Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.     

Both protein kinase A and mitogen-activated protein kinase are required in the amygdala for the macromolecular synthesis-dependent late phase of long-term potentiation.

The lateral amygdala (LA) is thought to be critical for the specific acquisition of conditioned fear, and the emotionally charged memories related to fear are thought to require a form of synaptic plasticity related to long-term potentiation (LTP). Is LTP in the lateral amygdala enduring, and, if so, does it require gene expression and the synthesis of new protein? Using brain slices, we have examined the molecular-signaling pathway of LTP in the cortico-amygdala and the thalamo-amygdala pathways. We find that a single high-frequency train of stimuli induces a transient LTP (E-LTP); by contrast, five repeated high-frequency trains induce an enduring late phase of LTP (L-LTP), which is dependent on gene expression and on new protein synthesis. In both pathways the late phase of LTP is mediated by protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). Application of the adenylyl cyclase activator forskolin induced L-LTP in both pathways, and this potentiation is blocked by inhibitors of protein synthesis. The late phase of LTP also is modulated importantly by beta-adrenergic agonists. An inhibitor of beta-adrenergic receptors blocks L-LTP; conversely, application of a beta-adrenergic agonist induces the L-LTP. Immunocytochemical studies show that both repeated tetanization and application of forskolin stimulate the phosphorylation of cAMP response element-binding proteins (CREB) in cells of the lateral nucleus of the amygdala. These results suggest that PKA and MAPK are critical for the expression of a persistent phase of LTP in the lateral amygdala and that this late component requires the synthesis of new protein and mRNA.     

Role of phosphorylation of ERK in induction and maintenance of LTP of the C-fiber evoked field potentials in spinal dorsal horn

Previous works have shown that activation of extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway is essential for long-term potentiation (LTP) in hippocampus. In the present study, the role of the ERK/CREB pathway in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathologic pain, was investigated in adult rats. Western blotting analysis showed that the protein level of phosphorylated ERK (p-ERK) in ipsilateral spinal dorsal horn was transiently increased after LTP induction, starting at 15 min and returning to control at 60 min after tetanic stimulation and that the protein level of p-CREB increased at 30 min, persisting for at least 3 hr after LTP induction. Double immunofluorescence staining showed that p-ERK and p-CREB were only located in neurons but not in glial cells in the spinal dorsal horn after LTP induction. More importantly, we found that spinal application of PD 98059 (100 microM), a selective MEK inhibitor, at 30 min before tetanic stimulation blocked LTP induction and prevented the increase in p-ERK and p-CREB in spinal dorsal horn. When applied 15 min after LTP induction, PD98059 reversed established LTP. The drug, however, did not affect the spinal LTP, when applied at 30 min after LTP. Our results suggested that activation of ERK/CREB pathway in spinal dorsal neurons is necessary for induction and maintenance of long-term potentiation of the C-fiber evoked field potentials.     

Genetic and pharmacological demonstration of a role for cyclic AMP-dependent protein kinase-mediated suppression of protein phosphatases in gating the expression of late LTP

Protein kinases and phosphatases play antagonistic roles in regulating hippocampal long-term potentiation (LTP), with kinase inhibition and phosphatase activation both impairing LTP. The late phase of LTP (L-LTP) requires activation of cAMP-dependent protein kinase (PKA) for its full expression. One way in which PKA may critically modulate L-LTP is by relieving an inhibitory constraint imposed by protein phosphatases. Using mutant PKA mice [R(AB) transgenic mice] that have genetically reduced hippocampal PKA activity, we show that deficient L-LTP in area CA1 of mutant hippocampal slices is rescued by acute application of two inhibitors of protein phosphatase-1 and protein phosphatase-2A (PP1/2A) (okadaic acid and calyculin A). Furthermore, synaptic facilitation induced by forskolin, an adenylyl cyclase activator, was impaired in R(AB) transgenics and was also rescued by a PP1/2A inhibitor in mutant slices. Inhibition of PP1/2A did not affect early LTP (E-LTP) or basal synaptic transmission in mutant and wildtype slices. Our data show that genetic inhibition of PKA impairs L-LTP by reducing PKA-mediated suppression of PP1/2A.     

BDNF modulates sensory neuron synaptic activity by a facilitation of GABA transmission in the dorsal horn

Topical application of brain-derived neurotrophic factor (BDNF) to the adult rat isolated dorsal horn with dorsal root attached preparation inhibited the electrically evoked release of substance P (SP) from sensory neurons. This effect of BDNF was dose dependent (EC(50) 250 pM) and reversed by the tyrosine kinase inhibitor, K-252a. BDNF-induced inhibition of SP release was blocked by the GABA(B) receptor antagonist CGP 55485 but not by naloxone. Acute application of BDNF significantly increased potassium-stimulated release of GABA in the dorsal horn isolated in vitro and this effect was blocked by K-252a. Intrathecal injection of BDNF into the rat lumbar spinal cord induced a short-lasting increase in hindpaw threshold to noxious thermal stimulation that was blocked by CGP 55485 and was associated with activation of ERK in dorsal horn. These data suggest that exogenous BDNF can indirectly modulate primary sensory neuron synaptic efficacy via facilitation of the release of GABA from dorsal horn interneurons.     

PLCgamma signaling underlies BDNF potentiation of Purkinje cell responses to GABA

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, neurite outgrowth, and excitatory synaptic transmission. We reported recently that acute BDNF exposure decreased gamma-aminobutyric acid (GABA) responses in cultured mouse cerebellar granule cells through tyrosine receptor kinase B (TrkB) receptor-mediated signaling. In the present study, we extend this work to investigate BDNF-induced modulation of GABA responses and GABA(A) receptor-mediated synaptic events in cerebellar slices. Thin (200 microm) parasagittal slices of cerebellum were prepared from postnatal Day 7 and 14 mice. Purkinje cells and granule cells, both of which express TrkB-like immunoreactivity, were identified for whole-cell recording. BDNF promptly enhanced GABA responses in Purkinje cells but, consistent with our previous finding in culture, attenuated those recorded in granule cells. In Purkinje cells, BDNF exposure shifted rightward the cumulative peak amplitude distribution for miniature inhibitory postsynaptic currents (mIPSCs) without changing the mIPSC frequency. BDNF-induced potentiation of Purkinje cell responses to GABA was blocked by TrkB-Fc (receptor body that sequesters BDNF), K252a (inhibitor of TrkB receptor autophosphorylation), U73122 (inhibitor of phospholipase-Cgamma [PLCgamma]), KN62 (specific inhibitor of calcium/calmodulin-dependent kinase), KT5720 (specific cyclic AMP-dependent kinase inhibitor), and by intracellular dialysis of Rp-cyclic AMP or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid). Overall, our results indicate that BDNF acutely potentiates GABA(A) receptor function in cerebellar Purkinje cells via the TrkB receptor-PLCgamma signal transduction cascade. In addition, we propose that cyclic AMP-mediated intracellular signaling mechanisms may facilitate manifestation of the BDNF-induced modulatory outcome.     

Alterations in the balance of protein kinase and phosphatase activities and age-related impairments of synaptic transmission and long-term potentiation

Aging is associated with an impaired ability to maintain long-term potentiation (LTP), but the underlying cause of the impairment remains unclear. To gain a better understanding of the cellular and molecular mechanisms responsible for this impairment, the synaptic transmission and plasticity were studied in the CA1 region of hippocampal slices from adult (6-8 months) and poor-memory (PM)-aged (23-24 months) rats. The one-way inhibitory avoidance learning task was used as the behavioral paradigm to screen PM-aged rats. With intracellular recordings, CA1 neurons of PM-aged rats exhibited a more hyperpolarized resting membrane potential, reduced input resistance, and increased amplitude of afterhyperpolarization and spike threshold, compared with those in adult rats. Although a reduction in the size of excitatory synaptic response was observed in PM-aged rats, no obvious differences were found between adult and PM-aged rats in the pharmacological properties of excitatory synaptic response, paired-pulse facilitation, or frequency-dependent facilitation, which was tested with trains of 10 pulses at 1, 5, and 10 Hz. Slices from the PM-aged rats displayed significantly reduced early-phase long-term potentiation (E-LTP) and late-phase LTP (L-LTP), and the entire frequency-response curve of LTP and LTD is modified to favor LTD induction. The susceptibility of time-dependent reversal of LTP by low-frequency afferent stimulation was also facilitated in PM-aged rats. Bath application of the protein phosphatase inhibitor, calyculin A, enhanced synaptic response in slices from PM-aged, but not adult, rats. In contrast, application of the cAMP-dependent protein kinase inhibitors, Rp-8-CPT-cAMPS and KT5720, induced a decrease in synaptic transmission only in slices from the adult rats. Furthermore, the selective beta-adrenergic receptor agonist, isoproterenol, and pertussis toxin-sensitive G-protein inhibitor, N-ethylmaleimide, effectively restored the deficit in E-LTP and L-LTP of PM-aged rats. These results demonstrate that age-related impairments of synaptic transmission and LTP may result from alterations in the balance of protein kinase/phosphatase activities.     

Ankyrin-rich membrane spanning protein plays a critical role in nuclear factor-kappa B signaling

Activation of nuclear factor-κB (NF-κB), a key feature of the neurotrophin signaling, has been shown to be critical for neuronal survival under pathologic settings. However, the precise mechanism by which neurotrophins activate NF-κB is not well understood. Here we report that the Ankyrin-rich Membrane Spanning (ARMS/Kidins220) protein, a novel transmembrane substrate of tropomyosin receptor kinase B (TrkB), plays an important role in NF-κB signaling elicited by brain-derived neurotrophic factor (BDNF). Accordingly, depletion of ARMS by specific RNA interference, or disruption of ARMS-TrkB interaction with expression of dominant-negative ARMS mutant, abolished BDNF-induced signaling to NF-κB. Our data further suggests that ARMS may promote NF-κB signaling via activation of mitogen-activated kinase (MAPK) and IκB kinase (IKK), thereby facilitating phosphorylation of RelA (major NF-κB subunit) at an IKK-sensitive site. The results shown here identify ARMS as a major factor that links neurotrophin signaling to NF-κB.     

Repetitive induction of late-phase LTP produces long-lasting synaptic enhancement accompanied by synaptogenesis in cultured hippocampal slices

Long-term plasticity of synaptic transmission is assumed to underlie the formation of long-term memory. Although the cellular mechanisms underlying short-term plasticity have been analyzed in detail, the mechanisms underlying the transformation from short-term to long-term plasticity remain largely unrevealed. We propose the novel long-lasting phenomenon as a model system for the analysis of long-term plasticity. We previously reported that the repetitive activation of cAMP-dependent protein kinase (PKA) by forskolin application led to an enhancement in synaptic strength coupled with synaptogenesis that lasted more than 3 weeks in cultured rat hippocampal slices. To elucidate whether this long-lasting synaptic enhancement depended on the induction of long-term potentiation (LTP) or on the pharmacological effect of forskolin, we applied glutamate (Glu) and correlated its dose with the production of the long-lasting synaptic enhancement. When the dose of Glu was low (10, 30 muM), only transient excitation or early-phase LTP (E-LTP) was induced by a single application and no long-lasting synaptic enhancement was produced by three applications. When the dose was raised to 100 or 300 muM, late-phase LTP (L-LTP) was induced by a single application and long-lasting synaptic enhancement was produced by three applications. The Glu-produced enhancement was accompanied by an increase in the frequency (but not the amplitude) of miniature EPSC and the number of synaptic structures. The enhancement depended on the interval of repetition and protein synthesis immediately after the Glu applications. These results indicate that the repetitive induction of L-LTP, but not E-LTP or transient excitation, triggers cellular processes leading to the long-lasting synaptic enhancement and the formation of new synapses.