Endogenous BDNF augments NMDA receptor phosphorylation in the spinal cord via PLCγ, PKC, and PI3K/Akt pathways during colitis

Background Spinal central sensitization is an important process in the generation and maintenance of visceral hypersensitivity. The release of brain-derived neurotrophic factor (BDNF) from the primary afferent neurons to the spinal cord contributes to spinal neuronal plasticity and increases neuronal activity and synaptic efficacy. The N-Methyl-D-aspartic acid (NMDA) receptor possesses ion channel properties, and its activity is modulated by phosphorylation of its subunits including the NMDA receptor 1 (NR1). Methods Colonic inflammation was induced by a single dose of intracolonic instillation of tri-nitrobenzene sulfonic acid (TNBS). NR1 phosphorylation by BDNF in vivo and in culture was examined by western blot and immunohistochemistry. Signal transduction was studied by direct examination and use of specific inhibitors. Results During colitis, the level of NR1 phospho-Ser896 was increased in the dorsal horn region of the L1 and S1 spinal cord; this increase was attenuated by injection of BDNF neutralizing antibody to colitic animals (36 μg/kg, intravenous (i.v.)) and was also reduced in BDNF+/− rat treated with TNBS. Signal transduction examination showed that the extracellular signal-regulated kinase (ERK) activation was not involved in BDNF-induced NR1 phosphorylation. In contrast, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediated BDNF-induced NR1 phosphorylation in vivo and in culture; this is an additional pathway to the phospholipase C-gamma (PLCγ) and the protein kinase C (PKC) that was widely considered to phosphorylate NR1 at Ser896. In spinal cord culture, the inhibitors to PLC (U73122), PKC (bisindolylmaleimide I), and PI3K (LY294002), but not MEK (PD98059) blocked BDNF-induced NR1 phosphorylation. In animals with colitis, treatment with LY294002 (50 μg/kg, i.v.) blocked the Akt activity as well as NR1 phosphorylation at Ser896 in the spinal cord. Conclusion BDNF participates in colitis-induced spinal central sensitization by up-regulating NR1 phosphorylation at Ser896. The PI3K/Akt pathway, in addition to PLCγ and PKC, mediates BDNF action in the spinal cord during colitis

Effects of contusion injury on voluntary wheel-running behavior, not split by linetype (contusion experiment).

Effects of contusion injury on voluntary wheel-running behavior, not split by linetype (contusion experiment).</p

Effects of contusion injury on voluntary wheel-running behavior, split by linetype (contusion experiment).

Effects of contusion injury on voluntary wheel-running behavior, split by linetype (contusion experiment).</p

Percentage of fibers that contained centrally located nuclei in different muscles (exercise experiment).

(A) C vs HR mice with and without wheel access. (B) Normal vs mini-muscle individuals with or without wheel access. Values are 1 + log10 transformed LS means ± standard errors from SAS Procedure Mixed ANCOVA. DG = deep gastrocnemius, SG = superficial gastrocnemius, PL = plantaris, SL = soleus.</p

Fig 1 -

H&E staining of (A) myofibers in the deep gastrocnemius with invading cells (green arrows), (B) myofibers in the superficial gastrocnemius that exhibit centrally located nuclei (green arrows), (C) myofiber exhibiting pale staining cytoplasm in the deep gastrocnemius (green arrow), (D) areas in the plantaris showing signs of regeneration and do not contain central nuclei (green boxes), (E) soleus with necrotic fibers (green boxes), and (F) soleus with perimysial infiltration (green boxes). All representative pictures come from normal-muscled mice (not mini-muscle individuals) at 10x.</p

Comparisons of voluntary wheel-running behavior between high runner and control lines of mice during the exercise-induced injury study (exercise experiment).

Comparisons of voluntary wheel-running behavior between high runner and control lines of mice during the exercise-induced injury study (exercise experiment).</p

Fig 6 -

Proportion of the population of C and HR (A-E) and normal- and mini-muscled (F-J mice from the Exercise Experiment (with or without wheels) that has at least one myofiber containing (A and F) areas of regeneration, (B and G) perimysial infiltration, (C and H) pale staining cytoplasm, (D and I) cellular invasion, and (E and J) necrotic fibers. Values are percentage of the population. DG = deep gastrocnemius, SG = superficial gastrocnemius, PL = plantaris, SL = soleus.</p

H&E staining of a normal-muscled superficial gastrocnemius and a mini-muscle superficial gastrocnemius taken at 10x.

Green arrows indicate centrally located nuclei. These individuals had no wheel access.</p

Fig 2 -

Average wheel running metrics across six days for the both the Contusion Experiment (A-D) and the Exercise Experiment (E-H) shown in Tables 3 and 4. (A) Total revolutions (n = 42). (B) Number of one-minute intervals with at least one revolution (running duration; n = 42). (C) Revolutions per minute (average speed; n = 42). (D) Maximum number of revolutions in any one-minute interval (n = 41). (E) Total revolutions (n = 61). (F) Revolutions per minute (average speed; n = 60). (G) Number of one-minute intervals with at least one revolution (running duration; n = 61). (H) Maximum number of revolutions in any one-minute interval (n = 61). Values are LS means ± standard errors from SAS Procedure Mixed repeated-measures ANCOVA.</p