby (red) was iontophoretically delivered into the left rVRG and PRV152 (green) was administered to the ipsilateral diaphragm. Descending Miniruby-positive fibers from the medulla were observed forming boutons on and around PRV-labeled phrenic motoneurons (A & B). Many of these contacts were seen on the dendrites running rostro-caudally between motoneurons (A) and around the soma of these cells (B). Similar contacts were observed on the dendrites (arrowhead in C) and soma (arrowhead in D) of PRV-positive cervical interneurons distributed bilaterally around the central canal. Rostro-caudal orientation is from top to bottom. Scale bar is 50 (A) and 25 micrometers (B-D). NB. A magenta-green version of this image is available as a Supplementary Figure (Supp. Fig. 4).

Figure 10

Longitudinal sections are illustrated of the cervical spinal cord 64 hours following dual-PRV labeling. PRV152 (green) was applied to the left diaphragm and PRV614 (red) to the left intercostal muscles. Interneurons labeled for both tracers were seen bilaterally throughout the cervical spinal cord, around the central canal. While the majority of cells were individually labeled for either PRV152 or PRV614, there was a sub-population of interneurons that were double-labeled (yellow, arrowheads). Rostro-caudal orientation is from top to bottom. Scale bar is 25 micrometers. cc = central canal. NB. A magenta-green version of this image is available as a Supplementary Figure (Supp. Fig. 5).

Figure 11

Longitudinal sections are shown from animals 2 weeks following a lateral C2 hemisection and 64 hours following PRV delivery to the ipsilateral hemidiaphragm. Although there is an apparent decrease in the number of labeled cells compared with uninjured animals (Table 1), this difference was not statistically significant. The distribution of labeled phrenic motoneurons (A) and cervical interneurons (B) was comparable with uninjured animals. Labeled interneurons were also within close proximity to the lesion cavity (C). Rostro-caudal orientation is from right to left. Scale bar is 200 (A, B and inset in C) and 400 micrometers (C). cc = central canal