OBJECTIVE

The 2 prominent features of interstitial cystitis are pain and increased numbers of mast cells in the bladder. In this pilot study we determined the concentration of soluble mediators associated with activation of sensory neurons and/or mast cells that were present in the urine.

METHODS

The study groups included 4 interstitial cystitis patients, 7 kidney donors with no history of bladder disease as negative controls, 6 bladder cancer patients and 7 patients with urinary tract infection as reference controls. Urine samples were assayed for different soluble mediators using immunoassays for tryptase (a marker for mast cell activation), neurotrophic factors (markers of neuronal plasticity) and chemokines (markers of inflammatory cell activity). Results were normalized based on creatinine concentration.

RESULTS

There was a marked increase in the average amounts of tryptase and 3 neurotrophic factors in patient urine. Interestingly, the mediator profile in the urine of bladder cancer patients was indistinguishable from that of interstitial cystitis patients with respect to these same 4 proteins. There was no difference between normal control and urinary tract infection urine samples.

CONCLUSIONS

These findings may account for several clinical and pathological features found in interstitial cystitis and bladder cancer. Although preliminary due to the limited numbers of patients, they also suggest that increased levels of neurotrophin-3, nerve growth factor, glial cell line-derived neurotrophic factor and tryptase in the urine could serve as a basis for adjunct diagnosis, monitoring and treatment of interstitial cystitis.

The retrograde communication of neurotrophic signals from axon terminals to neuron cell bodies is crucial for neuron survival and plasticity. Several mechanisms have been proposed in the past, but recent evidence strongly supports the hypothesis that the retrograde propagation of self-regenerating signaling organelles, derived from the endocytosis of activated neurotrophin-bound receptor tyrosine kinases, is the primary mechanism responsible for this long-distance communication.

OBJECTIVE

To determine the chronology of optic nerve head and retinal responses to elevated intraocular pressure (IOP).

METHODS

After 1 to 39 days of unilaterally elevated IOP, experimental and fellow rat eyes were examined for morphology and immunohistochemical labeling alterations and for ganglion cell DNA fragmentation.

RESULTS

Mean IOP for the experimental eyes was 36 +/- 8 mm Hg, an approximately 15-mm Hg elevation above normal values. By 7 days of pressure elevation above 40 mm Hg, endogenous immunostaining for brain-derived neurotrophic factor and neurotrophin 4/5 was absent from the nerve head and superior retina, whereas normal labeling was present in the inferior retina and distal optic nerve of these same eyes. These changes were preceded by a loss of gap junctional connexin43 labeling and astrocytic proliferation in the nerve head and by increased retinal ganglion cell layer apoptosis in the retina. Nerve head depletion of neurotrophins coincided with evidence of axonal degeneration, loss of astrocytic glial fibrillary acidic protein staining, and spread of collagen VI vascular immunolabeling. After longer durations at these same pressures, neurotrophin labeling returned to nerve head glia and scattered retinal ganglion cells.

CONCLUSIONS

Optic nerve head and retinal responses, including the depletion of endogenous neurotrophins, are readily identified in the rat eye after experimental IOP elevation. However, the apparent chronology of these responses suggests that the withdrawal of neurotrophic support was not the only determinant of retinal ganglion cell apoptosis and axonal degeneration in response to pressure.

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor-1 (FGF-1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co-cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF-1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF-induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co-cultured motor neurons. FGF-1 also activates the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR-dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF-1 and t-butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA-NONOate to the co-cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO-dependent toxicity to motor neurons by increasing GSH biosynthesis.

Nerve growth factor (NGF) and its TrkA receptor exert important bioactivities on neuronal cells such as promoting survival and neurite outgrowth. Activated TrkA receptors are not only localized on the cell surface but also in signaling endosomes, and internalized TrkA receptors are important for the mediation of neurite outgrowth. The regulation of the endosomal trafficking of TrkA is so far unknown. Because the endosome-associated GTPase Rab7 coimmunoprecipitated with TrkA, we examined whether the endosomal trafficking of TrkA might be under the control of Rab7. Inhibiting Rab7 by expression of a green fluorescent protein-tagged, dominant-negative Rab7 variant resulted in endosomal accumulation of TrkA and pronounced enhancement of TrkA signaling in response to limited stimulations with NGF, such as increased activation of Erk1/2 (extracellular signal-regulated kinase 1/2), neurite outgrowth, and expression of GAP-43 (growth-associated protein 43). Our studies show that the endosomal GTPase Rab7 controls the endosomal trafficking and neurite outgrowth signaling of TrkA. Because mutations of Rab7 are found in patients suffering from hereditary polyneuropathies, dysfunction of Rab7 might contribute to neurodegenerative conditions by affecting the trafficking of neurotrophins. Moreover, strategies aimed at controlling Rab7 activity might be useful for the treatment of neurodegenerative diseases.

The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.

The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects on memory processes are mediated by alterations in the expression of the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus. First, studies are described investigating the effect of corticosterone on BDNF expression in the rat hippocampus. It appears that corticosterone suppresses the BDNF expression at the mRNA and protein level in a subfield-specific way. Second, a model for the mechanism of action is proposed. In this model, activated mineralocorticoid and glucocorticoid receptors repress transcriptional activity of the BDNF promoter site-specifically via interaction with other transcription factors. Third, the implications for learning and memory are discussed. Studies show that during water maze training, corticosterone levels rise significantly, but the BDNF expression is not suppressed in any hippocampal subfield. Furthermore, high BDNF expression levels in specific subfields correlate with a good memory performance. Therefore, we suggest that the resistance of the hippocampal BDNF expression to suppression by corticosterone, as seen after water maze training, may contribute to an optimal memory performance.

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.

The capacity of CNS neurons for axonal regrowth after injury decreases as the age of the animal at time of injury increases. After spinal cord lesions at birth, there is extensive regenerative growth into and beyond a transplant of fetal spinal cord tissue placed at the injury site. After injury in the adult, however, although host corticospinal and brainstem-spinal axons project into the transplant, their distribution is restricted to within 200 micron of the host/transplant border. The aim of this study was to determine if the administration of neurotrophic factors could increase the capacity of mature CNS neurons for regrowth after injury. Spinal cord hemisection lesions were made at cervical or thoracic levels in adult rats. Transplants of E14 fetal spinal cord tissue were placed into the lesion site. The following neurotrophic factors were administered at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary-derived neurotrophic factor (CNTF), or vehicle alone. After 1-2 months survival, neuroanatomical tracing and immunocytochemical methods were used to examine the growth of host axons within the transplants. The neurotrophin administration led to increases in the extent of serotonergic, noradrenergic, and corticospinal axonal ingrowth within the transplants. The influence of the administration of the neurotrophins on the growth of injured CNS axons was not a generalized effect of growth factors per se, since the administration of CNTF had no effect on the growth of any of the descending CNS axons tested. These results indicate that in addition to influencing the survival of developing CNS and PNS neurons, neurotrophic factors are able to exert a neurotropic influence on injured mature CNS neurons by increasing their axonal growth within a transplant.

In situ hybridization was used to study the site and timing of the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 5 (NT-5) mRNAs in the developing inner ear of the rat. In the sensory epithelia, the levels of NGF and NT-5 mRNAs were below the detection limit. NT-3 and BDNF mRNAs were expressed in the otic vesicle in overlapping but also in distinct regions. Later in development, NT-3 transcripts were localized to the differentiating sensory and supporting cells of the auditory organ and vestibular maculae. In these sensory epithelia, the intensity of NT-3 mRNA expression decreased in parallel with maturation. The expression of BDNF mRNA was restricted to the sensory cells of both the auditory and vestibular organs, including ampullary cristae. In bioassays, BDNF and NT-3, but not NGF, at physiological concentrations induced neurite outgrowth from the statoacoustic ganglion explants. These results demonstrate that NT-3 and BDNF, rather than NGF and NT-5, are the primary neurotrophins present in the target fields of the cochlear and vestibular neurons. Expression of NT-3 and BDNF mRNAs in the otic vesicle before and during the ingrowth of neurites from the statoacoustic ganglion suggests that NT-3 or BDNF or both may serve as chemoattractants for the early nerve fibers. The results also suggest that these neurotrophins have a role in later development of the cochlear and vestibular neurons.

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are important mediators of brain and neuronal development, the maintenance of homeostatic conditions in the adult nervous system, and the complex interplay of central and peripheral physiological and pathophysiological factors. To date there are few studies examining blood concentrations of neurotrophic factors in large samples of healthy and diseased individuals and no published study specifically addresses peripheral BDNF and NGF levels in late life. Using improved highly sensitive and specific fluorometric two-site enzyme-linked immunosorbent assays we examined BDNF (n=465) and NGF (n=175) serum levels in a large cohort of elderly individuals (age range: 70-103 years). Neither BDNF nor NGF serum levels proved to be normally distributed, indicating that previously published studies with small sample sizes using parametric testing may be misleading. A significant correlation was found between BDNF and platelet count (r=0.344, p<0.01), age and BDNF protein (r=-0.101, p=0.029) and BDNF and NGF serum levels (r=0.152, p=0.04). No other major influencing factors were found including gender, depression, and dementia.

Mutations of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a protein and lipid phosphatase, have been associated with gliomas, macrocephaly, and mental deficiencies. We have assessed PTEN's role in the nervous system and find that PTEN is expressed in mouse brain late in development, starting at approximately postnatal day 0. In adult brain, PTEN is preferentially expressed in neurons and is especially evident in Purkinje neurons, olfactory mitral neurons, and large pyramidal neurons. To analyze the function of PTEN in neuronal differentiation, we used two well established model systems-pheochromocytoma cells and cultured CNS stem cells. PTEN is expressed during neurotrophin-induced differentiation and is detected in both the nucleus and cytoplasm. Suppression of PTEN levels with antisense oligonucleotides does not block initiation of neuronal differentiation. Instead, PTEN antisense leads to death of the resulting, immature neurons, probably during neurite extension. In contrast, PTEN is not required for astrocytic differentiation. These observations indicate that PTEN acts at multiple sites in the cell, regulating the transition of differentiating neuroblasts to postmitotic neurons.

Hippocampal interneurons inhibit pyramidal neurons through the release of the neurotransmitter GABA. Given the importance of this inhibition for the proper functioning of the hippocampus, the development of inhibitory synapses must be tightly regulated. In this study, the possibility that neuronal activity and neurotrophins regulate the density of GABAergic inhibitory synapses was investigated in organotypic slice cultures taken from postnatal day 7 rats. In hippocampal slices cultured for 13 d in the presence of the GABA(A) receptor antagonist bicuculline, the density of glutamic acid decarboxylase (GAD) 65-immunoreactive terminals was increased in the CA1 area when compared with control slices. Treatment with the glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione decreased the density of GAD65-immunoreactive terminals in the stratum oriens of CA1. These treatments had parallel effects on the density of GABA-immunoreactive processes. Electron microscopic analysis after postembedding immunogold labeling with antibodies against GABA indicated that bicuculline treatment increased the density of inhibitory but not excitatory synapses. Application of exogenous BDNF partly mimicked the stimulatory effect of bicuculline on GAD65-immunoreactive terminals. Finally, antibodies against BDNF, but not antibodies against nerve growth factor, decrease the density of GAD65-immunoreactive terminals in bicuculline-treated slices. Thus, neuronal activity regulates the density of inhibitory synapses made by postnatal hippocampal interneurons, and BDNF could mediate part of this regulation. This regulation of the density of inhibitory synapses could represent a feedback mechanism aimed at maintaining an appropriate level of activity in the developing hippocampal networks.

Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, share substantial amino acid sequence identity. We have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene, neurotrophin-3, that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development.

In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

Neurotrophins influence growth and survival of specific populations of neurons through activation of Trks, members of the receptor tyrosine kinase (RTK) family. In this report, we describe the identification and characterization of two substrates of Trk kinases, rAPS and SH2-B, which are closely related Src homolog 2 (SH2) domain-containing signaling molecules. rAPS and SH2-B are substrates of TrkB and TrkC in cortical neurons and SH2-B is a substrate of TrkA in sympathetic neurons. Moreover, rAPS and SH2-B bind to Grb2, and both are sufficient to mediate NGF induction of Ras, MAP kinase (MAPK), and morphological differentiation of PC12 cells. Lastly, antibody perturbation and transient transfection experiments indicate that SH2-B, or a closely related molecule, is necessary for NGF-dependent signaling in neonatal sympathetic neurons. Together, these observations indicate that rAPS and SH2-B mediate Trk signaling in developing neurons.

Brain-derived neurotrophic factor (BDNF) contributes to the induction of long-term potentiation (LTP) by theta-pattern stimulation, but the specific processes underlying this effect are not known. Experiments described here, using BDNF concentrations that have minor effects on baseline responses, show that the neurotrophin both reduces the threshold for LTP induction and elevates the ceiling on maximal potentiation. The enhanced LTP proved to be as stable and resistant to reversal as that recorded under control conditions. BDNF markedly increased the facilitation of burst responses that occurs within a theta train. This suggests that the neurotrophin acts on long-lasting events that (1) are set in motion by the first burst in a train and (2) regulate the amplitude of subsequent bursts. Whole-cell recordings established that BDNF causes a rapid reduction in the size of the long-lasting afterhyperpolarization (AHP) that follows individual theta bursts. Apamin, an antagonist of type 2 small-conductance Ca2+-activated potassium (SK2) channels, also reduced hippocampal AHPs and closely reproduced the effects of BDNF on theta-burst responses and LTP. The latter results were replicated with a newly introduced, highly selective inhibitor of SK2 channels. Immunoblot analyses indicated that BDNF increases SK2 serine phosphorylation in hippocampal slices. These findings point to the conclusion that BDNF-driven protein kinase cascades serve to depress the SK2 component, and possibly other constituents, of the AHP. It is likely that this mechanism, acting with other factors, promotes the formation and increases the magnitude of LTP.

Epilepsy is the most common neurologic disorder in young humans. Antiepileptic drugs (AEDs), used to treat seizures in children, infants, and pregnant women, cause cognitive impairment, microcephaly, and birth defects by unknown mechanisms. We tested whether common AEDs cause neurodegeneration in the developing rat brain. Rats aged 3-30 days received phenytoin, phenobarbital, diazepam, clonazepam, vigabatrin, or valproic acid. Histologic examination of the brains revealed that these drugs cause widespread and dose-dependent apoptotic neurodegeneration in the developing rat brain during the brain growth spurt period. Apoptotic neurodegeneration was triggered at plasma drug levels relevant for seizure control in humans. Antiepileptic drugs lead to reduced expression of neurotrophins and decreased concentrations of the active forms of ERK1/2, RAF, and AKT. beta-Estradiol, which stimulates pathways that are activated by neurotrophins, ameliorated AEDs-induced apoptotic neurodegeneration. Our findings present one possible mechanism to explain cognitive impairment and reduced brain mass associated with pre- or postnatal exposure of humans to antiepileptic therapy.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family which interacts with high-affinity protein kinase receptors (Trk) and the unselective p75(NGFR) receptor. The BDNF gene has a complex structure with multiple regulatory elements and four promoters that are differentially expressed in central or peripheral tissue. BDNF expression is regulated by neuronal activity or peripheral hormones. Neurotrophins regulate the survival and differentiation of neurons during development but growing evidence indicates that they are also involved in several functions in adulthood, including plasticity processes. BDNF expression in the central nervous system (CNS) is modified by various kinds of brain insult (stress, ischemia, seizure activity, hypoglycemia, etc.) and alterations in its expression may contribute to some pathologies such as depression, epilepsy, Alzheimer's, and Parkinson's disease. Apart from very traumatic situations, the brain functioning is resilient to stress and capable of adaptive plasticity. Neurotrophins might act as plasticity mediators enhancing this trait which seems to be crucial in adaptive processes. In addition to documenting all of the topics mentioned above in the CNS, we review the state of the art concerning neurotrophins and their receptors, including our personal contribution which is essentially focused on the stress response.

One cardinal feature of Huntington's disease (HD) is the degeneration of striatal neurons, whose survival greatly depends on the binding of cortical brain-derived neurotrophic factor (BDNF) with high-affinity (TrkB) and low-affinity neurotrophin receptors [p75 pan-neurotrophin receptor (p75(NTR))]. With a few exceptions, results obtained in HD mouse models demonstrate a reduction in cortical BDNF mRNA and protein, although autopsy data from a limited number of human HD cortices are conflicting. These studies indicate the presence of defects in cortical BDNF gene transcription and transport to striatum. We provide new evidence indicating a significant reduction in BDNF mRNA and protein in the cortex of 20 HD subjects in comparison with 17 controls, which supports the hypothesis of impaired BDNF production in human HD cortex. Analyses of the BDNF isoforms show that transcription from BDNF promoter II and IV is down-regulated in human HD cortex from an early symptomatic stage. We also found that TrkB mRNA levels are reduced in caudate tissue but not in the cortex, whereas the mRNA levels of T-Shc (a truncated TrkB isoform) and p75(NTR) are increased in the caudate. This indicates that, in addition to the reduction in BDNF mRNA, there is also unbalanced neurotrophic receptor signaling in HD.

Distinct classes of primary sensory neurons in dorsal root ganglia subserve different sensory modalities, terminate in different dorsoventral locations in the spinal cord, and display different neurotrophin response profiles. Large diameter muscle afferents that terminate in the ventral spinal cord are NT-3 responsive, whereas small diameter afferents subserving pain and temperature are NGF responsive and terminate in the dorsal spinal cord. Previous in vitro studies showed that the developing ventral spinal cord secretes a diffusible factor that inhibits the growth of sensory axons. Here we show that this factor repels NGF-responsive axons but has little effect on NT-3-responsive axons. We also provide evidence implicating semaphorin III/collapsin, a diffusible guidance molecule expressed by ventral spinal cord cells, in mediating this effect. These results suggest that semaphorin III functions to pattern sensory projections by selectively repelling axons that normally terminate dorsally.

The mammalian neurotrophins (NTs) NGF, BDNF, NT-3, and NT-4 constitute a family of secreted neuronal growth factors. In addition, NTs are implicated in several forms of activity-dependent synaptic plasticity. Although synaptic secretion of NTs has been described, the intracellular signaling cascades that regulate synaptic secretion of NTs are far from being understood. Analysis of NT secretion at the subcellular level is thus required to resolve the role of presynaptic and postsynaptic NT secretion for synaptic plasticity. Here, we transfected cultures of dissociated rat hippocampal neurons with green fluorescent protein-tagged versions of BDNF and NT-3, respectively, and identified NT vesicles at glutamatergic synapses by colocalization with the cotransfected postsynaptic marker PSD-95 (postsynaptic density-95)-DsRed. Depolarization-induced secretion of BDNF and NT-3 was monitored with live cell imaging. Direct postsynaptic depolarization with elevated K+ in the presence of blockers of synaptic transmission allowed us to investigate the signaling cascades that are involved in the postsynaptic NT vesicle secretion process. We show that depolarization-induced postsynaptic NT secretion is elicited by Ca2+ influx, either via L-type voltage-gated calcium channels or via NMDA receptors. Subsequent release of Ca2+ from internal stores via ryanodine receptors is required for the secretion process. Postsynaptic NT secretion is inhibited in the presence of KN-62 ([4(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester) and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide), indicating a critical dependence on the activation of alpha-calcium-calmodulin-dependent protein kinase II (CaMKII). The cAMP/protein kinase A (PKA) signaling inhibitor Rp-cAMP-S impaired NT secretion, whereas elevation of intracellular cAMP levels was without effect. Using the Trk inhibitor k252a, we show that NT-induced NT secretion does not contribute to the NT release process at synapses, and BDNF does not induce its own secretion at postsynaptic sites. Release experiments in the presence of the fluorescence quencher bromphenol blue provide evidence for asynchronous and prolonged fusion pore opening of NT vesicles during secretion. Because fusion pore opening is fast compared with compound release, the speed of NT release seems to be limited by diffusion of NTs out of the vesicle. Together, our results reveal a strong dependence of activity-dependent postsynaptic NT secretion on Ca2+ influx, Ca2+ release from internal stores, activation of CaMKII, and intact PKA signaling, whereas Trk signaling and activation of Na+ channels is not required.

Emotionally important events are well remembered. Although memories of emotional experiences are known to be mediated and modulated by stress hormones such as glucocorticoids, little is known about the underlying molecular mechanisms. We found that the hippocampal glucocorticoid receptors that are critically engaged during the formation of long-term inhibitory avoidance memory in rats were coupled to the activation of CaMKIIα, TrkB, ERK, Akt, PLCγ and CREB, as well as a to a substantial induction of Arc and synaptic GluA1. Most of these changes, which are initiated by a nongenomic effect of glucocorticoid receptors, were also downstream of the activation of brain-derived neurotrophic factor (BDNF). Hippocampal administration of BDNF, but not of other neurotrophins, selectively rescued both the amnesia and the molecular impairments produced by glucocorticoid receptor inhibition. Thus, glucocorticoid receptors mediate long-term memory formation by recruiting the CaMKIIα-BDNF-CREB-dependent neural plasticity pathways.