The brain-derived neurotrophic factor receptor TrkB is critical for the acquisition but not expression of conditioned incentive value

Stimuli paired with reward acquire incentive properties that are important for many aspects of motivated behavior, such as feeding and drug-seeking. Here we used a novel chemical–genetic strategy to determine the role of the brain-derived neurotrophic factor (BDNF) receptor TrkB, known to be critical to many aspects of neural development and plasticity, during acquisition and expression of positive incentive value by a cue paired with food. We assessed that cue's learned incentive value in a conditioned reinforcement task, in which its ability to reinforce instrumental responding later, in the absence of food itself, was examined. In TrkB ^F616A knock-in mice, TrkB kinase activity was suppressed by administering the TrkB inhibitor 1NMPP1 during the period of initial cue incentive learning only (i.e. Pavlovian training), during nose-poke conditioned reinforcement testing only, during both phases, or during neither phase. All mice acquired cue–food associations as indexed by approach responses. However, TrkB ^F616A mice that received 1NMPP1 during initial cue incentive learning failed to show conditioned reinforcement of nose-poking, regardless of their treatment in testing, whereas administration of 1NMMP1 only during the testing phase had no effect. The effects of 1NMPP1 administration were due to inhibition of TrkB^F616A, because the performance of wild-type mice was unaffected by administration of the compound during either phase. These data indicate that BDNF or NT4 signaling through TrkB receptors is required for the acquisition of positive incentive value, but is not needed for the expression of previously acquired incentive value in the reinforcement of instrumental behavior.     

Immunohistochemical study of brain-derived neurotrophic factor and its receptor,TrkB, in the hippocampal formation of schizophrenic brains

Recently, the pathogenesis of schizophrenia has been investigated from the perspective of neurodevelopmental dysfunction theory. On the other hand, it has been indicated that neurotrophic factors, such as nerve growth factors, brain-derived neurotrophic factor (BDNF), and neurotrophin-3, are significantly involved in the development and functional differences of central nervous system (CNS). Some reports proposed that the dysfunction of these factors could explain the pathogenesis of schizophrenia possibly. In this study, the authors investigated immunohistochemically the distribution and/or morphology of BDNF and TrkB, its peculiar receptor, in the hippocampal formation of schizophrenic brain. As a result, BDNF-positive pyramidal cells in the CA2 and neurons in the CA3 and the field of the CA4 were intensely stained compared to those of normal control. Staining of TrkB-positive neurons showed a signet-ring like shape in the hippocampus of normal control brains. Such figures were not observed on staining of those neurons from schizophrenic brains. In the control cases, TrkB-immunopositive varicose fibers were frequently seen. Those observed differences between schizophrenic and normal cases may indicate the existence of dysfunction of BDNF and TrkB in schizophrenic brain, and this dysfunction may be one of the factors involved in the pathogenesis of schizophrenia.     

Induction of functional and morphological expression of neuropeptide Y (NPY) in cortical cultures by brain-derived neurotrophic factor (BDNF): evidence for a requirement for extracellular-regulated kinase (ERK)-dependent and ERK-independent mechanisms

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) induces expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from the fetal rat cortex. Using BDNF induction of NPY production and neurite extension of NPY neurons as functional and morphological criteria, respectively, we addressed the question: Does BDNF activate the extracellular-regulated kinase (ERK) pathway and if so, is activated (phosphorylated, P)-ERK required for the induction of both the functional and morphological expression of NPY? BDNF led to a rapid (30 min) and sustained (6 h) phosphorylation of ERK. PD98059 (PD, a specific inhibitor of the ERK kinase MEK), drastically inhibited, LY294002 (LY, a specific inhibitor of phosphatidylinositol-3-kinase, PI-3K) partially inhibited, and GF 109203X (GF, a specific inhibitor of protein kinase C) did not inhibit phosphorylation of ERK. A 24-h exposure to BDNF led to approximately 2-fold increase in the total culture content of NPY ( approximately 60% of which was secreted and approximately 40% remained in the aggregates) and to an abundance of neurite-bearing NPY neurons. BDNF-induced NPY produced and secreted into the medium was inhibited 73% by PD, 52% by LY and not at all by GF. In contrast, BDNF-induced NPY produced and sequestered in the aggregates was not inhibited by any of these inhibitors, suggesting a role for the ERK pathway in induced secretion of NPY. PD or LY did not inhibit BDNF-induced abundance of neurite-bearing NPY neurons. K252a (an inhibitor of TrkB-tyrosine kinase) abolished all the effects of BDNF assessed in our cultures. In summary, we demonstrate that TrkB-mediated activation of the ERK pathway is preferentially required for BDNF induction of NPY produced and secreted but not for the induction of the expression of neurite-bearing NPY neurons. Thus, BDNF induction of the functional and morphological expression of NPY is brought about by ERK-dependent and ERK-independent mechanisms.     

The distribution of neuropeptide Y and brain-derived neurotrophic factor immunoreactivity in hippocampal formation of the monkey and rat

The distribution of neuropeptide Y (NPY) and Brain-Derived Neurotrophic Factor (BDNF) in the hippocampal formation of monkey and rat brains was studied immunohistochemically. The NPY-neuronal system is more highly developed in the monkey compared to that in the rat. The distribution of NPY-positive products was coincident with that of abundant BDNF-positive deposits. These observations suggest that the role of BDNF and the interaction of BDNF-NPY may differ between species.     

CSF levels of a set of neurotrophic factors (brain-derived neurotrophic factor,nerve growth factor) and neuropeptides (neuropeptide Y,galanin) in epileptic children

This paper aims to investigate the possible roles of a set of neurotrophic factors (brain-derived neurotrophic factor-BDNF, nerve growth factor-NGF) and neuropeptides (neuropeptide Y-NPY, and galanin) in children with active epileptogenesis. The cerebrospinal fluid (CSF) levels of BDNF, NPY, NGF and galanin were measured with enzyme-linked immunosorbent assays in epileptic children (n = 73) and controls (n = 64). There were no significant alterations in the CSF levels of BDNF, NPY and NGF in epileptic children with active clinical seizures compared with the levels of controls. However profoundly depressed galanin levels were found in infants with epileptic encephalopathy (mean ± SD:0.63 ± 0.19 pg/ml) and significantly increased galanin levels were measured in children with drug resistant epilepsy during the period of status epilepticus (mean ± SD: 6.92 ± 1.19, pg/ml pg/ml) compared with the levels of controls. Depressed levels of galanin might reflect a defective anti-epileptogenic effect of galanin in infants with epileptic encephalopathy. On the contrary, increased CSF levels of galanin might be a result of anti-epileptogenic effects of this peptide in epileptic children with status epilepticus.     

Involvement of a spinal brain-derived neurotrophic factor/full-length TrkB pathway in the development of nerve injury-induced thermal hyperalgesia in mice

Partial sciatic nerve ligation in mice caused a marked and persistent decrease in the latency of paw withdrawal from a thermal stimulus only on the ipsilateral side. This thermal hyperalgesia was abolished by repeated intrathecal pretreatment with a specific antibody to brain-derived neurotrophic factor (BDNF), but not neurotrophin-4, just before and after the nerve ligation. These results provide direct evidence that BDNF within the spinal cord may contribute to the development of thermal hyperalgesia caused by nerve injury in mice. We previously reported that protein level of full-length TrkB, which contains the cytoplasmic protein tyrosine kinase domain, were clearly increased on the ipsilateral side of spinal cord membranes obtained from sciatic nerve-ligated mice. In the present study, we further demonstrated that the increased in the protein level of full-length TrkB is completely reversed by concomitant intrathecal injection of BDNF antibody. Furthermore, thermal hyperalgesia induced by nerve ligation was completely suppressed by repeated intrathecal injection of a specific antibody to full-length TrkB and an inhibitor of the protein tyrosine kinase activity for the neurotrophin receptor, K-252a. However, repeated intrathecal injection of a specific antibody to truncated TrkB, which lacks the cytoplasmic protein tyrosine kinase domain, failed to reverse thermal hyperalgesia observed in nerve-ligated mice. These findings suggest the possibility that the binding of BDNF to full-length TrkB and subsequent its activation may play a critical role in the development of neuropathic pain-like thermal hyperalgesia induced by nerve injury in mice.     

Distribution of brain-derived neurotrophic factor and TrkB receptor proteins in the fetal and postnatal hippocampus and cerebellum of the guinea pig

This study investigates the distribution of brain-derived neurotrophic factor protein (BDNF) and its receptor, TrkB, during the development of hippocampus and cerebellum in a long-gestation species, the guinea pig. In the granule cell populations of both structures, BDNF immunoreactivity (-IR) was exclusive to postmigratory, mature neurons. In dentate granule cells, TrkB-IR was coexpressed with BDNF-IR, suggesting that the ligand-receptor interaction could occur by means of an autocrine/paracrine mechanism. In cerebellar granule cells, TrkB-IR was detected in both pre- and postmigratory cells, indicating that immature neurons are also BDNF-responsive. With advancing gestational age an increase in the intensity of BDNF-IR in granule cells was accompanied by concomitant increases in the staining and areal growth of the associated mossy fiber layer in the hippocampus, and the molecular layer in the cerebellum. The developmental increase in BDNF- and TrkB-IR in the neuropil of both structures coincided with periods of significant growth in all strata, indicating a role for BDNF and TrkB in process outgrowth. In the hippocampus, CA2, CA3, and hilar, neurons demonstrated both BDNF- and TrkB-IR during development and maturation, whereas CA1 neurons showed TrkB-IR throughout this period but only transient BDNF-IR in early gestation. In the fetal cerebellum, Purkinje cell bodies coexpressed BDNF-IR and TrkB-IR. In the postnatal period, BDNF-IR was down-regulated but TrkB-IR persisted, indicating that mature Purkinje cells might retain their responsiveness to BDNF. Thus, we have demonstrated in both the hippocampus and cerebellum that the spatiotemporal distribution of BDNF-IR and TrkB-IR coincides with the maturation of granule cells prenatally and with significant periods of neuropil growth, both prenatally and in the immediate postnatal period.     

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons

Neurotrophins (NTs) play an important role in the modulation of synaptic transmission and in morphological changes in synaptic structures. Although there is agreement that brain-derived neurotrophic factor (BDNF) is sorted to large dense-core vesicles (LDCVs) and released via the regulated secretory pathway, there has been some dispute regarding the mode of secretion of nerve growth factor (NGF) and neurotrophin-3 (NT-3), two structurally related members of the NT family. In this study, we examined the subcellular localization and release characteristics of NGF, BDNF, and NT-3 in adenovirus-infected primary cortical neurons. We found that all members of the NT family colocalized with markers for the endoplasmic reticulum and Golgi within cell bodies and in a punctate manner with a marker for LDCVs within processes. Moreover, their release was triggered by depolarization, indicating that NGF, BDNF, and NT-3 are released via the regulated secretory pathway. When neurons were coinfected with two separate adenoviruses coding for NGF or BDNF, both NTs showed almost complete vesicular colocalization within single cells, suggesting that different NTs might be packaged into shared vesicles. We also examined whether the two splice variants of NGF, the short and long precursors, differ in their release characteristics. We found that neurons infected with viruses coding for either splice variant released NGF in a regulated way. Overall, our study supports the notion that all members of the NT family undergo activity-dependent regulated release from neurons, enabling them to act as "synaptotrophins" on electrically active neurons.     

Dose-dependent induction of mRNAs encoding brain-derived neurotrophic factor and heat-shock protein-72 after cortical spreading depression in the rat

Previous studies have demonstrated that cortical spreading depression (CSD) increases the expression of putative neuroprotective proteins. The objective of the present study was to elucidate the relationship between the number of episodes of CSD and steady-state levels of mRNAs encoding brain-derived neurotrophic factor (BDNF), heat-shock protein-72 (hsp72) and c-fos. Wistar rats were administered one, five, or twenty-five episodes of CSD evoked by application of 2 M KCl to the frontal cortex of one hemisphere. Animals were permitted to recover for 30 min, 2 h or 24 h prior to sacrifice. Total RNA was isolated from the parietal cortex of each hemisphere and analyzed using Northern blots. At 30 min recovery, levels of BDNF mRNA were not significantly elevated after 1 episode of CSD, but were increased 4-fold after five episodes of CSD and 11-fold after twenty-five episodes of CSD, relative to levels in the contralateral hemisphere. At 2 h recovery, BDNF mRNA levels increased 2-, 3- and 9-fold, respectively. At 24 h, BDNF mRNA had returned to control levels in all groups. Thus, CSD increased levels of BDNF mRNA in a dose-dependent fashion at the early recovery times. Hsp72 mRNA was below the level of detection after 1 and 5 episodes of CSD. However, after twenty-five episodes of CSD, hsp72 mRNA levels were increased in the ipsilateral hemisphere at 30 min and 2 h recovery. Unlike levels of BDNF and hsp72 mRNA, levels of c-fos mRNA were increased nearly to the same extent at 30 min and 2 h after one, five or twenty-five episodes of CSD before returning to control by 24 h recovery. These results demonstrate that CSD triggers a dose-dependent increase in the expression of genes encoding neuroprotective proteins, which may mediate tolerance to ischemia induced by CSD.     

The neurotrophic activities of brain-derived neurotrophic factor are potentiated by binding with apigenin,a common flavone in vegetables,in stimulating the receptor signaling

Aims

We aimed to identify the neurotrophic activities of apigenin (4′,5,7-trihydroxyflavone) via its coordination with brain-derived neurotrophic factor (BNDF) and an elevated signaling of tyrosine kinase receptor B (Trk B receptor).

Methods

The direct binding of apigenin to BDNF was validated by ultrafiltration and biacore assay. Neurogenesis, triggered by apigenin and/or BDNF, was determined in cultured SH-SY5Y cells and rat cortical neurons. The amyloid-beta (Aβ)_25-35-induced cellular stress was revealed by propidium iodide staining, mitochondrial membrane potential, bioenergetic analysis, and formation of reactive oxygen species levels. Activation of Trk B signaling was tested by western blotting.

Results

Apigenin and BDNF synergistically maintained the cell viability and promoted neurite outgrowth of cultured neurons. In addition, the BDNF-induced neurogenesis of cultured neurons was markedly potentiated by applied apigenin, including the induced expressions of neurofilaments, PSD-95 and synaptotagmin. Moreover, the synergy of apigenin and BDNF alleviated the (Aβ)_25-35-induced cytotoxicity and mitochondrial dysfunction. The synergy could be accounted by phosphorylation of Trk B receptor, and which was fully blocked by a Trk inhibitor K252a.

Conclusion

Apigenin potentiates the neurotrophic activities of BDNF through direct binding, which may serve as a possible treatment for its curative efficiency in neurodegenerative diseases and depression.     

Melanocortin-4 receptor activation stimulates hypothalamic brain-derived neurotrophic factor release to regulate food intake, body temperature and cardiovascular function

In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.     

Neurotrophin receptor TrkB activation is not required for the postnatal survival of retinal ganglion cells in vivo.

During early postnatal development, apoptosis of retinal ganglion cells (RGCs) is regulated by target contact with the optic tectum. The neurotrophins BDNF and NT-4, but not NGF, prevent the apoptosis of retinal ganglion cells that is otherwise observed after target ablation or axotomy. Thus receptors activated by BDNF and NT-4 are candidates to mediate the early postnatal survival of RGCs. BDNF and NT-4, but not NGF, bind to all isoforms of the receptor TrkB, whether or not they contain a tyrosine kinase domain. To examine the roles of TrkB receptor isoforms in early postnatal survival, we compared RGC numbers in wild-type mice to those in a mutant lacking all isoforms of TrkB. Surprisingly, no reduction in RGCs was observed in the mutant at postnatal day 16, the latest age at which these animals are consistently viable, so TrkB signaling is not essential for target-dependent survival of these cells. In wild-type mice, RGCs also are lost gradually during adulthood, possibly due to oxidative stress. To determine whether TrkB signaling regulates this phase of RGC degeneration, RGC numbers were examined in a viable mutant of TrkB that expresses only about 25% the normal level of TrkB receptor kinase. Compared to controls, approximately 20% of the RGC were lost in mutant 3-month-old-animals. Thus, TrkB signaling is not required for survival of RGCs during the period of target-dependent survival, but does appear to reduce degeneration of RGCs in adult animals.     

Transforming growth factor-beta1 enhances expression of brain-derived neurotrophic factor and its receptor, TrkB, in neurons cultured from rat cerebral cortex.

The effects of transforming growth factor (TGF)-beta1 on expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, in neurons cultured from the cerebral cortex of 18-day-old embryonic rats were examined. BDNF mRNA was significantly increased from 24-48 hr after the TGF-beta1 treatment over 20 ng/ml. Accumulation of BDNF protein in the culture medium was also potentiated by TGF-beta1, although the intracellular content of BDNF was nearly unchanged. The enhancement of BDNF mRNA expression was suppressed by the co-presence of decorin, a small TGF-beta-binding proteoglycan that inhibits the biological activities of TGF-betas. mRNA expression of full-length TrkB, the bioactive high-affinity receptor for BDNF, was also upregulated after treatment with TGF-beta1. These observations suggest that: 1) TGF-beta1 potentiates BDNF/TrkB autocrine or local paracrine system; and 2) the neurotrophic activity of TGF-beta1 is partly responsible for the BDNF induced by TGF-beta1 itself. To test this latter possibility, we examined the neuronal survival activity of TGF-beta1 with or without K252a, a selective inhibitor of Trk family tyrosine kinases. TGF-beta1 significantly enhanced neuronal survival, but the co-presence of K252a completely suppressed the activity, demonstrating the involvement of Trk receptor signaling in TGF-beta1-mediated neuronal survival in cultured rat cortical neurons. These results seem to be in line with recent findings by other investigators that some neurotrophic factors including BDNF require TGF-betas as a cofactor to exert their neurotrophic activities.     

Ghrelin-induced stimulation of colonic propulsion is dependent on hypothalamic neuropeptide Y1- and corticotrophin-releasing factor 1 receptor activation

Peptides participating in the hypothalamic control of feeding behaviour are also involved in the central autonomic control of gastrointestinal functions, such as secretion and motility. An anatomical interaction and functional relationship in the central nervous system between the feeding-related peptides neuropeptide Y and ghrelin is well documented. Furthermore, it has been shown that feeding-related peptides can influence digestive function via central corticotrophin-releasing factor (CRF) pathways. In the present study, we investigated the role of ghrelin in the central autonomic control of colonic motility. Furthermore, we addressed the hypothesis that ghrelin is involved in the hypothalamic control of colonic motor function, utilizing central neuropeptide Y receptors and hypothalamic CRF pathways. Ghrelin (0.03, 0.06 and 0.12 nmol) bilaterally microinjected into the paraventricular nucleus (PVN) induced a significant stimulation of colonic propulsion. In particular, the colonic transit time decreased from 312+/-7 min to 198+/-12 min. Microinjection of the neuropeptide Y1 receptor antagonist, BIBP-3226 (200 pmol), or the nonselective CRF receptor antagonist, astressin (30 pmol), into the PVN abolished the stimulatory effect of ghrelin injected into the PVN on colonic transit time, whereas pretreatment with the selective CRF2 receptor, antisauvagine-30 (28 pmol), failed to affect the effect of PVN-ghrelin injection on colonic propulsion. These results suggest that ghrelin can act as central modulator of gastrointestinal motor functions at the level of the PVN via neuropeptide Y1- and CRF1 receptor-dependent mechanisms.     

Ethanol exposure enhances cell death in the developing cerebral cortex: role of brain-derived neurotrophic factor and its signaling pathways

Exposure to ethanol during fetal development induces brain damage, causing cell loss in several brain areas and affecting synaptic connections. Because neurotrophin signaling plays an important role in neuronal survival and differentiation, we have investigated the effect of ethanol exposure on cell death in the developing cerebral cortex and whether this effect correlates with alterations in brain-derived neurotrophic factor (BDNF) levels, expression of its receptors, TrkB, and its signaling. We report that chronic ethanol intake during gestation and lactation enhances natural cell death and induces cell necrosis, decreases BDNF levels, and increases the ratio of the truncated to full-length TrkB mRNA receptors during postnatal developing cerebral cortex. Furthermore, we provide evidence that during brain development BDNF activates the extracellular signal-regulated kinases (ERK1 and ERK2) and the phosphoinoside-3-kinase (PI-3-K/Akt) pathways. However, BDNF-induced cell signaling throughout the above-mentioned survival pathways is significantly reduced by ethanol exposure. These findings suggest that ethanol-induced alterations in BDNF availability and in its receptor function might impair intracellular signaling pathways involved in cell survival, growth, and differentiation, leading to enhanced natural cell death during cerebral cortex development.