Disruption of BDNF/TrkB Signaling Alters Glutamatergic mRNA Expression at Phrenic Motor Neurons
Authors: Rana Sabhya , Zhan Wen-Zhi , Sieck Gary C. , Mantilla Carlos B. 
Published: April 2020

Brain‐derived neurotrophic factor (BDNF) signaling via its high affinity tropomyosin‐related kinase sub‐type B (TrkB) receptor is widely implicated in regulating glutamatergic synaptic transmission in the context of neuroplasticity. At phrenic motor neurons innervating the main inspiratory muscle in mammals (the diaphragm muscle), glutamate is the main excitatory neurotransmitter and acts primarily via ionotropic AMPA and NMDA receptors. In various regions in the CNS, BDNF/TrkB signaling influences rapid phosphorylation of AMPA and NMDA receptor subunits, supporting a main role in neuroplasticity. BDNF/TrkB signaling also plays an important role across various models of injury and respiratory neuroplasticity. In addition, BDNF/TrkB signaling increases AMPA and NMDA receptor expression in the CNS, but whether this effect is also present in motor neurons is presently unknown. BDNF/TrkB signaling modulates motor neuron excitability, which may reflect changes in glutamatergic receptor expression. In the present study, AMPA and NMDA mRNA expression in retrogradely‐labeled phrenic motor neurons was measured using RNAscope in situ hybridization (ACDBio). Adult male rats were randomly assigned to control (untreated) and 11‐day treatment groups (12 ml/day) with intrathecal infusion of the fusion‐protein TrkB‐Fc or artificial cerebrospinal fluid (aCSF) in the region of the phrenic motor neuron pool. Intrathecal catheter tip position was verified at the terminal experimental at ~C4 segment, and the infusion rate was confirmed to restrict delivery to the cervical spinal cord. Expression of both AMPA and NMDA receptor mRNA was evident as puncta in phrenic motor neurons (identified by cholera toxin sub‐unit B – CTB‐labeling) as well as in other large neurons in the ventral horn. Our analysis of mRNA puncta density was restricted to clearly‐identified, retrogradely (CTB)‐labeled phrenic motor neurons containing the mid‐nuclear region in frozen, thin (10μm) longitudinal sections of the spinal cord. At least 25 phrenic motor neurons were stereologically selected for analysis in each animal. No differences in AMPA or NMDA mRNA density were evident between the untreated control and aCSF treated groups, and these results were combined. Across phrenic motor neurons in the control and aCSF groups, there was ~30% variance in AMPA and NMDA mRNA density within each animal, reflecting heterogeneity across motor neurons of varying size, expected to be recruited for the various levels of force production by the diaphragm muscle. Following TrkB‐Fc infusion, there was evidence of decreased AMPA mRNA density in phrenic motor neurons following but no change in NMDA mRNA density. These results support a role for BDNF/TrkB signaling in modulating glutamatergic receptor expression at phrenic motor neurons, which may thus contribute to the regulation of motor neuron excitability and neuroplasticity following injury or disease.Support or Funding InformationNIH grants R01 HL96750 and R01 HL146114