Brain-Derived Neurotrophic Factor–Mediated Downregulation of Brainstem K⁺–Cl^– Cotransporter and Cell-Type–Specific GABA Impairment for Activation of Descending Pain Facilitation

Abstract

Chronic pain is thought to be partly caused by a loss of GABAergic inhibition and resultant neuronal hyperactivation in the central pain-modulating system, but the underlying mechanisms for pain-modulating neurons in the brain are unclear. In this study, we investigated the cellular mechanisms for activation of brainstem descending pain facilitation in rats under persistent pain conditions. In the nucleus raphe magnus (NRM), a critical relay in the brain's descending pain-modulating system, persistent inflammatory pain induced by complete Freund's adjuvant decreased the protein level of K 1 -Cl 2 cotransporter (KCC2) in both total and synaptosomal preparations. Persistent pain also shifted the equilibrium potential of GABAergic inhibitory postsynaptic current (E IPSC ) to a more positive level and increased the firing of evoked action potentials selectively in m-opioid receptor (MOR)-expressing NRM neurons, but not in MOR-lacking NRM neurons. Microinjection of brain-derived neurotrophic factor (BDNF) into the NRM inhibited the KCC2 protein level in the NRM, and both BDNF administration and KCC2 inhibition by furosemide mimicked the pain-induced effects on E IPSC and excitability in MOR-expressing neurons. Furthermore, inhibiting BDNF signaling by NRM infusion of tyrosine receptor kinase B-IgG or blocking KCC2 with furosemide prevented these pain effects in MOR-expressing neurons. These findings demonstrate a cellular mechanism by which the hyperactivity of NRM MOR-expressing neurons, presumably responsible for descending pain facilitation, contributes to pain sensitization through the signaling cascade of BDNF-KCC2-GABA impairment in the development of chronic pain.

Compensatory Activation of Cannabinoid CB2 Receptor Inhibition of GABA Release in the Rostral Ventromedial Medulla in Inflammatory Pain

“…Opioid-induced inhibition of GABA release disinhibits PAG and RVM output neurons (Heinricher and Ingram, 2008;Lau and Vaughan, 2014) and microinjection of GABA A receptor antagonists in either area results in antinociception (Moreau and Fields, 1986;Heinricher and Tortorici, 1994;Bobeck et al, 2009). Altered GABA release in the PAG and RVM is associated with chronic inflammatory or neuropathic pain models, although both increases and decreases in GABAergic mIPSCs have been reported (Hahm et al, 2011;Zhang et al, 2013) indicating the complexity of the changes in the descending pain circuit. One key variable in these studies may be age of animals that are used for recording as we have previously reported a marked decrease in GABA release probability between neonatal and adult RVM neurons that is the result of increased endocannabinoid tone in the adult rat RVM (Li et al, 2015).…”

“…RVM slice preparation was performed as described previously (Li et al, 2015). Rats were deeply anesthetized with isoflurane and the brains were rapidly removed and placed in N-methyl-D-glucamine-sucrose-based "cutting buffer" containing the following (in mM): 52 N-methyl-D-glucamine, 2.5 KCl, 0.5 CaCl 2 , 10 MgSO 4 , 1.2 NaH 2 PO 4 , 30 NaHCO 3 , 25 D-dextrose, 75 sucrose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, pH 7.4, adjusted with HCl and 300 -310 mOsm (Zhao et al, 2011;Ting et al, 2014). Coronal slices (180 -200 m) were cut in 95% O 2 -and 5% CO 2 -oxygenated cutting buffer.…”

Persistent Inflammation-induced Up-regulation of Brain-derived Neurotrophic Factor (BDNF) Promotes Synaptic Delivery of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor GluA1 Subunits in Descending Pain Modulatory Circuits

“…All NRM microinjection sites were verified histologically after the experiment by injection of 0.5 l of a blue dye, and controls of off-site injections were performed as described elsewhere (36). The confined effect of an injected drug within the NRM with this microinjection method has been demonstrated in previous studies (21,33,34). The pain threshold was measured every 5 min by paw withdrawal test on a freely moving rat using a Hargreaves analgesia instrument (Stoelting, Wood Dale, IL).…”

“…Synaptosome Preparations and Western Blot Analysis-The synaptosome preparations and Western blot analysis were performed as described in previous reports (21,38). For synaptosome preparations, NRM tissues from saline-and CFA-injected rats were homogenized gently in ice-cold 0.32 M sucrose buffer at pH 7.4 and then centrifuged for 10 min at 1000 ϫ g (4°C).…”

“…A study by Guo et al (20) showed that BDNF in the RVM may have originated from BDNF-containing neurons in the PAG and that BDNF activation of TrkB signaling in the RVM induces descending pain facilitation, suggesting that the signaling cascade of BDNFTrkB receptors in the RVM circuitry plays a critical role in the development of persistent pain after inflammation. Previous studies have shown that persistent inflammation up-regulates BDNF, which decreases the KCC2 function of maintaining the chloride gradient for inhibitory GABA synapses, resulting in impaired GABA inhibition and neuronal hyperexcitability in the nucleus raphe magnus (NRM), which is the major structure of the RVM, contributing to pain sensitization (21). As a pain modulator, BDNF can modulate excitatory glutamatergic and inhibitory GABAergic/glycinergic signals (17,22).…”