Identification of the neural pathway underlying spontaneous crossed phrenic activity in neonatal rats

Abstract

Cervical spinal cord hemisection at C2 leads to paralysis of the ipsilateral hemidiaphragm in rats. Respiratory function of the paralyzed hemidiaphragm can be restored by activating a latent respiratory motor pathway in adult rats. This pathway is called the crossed phrenic pathway and the restored activity in the paralyzed hemidiaphragm is referred to as crossed phrenic activity. The latent neural pathway is not latent in neonatal rats as shown by the spontaneous expression of crossed phrenic activity. However, the anatomy of the pathway in neonatal rats is still unknown. In the present study, we hypothesized that the crossed phrenic pathway may be different anatomically in neonatal and adult rats. To delineate this neural pathway in neonates, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), a retrograde transsynaptic tracer, into the phrenic nerve ipsilateral to hemisection. We also injected cholera toxin subunit B-horseradish peroxidase (BHRP) into the ipsilateral hemidiaphragm following hemisection in other animals to determine if there are midline-crossing phrenic dendrites involved in the crossed phrenic pathway in neonatal rats. The WGA-HRP labeling was observed only in the ipsilateral phrenic nucleus and ipsilateral rostral ventral respiratory group (rVRG) in the postnatal day (P) 2, P7, and P28 hemisected rats. Bilateral labeling of rVRG neurons was shown in P35 rats. The BHRP study showed that many phrenic dendrites cross the midline in P2 neonatal rats at both rostral and caudal parts of the phrenic nucleus. There was a marked reduction of crossing dendrites observed in P7 and P28 animals and no crossing dendrites observed in P35 rats. The present results suggest that the crossed phrenic pathway in neonatal rats involves the parent axons from ipsilateral rVRG premotor neurons that cross at the level of obex as well as decussating axon collaterals that cross over the spinal cord midline to innervate ipsilateral phrenic motoneurons following C2 hemisection. In addition, midline-crossing dendrites of the ipsilateral phrenic motoneurons may also contribute to the crossed phrenic pathway in neonates.

Figure 1

Electromyographic (EMG) recordings from the left and right ventral hemidiaphragm of postnatal rats before and after a complete left C2 spinal cord hemisection showing activity on both sides of the diaphragm. A, B) The spontaneous crossed phrenic activity which drives the left ventral hemidiaphram after left C2 hemisection was expressed in a P2 and P7 neonatal rat. C) No spontaneous crossed activity was observed in a P35 hemisected rat. This reflects the adult rat pattern in which no crossed phrenic activity is expressed after ipsilateral spinal cord hemisection.

Figure 2

Phrenic motoneurons are labeled ipsilateral to the C2 spinal hemisection. Low power (A) and high power photomicrographs (B, C) of a transverse section of the C4 spinal cord in a P2 rat showing that the left phrenic nucleus, B (dark circled area in A) was retrogradely labeled with WGA-HRP after left phrenic nerve injection. C). No labeling in the phrenic nucleus contralateral to hemisection (light circled area in A). Low power (D) and higher power photomicrographs (E, F) of a transverse section of the C4 spinal cord at P7. Low power (G) and higher power photomicrographs (H, I) of a transverse section of the C4 spinal cord at P28. Low power (J) and higher power photomicrographs (K, L) of a transverse section of the C4 spinal cord at P35. The rest of the sections examined also show the same pattern of labeling shown by A-L. P, pinhole on right side in A, D, G and J. Magnification in A is the same as in D, G and J. Magnification in B is the same as in C, E, F, H, I, K and L.

Figure 3

rVRG neurons in the medulla labeled with WGA-HRP in the hemisected rats at different ages. Low-power (A) and higher power photomicrographs (B, C) of a transverse section through the medulla in a P2 rat showing that the ipsilateral rVRG neurons (dark circled area in A) were transynaptically labeled with WGA-HRP. This area is enlarged in B. (C). No labeled rVRG neurons noted on the right side (light circled area in A). P, pinhole marking right side in A, D, G, J. Low power (D) and higher power photomicrographs (E,F) of a transverse section through the medulla in a P7 rat showing that the labeled rVRG neurons are ipsilateral to hemisection (dark circled area in D magnified in E). (F). Contralateral rVRG neurons are not labeled (light circled area in D). Low power (G) and higher power photomicrographs (H, I) of a transverse section through the medulla in a P28 rat showing that the labeled rVRG neurons are ipsilateral to hemisection (dark circled area in G magnified in H). (I). No Contralateral rVRG labeling is observed (light circled area in G). Low power (J) and higher power photomicrographs (K, L) of a transverse section through the medulla in a P35 rat. Note, for the first time that bilateral rVRG premotor neurons are labeled by WGA-HRP. These neurons are delineated by the dark and light circled areas and magnified in K and J respectively. IV, fourth ventricle. Magnification in A is the same as in D, G and J. Magnification in B is the same as in C, E, F, H and I. Magnification in K is the same as in L.

Figure 4

Quantification of the labeled rVRG neurons in P2, P7 and P28 rats. The WGA-HRP labeled rVRG premotor neurons in the medulla were ipsilateral to hemisection in neonatal rats. ** represents that significantly more labeled rVRG neurons in P2 than P7 and P28. * represents that significantly less labeled rVRG neurons in P28 compared to P7 animals.

Figure 5

Horizontal sections of the cervical spinal cord in different aged rats showing the anatomy of phrenic dendrites during postnatal development. Lower power (A) and higher power photomicrographs (B, C) of phrenic motoneurons labeled with BHRP in a P2 rat. B shows the rostral half of the nucleus while C shows the caudal half. Note that phrenic dendrites (indicated by the solid arrows) cross the midline (dashed arrow in A) at both rostral and caudal levels of the cervical spinal cord. (D-F). Phrenic motoneurons labeled with BHRP in a P7 hemisected rat. (E), Note that phrenic dendrites cross the midline of the cervical spinal cord only at the rostral level. Arrows indicate the crossing dendrites. (F), Note that there are no midline-crossing dendrites at the caudal pole of the phrenic nucleus. P, pinhole marking the rostral part of the cervical spinal cord in A and D. Magnification in A is the same as in D. Magnification in B is the same as in C, E and F.

Figure 6

Horizontal sections of the cervical spinal cord in P28 (A-C) and P35 rats (D-F) showing the anatomy of phrenic dendrites during postnatal development. Lower power (A) and higher power photomicrographs (B, C) of phrenic motoneurons are labeled with BHRP in a P28 hemisected rat. Solid arrows (B) point to the dendrites approaching, but not crossing the midline. (D-F). Phrenic motoneurons labeled with BHRP in a P35 hemisected rat. None of the phrenic dendrites tend to cross the midline of the cervical spinal cord. Dashed arrows in A and D approximate the midline of the spinal cord. Magnification in A is the same as in D. Magnification in B is the same as in C, E and F.

Figure 7

Quantitative summary of BHRP labeled midline-crossing phrenic dendrites in P2, P7 and P28 rats. A) At the rostral level of the phrenic nucleus, the number of P2 crossing phrenic dendrites were significantly more than P7 and P28 crossing dendrites. ** represents significance between P2 and P7 as well as P28 following hemisection. * represents the same change in the control animals. ## represents significance between P7 than P28 following hemisection. # represents the same change in the control animals. B) At the caudal level of the phrenic nucleus, crossing phrenic dendrites in P7 were significantly fewer than P2. Caudal crossing phrenic dendrites were not found in P28. C) The number of the crossing phrenic dendrites was similar at both rostral and caudal spinal cord in P2 rats. D) In P7 animals, the rostral crossing dendrites were significantly more than caudal crossing dendrites. ** represents a significance in P7 hemisection group. * represents that a significance in P7 control animals.

Figure 8

Diagrams of the crossed phrenic pathway in neonatal rats and young rats at five postnatal weeks (P35). A). Diagram of the crossed phrenic pathway in neonatal rats. The pathway in neonatal rats is comprised of spinal cord midline-crossing collaterals from the rVRG descending axons that cross in the medulla as well as midline-crossing dendrites from the ipsilateral phrenic motoneurons. B). Diagram of the crossed phrenic pathway in P35 rats. The pathway involves bilateral rVRG respiratory premotoneuron axons with axon collaterals that cross the midline of the cervical spinal cord. Arrows indicate the direction of impulses along the crossed phrenic pathway.