Effects of p-CPA on plasticity in the phrenic nucleus and the expression of the crossed phrenic phenomenon four hours following C2 spinal cord hemisection

C2 spinal hemisection results in synaptic and astroglial changes in the ipsilateral phrenic nucleus, observed at the electron microscopic (EM) level, have been associated with the physiological recovery of the ipsilateral phrenic nerve and hemidiaphragm during the expression of the crossed phrenic phenomenon (CPP). The present study investigated the effects of the 5-HT synthesis inhibitor para-chlorophenylalanine (p-CPA) on synaptic and astrocytic plasticity in the rat phrenic nucleus as well as the physiological expression of the CPP four hours following C2 hemisection. Quantification of phrenic nerve activity indicated that the mean amplitude of respiratory bursts during the CPP was significantly lower in p-CPA-treated rats than in saline controls. 8/8 saline-injected rats expressed the CPP, while only 4/8 p-CPA-treated rats expressed the CPP. Recordings from the contralateral phrenic nerve in saline-injected controls, but not in p-CPA-treated rats, demonstrated significant increases in mean respiratory frequency and mean amplitude of integrated respiratory waveforms during asphyxia, compared to normocapnia. Quantitative EM morphometric analysis demonstrated that saline-treated hemisected rats showed typical hemisection-induced synaptic and astrocytic plasticity in the ipsilateral phrenic nucleus. p-CPA treatment three days prior to hemisection resulted in an attenuation of both synaptic and astrocytic plasticity in the ipsilateral phrenic nucleus observed four hours following hemisection. In addition, p-CPA treatment attenuated injury induced alterations in immunohistochemical staining of glial fibrillary acidic protein (GFAP). Western blot analysis of GFAP demonstrated that the overall levels of GFAP did not differ significantly between groups. HPLC analysis indicated that C4 spinal cord 5-HT levels were significantly higher and DA levels were significantly lower in saline-treated hemisected rats compared to uninjured controls. In p-CPA-treated hemisected rats, spinal cord levels of 5-HT, DA, NE, and the 5-HT metabolite, 5-MAA, were all significantly lower compared to saline-treated hemisected rats and uninjured controls. The results of the present study suggest that p-CPA treatment: (1) attenuates the CPP and asphyxia-induced increases in respiratory frequency and respiratory burst amplitude four hours following hemisection; and (2) attenuates hemisection-induced plasticity in the phrenic nucleus four hours following an ipsilateral C2 hemisection.