TrkB Receptor Activation Research Articles

Slow intracellular accumulation of GABA A receptor δ subunit is modulated by brain-derived neurotrophic factor

GABA A receptors composed of the γ2 and δ subunits have distinct properties, functions and subcellular localization, and pathological conditions differentially modulate their surface expression. Recent studies demonstrate that acute seizure activity accelerated trafficking of the γ2 and β2/3 subunits but not that of the δ subunit. The trafficking of the γ2 and β2/3 subunits is relatively well understood but that of the δ subunit has not been studied. We compared intracellular accumulation of the δ and γ2 subunits in cultured hippocampal neurons using an antibody feeding technique. Intracellular accumulation of the δ subunit peaked between 3 and 6 h, whereas, maximum internalization of the γ2 subunit took 30 min. In the organotypic hippocampal slice cultures internalization of the δ subunit studied using a biotinylation assay revealed highest accumulation between 3 and 5 h and that of the γ2 subunit between 15 and 45 min. The surface half-life of the δ subunit was 171 min in cultured hippocampal neurons and 102 min in the organotypic hippocampal slice cultures. In the subsequent studies, internalization of the δ subunit was found to be dependent on network activity but independent of ligand-binding. Brain-derived neurotrophic factor (BDNF) reduced buildup of the δ subunit in the cytoplasmic compartments and increased its surface expression, and this BDNF effect was independent of network activity. BDNF effect was mediated by activation of TrkB receptors, PLCγ and PKC. Increase in the basal PKC activity augmented cell surface stability of the δ subunit. These results suggest that rate of intracellular accumulation of the δ subunit was distinct and modulated by BDNF.

Positive AMPA Receptor Modulation Rapidly Stimulates BDNF Release and Increases Dendritic mRNA Translation

Brain-derived neurotrophic factor (BDNF) stimulates local dendritic mRNA translation and is involved in formation and consolidation of memory. 2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan-10-one (CX614), one of the best-studied positive AMPA receptor modulators (also known as ampakines), increases BDNF mRNA and protein and facilitates long-term potentiation (LTP) induction. Several other ampakines also improve performance in various behavioral and learning tasks. Since local dendritic protein synthesis has been implicated in LTP stabilization and in memory consolidation, this study investigated whether CX614 could influence synaptic plasticity by upregulating dendritic protein translation. CX614 treatment of primary neuronal cultures and acute hippocampal slices rapidly activated the translation machinery and increased local dendritic protein synthesis. CX614-induced activation of translation was blocked by K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], CNQX, APV, and TTX, and was inhibited in the presence of an extracellular BDNF scavenger, TrkB-Fc. The acute effect of CX614 on translation was mediated by increased BDNF release as demonstrated with a BDNF scavenging assay using TrkB-Fc during CX614 treatment of cultured primary neurons and was blocked by nifedipine, ryanodine, and lack of extracellular Ca(2+) in acute hippocampal slices. Finally, CX614, like BDNF, rapidly increased dendritic translation of an exogenous translation reporter. Together, our results demonstrate that positive modulation of AMPA receptors rapidly stimulates dendritic translation, an effect mediated by BDNF secretion and TrkB receptor activation. They also suggest that increased BDNF secretion and stimulation of local protein synthesis contribute to the effects of ampakines on synaptic plasticity.

Spinal NMDA receptor activation is necessary for de novo, but not the maintenance of, A2a receptor-mediated phrenic motor facilitation

Adenosine 2a (A2a) receptor agonists elicit persistent increases in phrenic nerve activity by transactivating the neurotrophin receptor, TrkB, near phrenic motoneurons. Our working model proposes that A2a receptor-mediated TrkB receptor activation strengthens glutamatergic synapses onto phrenic motoneurons. Activation of glutamate N-methyl d-aspartate (NMDA) receptors has been implicated in other models of phrenic motor plasticity. Thus we hypothesized that NMDA receptor activation also would contribute to A2a receptor-mediated phrenic motor facilitation. Adult male Sprague-Dawley rats were anesthetized with urethane, mechanically ventilated, neuromuscularly paralyzed, and bilaterally vagotomized. The A2a receptor agonist CGS-21680 and the NMDA receptor-channel blocker MK-801 were administered intrathecally over the C4 spinal segment. Phrenic nerve activity was recorded before, during, and after drug administration. MK-801 (concentration range 0.1, 1.0, 10.0, and 100 microM) was administered 30 min before CGS-21680 (50 microM). MK-801 dose-dependently blocked A2a receptor-mediated phrenic motor facilitation. When administered at 60 min post-CGS-21680, MK-801 prevented further increases in phrenic nerve activity compared with the CGS-21680 alone (CGS-21680 alone at 120 min: 114 +/- 19%; CGS-21680 and MK-801 at 60 min post-CGS-21680: 61 +/- 11%, above baseline, P < 0.05) but did not return phrenic motor output to baseline values. Our data suggest that NMDA receptor activation is necessary for de novo A2a receptor-mediated phrenic motor facilitation and that the maintenance of preexisting phrenic motor facilitation does not involve NMDA receptor-dependent mechanisms.

Between Clotho and Lachesis

Between Clotho and Lachesis

Tropomyosin-Related Kinase B in the Mesolimbic Dopamine System: Region-Specific Effects on Cocaine Reward

Previous studies found that brain-derived neurotrophic factor (BDNF) derived from nucleus accumbens (NAc) neurons can mediate persistent behavioral changes that contribute to cocaine addiction. To further investigate BDNF signaling in the mesolimbic dopamine system, we analyzed tropomyosin-related kinase B (TrkB) messenger RNA (mRNA) and protein changes in the NAc and ventral tegmental area (VTA) in rats following 3 weeks of cocaine self-administration. To study the role of BDNF-TrkB activity in the VTA and NAc in cocaine reward, we used localized viral-mediated Cre recombinase expression in floxed BDNF and floxed TrkB mice to knockdown BDNF or TrkB in the VTA and NAc in cocaine place conditioning tests and TrkB in the NAc in cocaine self-administration tests. We found that 3 weeks of active cocaine self-administration significantly increased TrkB protein levels in the NAc shell, while yoked (passive) cocaine exposure produced a similar increase in the VTA. Localized BDNF knockdown in either region reduced cocaine reward in place conditioning, whereas only TrkB knockdown in the NAc reduced cocaine reward. In mice self-administering cocaine, TrkB knockdown in the NAc produced a downward shift in the cocaine self-administration dose-response curve but had no effect on the acquisition of cocaine or sucrose self-administration. Together, these data suggest that BDNF synthesized in either VTA or NAc neurons is important for maintaining sensitivity to cocaine reward but only BDNF activation of TrkB receptors in the NAc mediates this effect. In addition, up-regulation of NAc TrkB with chronic cocaine use could promote the transition to more addicted biological states.

Spontaneous glutamatergic activity induces a BDNF‐dependent potentiation of GABAergic synapses in the newborn rat hippocampus

Spontaneous ongoing synaptic activity is thought to play an instructive role in the maturation of the neuronal circuits. However the type of synaptic activity involved and how this activity is translated into structural and functional changes is not fully understood. Here we show that ongoing glutamatergic synaptic activity triggers a long-lasting potentiation of gamma-aminobutyric acid (GABA) mediated synaptic activity (LLP(GABA-A)) in the developing rat hippocampus. LLP(GABA-A) induction requires (i) the activation of AMPA receptors and L-type voltage-dependent calcium channels, (ii) the release of endogenous brain-derived neurotrophic factor (BDNF), and (iii) the activation of postsynaptic tropomyosin-related kinase receptors B (TrkB). We found that spontaneous glutamatergic activity is required to maintain a high level of native BDNF in the newborn rat hippocampus and that application of exogenous BDNF induced LLP(GABA-A) in the absence of glutamatergic activity. These results suggest that ongoing glutamatergic synaptic activity plays a pivotal role in the functional maturation of hippocampal GABAergic synapses by means of a cascade involving BDNF release and downstream signalling through postsynaptic TrkB receptor activation.

Cystamine prevents haloperidol‐induced decrease of BDNF/TrkB signaling in mouse frontal cortex

The role of brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology as well as treatment outcome of schizophrenia. Rodent studies indicate that several antipsychotic drugs have time-dependent (and differential) effects on BDNF levels in the brain. Earlier studies from our laboratory have indicated that long-term treatment with haloperidol (HAL) decreases BDNF, reduced GSH and anti-apoptotic marker, Bcl-xl protein levels and increases the expression of pro-apoptotic proteins in rat frontal cortex. Furthermore, findings from human as well as rodent studies suggest that treatment of schizophrenia must involve the neuroprotective strategies to improve the neuropathology and thereby clinical outcome. In the present study, we investigated the potential of cystamine (CYS), an anti-oxidant and anti-apoptotic compound, to prevent HAL-induced reduction in BDNF, GSH, and Bcl-xl protein levels in mice and the signaling mechanism(s) involved in the beneficial effects of CYS. The results indicated that CYS as well as cysteamine (the FDA-approved precursor of CYS) increased BDNF protein levels in mouse frontal cortex 7 days after treatment. CYS co-treatment prevented chronic HAL treatment-induced reduction in BDNF, GSH, and Bcl-xl protein levels. CYS treatment enhanced TrkB-tyrosine phosphorylation and activated Akt and extracellular signal-regulated kinase (ERK)1/2, downstream molecules of TrkB signaling. In addition, in vitro experiments with mouse cortical neurons showed that CYS prevented the HAL-induced reduction in neuronal cell viability and BDNF protein levels, and increase in apoptosis. BDNF-neutralizing antibody as well as K252a, a selective inhibitor of neurotrophin signaling blocked the CYS-mediated neuroprotection. Moreover, CYS-mediated neuroprotection is also blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. Thus, CYS protects cortical neurons through a mechanism involving TrkB receptor activation, and a signaling pathway involving phosphatidylinositol 3-kinase and MAPK. The findings from the present study may be helpful for the development of novel neuroprotective strategies to improve the treatment outcome of schizophrenia.

Dopamine D1 Receptor-induced Signaling through TrkB Receptors in Striatal Neurons

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cgamma, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca(2+). These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.

Protective effect of BDNF against beta-amyloid induced neurotoxicity in vitro and in vivo in rats

We examined the potential protective effect of BDNF against β-amyloid-induced neurotoxicity in vitro and in vivo in rats. In neuronal cultures, BDNF had specific and dose–response protective effects on neuronal toxicity induced by Aβ 1–42 and Aβ 25–35. It completely reversed the toxic action induced by Aβ 1–42 and partially that induced by Aβ 25–35. These effects involved TrkB receptor activation since they were inhibited by K252a. Catalytic BDNF receptors (TrkB.FL) were localized in vitro in cortical neurons (mRNA and protein). In in vivo experiments, Aβ 25–35 was administered into the indusium griseum or the third ventricle and several parameters were measured 7 days later to evaluate potential Aβ 25–35/BDNF interactions, i.e. local measurement of BDNF release, number of hippocampal hilar cells expressing SRIH mRNA and assessment of the corpus callosum damage (morphological examination, pyknotic nuclei counting and axon labeling with anti-MBP antibody). We conclude that BDNF possesses neuroprotective properties against toxic effects of Aβ peptides.

BHLHB2 ControlsBdnfPromoter 4 Activity and Neuronal Excitability

Brain-derived neurotrophic factor (BDNF), via activation of TrkB receptors, mediates vital physiological functions in the brain, ranging from neuronal survival to synaptic plasticity, and has been implicated in the pathophysiology of neurodegenerative disorders. Although transcriptional regulation of the BDNF gene (Bdnf) has been extensively studied, much remains to be understood. We discovered a sequence within Bdnf promoter 4 that binds the basic helix-loop-helix protein BHLHB2 and is a target for BHLHB2-mediated transcriptional repression. NMDA receptor activation de-repressed promoter 4-mediated transcription and correlated with reduced occupancy of the promoter by BHLHB2 in cultured hippocampal neurons. Bhlhb2 gene -/- mice showed increased hippocampal exon 4-specific Bdnf mRNA levels compared with +/+ littermates under basal and activity-dependent conditions. Bhlhb2 knock-out mice also showed increased status epilepticus susceptibility, suggesting that BHLHB2 alters neuronal excitability. Together, these results support a role for BHLHB2 as a new modulator of Bdnf transcription and neuronal excitability.

Signaling Endosomes Point the Way

Brain-derived neurotrophic factor (BDNF) stimulates the migration of cerebellar granule cell precursors (GCPs) from the external granule layer to the internal granule layer. The movement of the GCPs is impaired in BDNF knockout mice. Zhou et al . isolated GCPs from postnatal day 6 (P6) mice and monitored their migration in culture in response to BDNF, applied uniformly or as a gradient. BDNF promoted the directional chemotaxis of the GCPs only when presented as a gradient. Although exogenously applied BDNF stimulated movement of GCPs from bdnf −/− mice, these cells lacked directional migration even in response to a gradient of BDNF, which suggests that an autocrine function for BDNF is also important for proper chemotaxis. To understand how autocrine BDNF may contribute to directional movement, cultured GCPs were stimulated with a related neurotrophin NT4, which also stimulates the TrkB receptor, and BDNF was found to be secreted, which could amplify the BDNF gradient and thus contribute to directional migration. GCPs have two receptors for BDNF: TrkB and p75 NTR . BDNF-induced chemotaxis (in cultured cells and organotypic slices) was blocked if the TrkB receptor activity was inhibited but was unaffected in cells defective for p75 NTR signaling. In the cultured GCPs, TrkB receptors colocalized with early endosomal markers at the leading edge toward the BDNF gradient, suggesting internalization of the receptor. In the P6 mouse cerebellum, TrkB was found in punctate structures that often colocalized with an endosomal marker; however, only in the wild-type mouse brains was TrkB consistently oriented toward the internal granule layer. Pharmacological inhibition of endocytosis inhibited cultured GCP migration toward a BDNF gradient, and impairment of dynamin function, which is required for clathrin-mediated endocytosis, blocked movement of GCPs from the external granule layer in organotypic slices. BDNF-induced GCP migration involved phosphoinositide 3-kinase (PI3K) signaling and activation of the Rac-specific guanine-nucleotide exchange factor Tiam1 (based on pharmacological inhibition and siRNA knockdown experiments). Thus, BDNF-induced migration appears to involve both the formation of oriented signaling endosomes and endogenous production of BDNF, which may amplify the BDNF gradient to promote motility (see Kaplan and Miller for commentary). P. Zhou, M. Porcionatto, M. Pilapil, Y. Chen, Y. Choi, K. F. Tolias, J. B. Bikoff, E. J. Hong, M. E. Greenberg, R. A. Segal, Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors. Neuron 55 , 53-68 (2007). [PubMed] D. R. Kaplan, F. D. Miller, Developing with BDNF: A moving experience. Neuron 55 , 1-2 (2007). [PubMed]

Interleukin-1β impairs brain derived neurotrophic factor-induced signal transduction

The expression of IL-1 is elevated in the CNS in diverse neurodegenerative disorders, including Alzheimer's disease. The hypothesis was tested that IL-1β renders neurons vulnerable to degeneration by interfering with BDNF-induced neuroprotection. In trophic support-deprived neurons, IL-1β compromised the PI3-K/Akt pathway-mediated protection by BDNF and suppressed Akt activation. The effect was specific as in addition to Akt, the activation of MAPK/ERK, but not PLCγ, was decreased. Activation of CREB, a target of these signaling pathways, was severely depressed by IL-1β. As the cytokine did not influence TrkB receptor and PLCγ activation, IL-1β might have interfered with BDNF signaling at the docking step conveying activation to the PI3-K/Akt and Ras/MAPK pathways. Indeed, IL-1β suppressed the activation of the respective scaffolding proteins IRS-1 and Shc; this effect might involve ceramide generation. IL-1-induced interference with BDNF neuroprotection and signal transduction was corrected, in part, by ceramide production inhibitors and mimicked by the cell-permeable C2-ceramide. These results suggest that IL-1β places neurons at risk by interfering with BDNF signaling involving a ceramide-associated mechanism.

Rapid regulation of dopamine transporters by tyrosine kinases in rat neuronal preparations

Termination of dopamine neurotransmission is primarily controlled by the plasma membrane-localized dopamine transporter. In this study, we investigated how this transporter is regulated by tyrosine kinases in neuronal preparations. In rat dorsal striatal synaptosomes, inhibition of tyrosine kinases by genistein or tyrphostin 23 resulted in a rapid (5-15 min), concentration-dependent decrease in [(3)H]dopamine uptake because of a reduction in maximal [(3)H]dopamine uptake velocity and dopamine transporter cell surface expression. The reduced transporter activity was associated with a decrease in phosphorylated p44/p42 mitogen-activated protein kinases. In primary rat mesencephalic neuronal cultures, the tyrosine kinase inhibitors similarly reduced [(3)H]dopamine uptake. When cultures were serum-deprived, acute activation of tyrosine kinase-coupled TrkB receptors by 100 ng/mL brain-derived neurotrophic factor significantly increased [(3)H]dopamine uptake; the effects were complex with increased maximal velocity but reduced affinity. The facilitatory effect of brain-derived neurotrophic factor on dopamine transporter activity depended on both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Taken together, our results suggest that striatal dopamine transporter function and cell surface expression is constitutively up-regulated by tyrosine kinase activation and that brain-derived neurotrophic factor can mediate this type of rapid regulation.

Endogenous Brain-Derived Neurotrophic Factor Triggers Fast Calcium Transients at Synapses in Developing Dendrites

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of the formation of functional neuronal networks. BDNF signaling regulates neuronal development not only globally, at the level of entire neurons or networks, but also at a subcellular level and with high temporal specificity; however, the spatiotemporal characteristics of intrinsic BDNF signaling are essentially unknown. Here, we used calcium imaging to directly observe intrinsic BDNF signaling in developing hippocampal neurons. We found that blocking intrinsic BDNF signaling with function-blocking BDNF antibodies (alphaBDNF) or K252-a reduced the frequency of spontaneously occurring fast and localized calcium rises in dendrites. Conversely, focal application of BDNF evoked fast and local dendritic calcium transients, which required activation of TrkB (tropomyosin-related kinase B) receptors as well as activation of voltage-gated sodium and calcium channels. Virus-mediated expression of PSD-95:CFP (postsynaptic density-95 tagged with cyan fluorescent protein) revealed that spontaneous local calcium transients occurred frequently at postsynaptic sites along the dendrite. The frequency of synaptically localized calcium transients was specifically reduced by blocking intrinsic BDNF signaling, whereas nonsynaptic calcium rises were not affected. Furthermore, focal BDNF delivery evoked localized and fast calcium elevations specifically at postsynaptic sites. Together, our results demonstrate that BDNF-dependent calcium signaling in developing hippocampal neurons is fast and occurs at synapses. These temporal and spatial characteristics of intrinsic BDNF signaling as well as its relative abundance renders BDNF an ideal signaling molecule in the establishment of specific synaptic connectivity and functional neuronal networks.

Distinct expression profiles of TrkB variants in the developing mouse nervous system

Brain-derived neurotrophic factor (BDNF) belongs to a family of small secreted proteins that also include nerve growth factor, neurotrophin 3, and neurotrophin 4. BDNF stands out among all neurotrophins by its high expression levels in the brain and its potent effects at synapses. Several aspects of BDNF biology such as transcription, processing, and secretion are regulated by synaptic activity. Such observations prompted the suggestion that BDNF may regulate activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP), a sustained enhancement of excitatory synaptic efficacy thought to underlie learning and memory. Here, we will review the evidence pointing to a fundamental role of this neurotrophin in LTP, especially within the hippocampus. Prominent questions in the field, including the release and action sites of BDNF during LTP, as well as the signaling and molecular mechanisms involved, will also be addressed. The diverse effects of BDNF at excitatory synapses are determined by the activation of TrkB receptors and downstream signaling pathways, and the functions, typically opposing in nature, of its immature form (proBDNF). The activation of p75NTR receptors by proBDNF and the implications for long-term depression will also be addressed. Finally, we discuss the synergy between TrkB and glucocorticoid receptor signaling to determine cellular responses to stress.

Injury‐induced axonal sprouting in the hippocampus is initiated by activation of trkB receptors

Penetrating head injuries are often accompanied by the delayed development of post-traumatic epilepsy. Schaffer collateral transection leads to axonal sprouting and hyperexcitability in area CA3 of hippocampal slice cultures. We used this model to test the hypothesis that the injury-induced axonal sprouting results from increased neurotrophin signaling via trkB receptors near the lesion. Using rats and mice, we established that sprouting CA3 pyramidal cell axons are labeled with an antibody to the growth-associated protein GAP-43. We observed two- to threefold increases in the level of brain-derived neurotrophic factor and trkB protein in area CA3 by 24-48 h after Schaffer collateral transection, preceding the onset of axonal sprouting. Finally, we demonstrated that injury-induced axonal sprouting of GAP-43-immunoreactive axons is impaired in hippocampal slice cultures from mice expressing low levels of trkB receptors. We conclude that injury-induced axonal sprouting is initiated by brain-derived neurotrophic factor-trkB signaling and suggest that this process may be critical for the genesis of post-traumatic epilepsy.

Activity‐dependent modulation of inhibition in Purkinje cells by TrkB ligands

In the cerebellum in vitro TrkB receptor ligands promote activity-dependent inhibitory synaptogenesis of Purkinje cells and also modulate inhibitory synaptic function. This mini review examines the roles of TrkB receptor activation by BDNF particularly in relation to activity-dependent synaptic plasticity on Purkinje cells and recent studies on the acute modulation of GABAergic synapses by BDNF.

Activity-Induced Rapid Synaptic Maturation Mediated by Presynaptic Cdc42 Signaling

Maturation of presynaptic transmitter secretion machinery is a critical step in synaptogenesis. Here we report that a brief train of presynaptic action potentials rapidly converts early nonfunctional contacts between cultured hippocampal neurons into functional synapses by enhancing presynaptic glutamate release. The enhanced release was confirmed by a marked increase in the number of depolarization-induced FM4-64 puncta in the presynaptic axon. This rapid presynaptic maturation can be abolished by treatments that interfered with presynaptic BDNF and Cdc42 signaling or actin polymerization. Activation of Cdc42 by applying BDNF or bradykinin mimicked the effect of electrical activity in promoting synaptic maturation. Furthermore, activity-induced increase in presynaptic actin polymerization, as revealed by increased concentration of actin-YFP at axon boutons, was abolished by inhibiting BDNF and Cdc42 signaling. Thus, rapid presynaptic maturation induced by neuronal activity is mediated by presynaptic activation of the Cdc42 signaling pathway.

Tyrosine Phosphorylation of Kir3.1 in Spinal Cord Is Induced by Acute Inflammation, Chronic Neuropathic Pain, and Behavioral Stress

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.

Brain‐derived neurotrophic factor induces a rapid dephosphorylation of tau protein through a PI‐3Kinase signalling mechanism

The microtubule-associated protein tau is essential for microtubule stabilization in neuronal axons. Hyperphosphorylation and intracellular fibrillar formation of tau protein is a pathology found in Alzheimer's disease (AD) brains, and in a variety of neurodegenerative disorders referred to as 'taupathies'. In the present study, we investigated how brain-derived neurotrophic factor (BDNF), an extracellular factor that is down-regulated in AD brains, affects tau phosphorylation. BDNF stimulation of neuronally differentiated P19 mouse embryonic carcinoma cells resulted in a rapid decrease in tau phosphorylation, at phosphorylation sites recognized by Tau 1, AT 8, AT 180 and p 262-Tau antibodies. K 252 a, a tyrosine receptor kinase (Trk) inhibitor, attenuated this dephosphorylation event, suggesting that BNDF activation of TrkB is responsible for the tau dephosphorylation. In addition, BDNF had no affect on tau phosphorylation in the presence of wortmannin, a PI-3 Kinase inhibitor, or lithium, a GSK 3 beta inhibitor, suggesting that these two kinases are part of the signaling transduction cascade leading from TrkB receptor activation to tau dephosphorylation. These results suggest a link between a correlate of AD, decrease in BDNF levels and an AD pathology, tau hyperphosphorylation.