Benedikt Berninger

cAMP-dependent growth cone guidance by netrin-1

Netrin-1 is known to function as a chemoattractant for several classes of developing axons and as a chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibited chemoattractive turning toward the source of netrin-1 but showed chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A. Both attractive and repulsive responses were abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP.

Phospholipase C-γ and Phosphoinositide 3-Kinase Mediate Cytoplasmic Signaling in Nerve Growth Cone Guidance

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.

GABAergic Stimulation Regulates the Phenotype of Hippocampal Interneurons through the Regulation of Brain-Derived Neurotrophic Factor

Gamma-Aminobutyric acid (GABA) switches from enhancing to repressing brain-derived neurotrophic factor (BDNF) mRNA synthesis during the maturation of hippocampal neurons in vitro. Interneurons do not produce BDNF themselves, but BDNF enhances their differentiation. Therefore, the question arose whether hippocampal interneurons regulate their phenotype by regulating BDNF expression and release from adjacent cells. The GABA(A) receptor agonist muscimol and BDNF increased the size and neuropeptide Y (NPY) immunoreactivity of hippocampal interneurons. However, GABAergic stimulation failed to increase NPY immunoreactivity in cultures from BDNF knockout embryos. At later developmental stages, when GABA represses BDNF synthesis, treatment with muscimol induced a decrease in cell size and NPY immunoreactivity of interneurons. Interneurons might thus control their phenotype through the regulation of BDNF synthesis in, and release from, their target neurons.

BDNF reduces miniature inhibitory postsynaptic currents by rapid downregulation of GABAA receptor surface expression

Changes in neurotransmitter receptor density at the synapse have been proposed as a mechanism underlying synaptic plasticity. Neurotrophic factors are known to influence synaptic strength rapidly. In the present study, we found that brain‐derived neurotrophic factor (BDNF) acts postsynaptically to reduce γ‐aminobutyric acid (GABA)‐ergic function. Using primary cultures of rat hippocampal neurons, we investigated the effects of BDNF on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and on the localization of GABAA receptors. Application of BDNF (100 ng/mL) led within minutes to a marked reduction (33.5%) of mIPSC amplitudes in 50% of neurons, recorded in the whole‐cell patch‐clamp mode, leaving frequency and decay kinetics unaffected. This effect was blocked by the protein kinase inhibitor K252a, which binds with high affinity to trkB receptors. Immunofluorescence staining with an antibody against trkB revealed that about 70% of cultured hippocampal pyramidal cells express trkB. In dual labelling experiments, use of neurobiotin injections to label the recorded cells revealed that all cells responsive to BDNF were immunopositive for trkB. Treatment of cultures with BDNF reduced the immunoreactivity for the GABAA receptor subunits‐α2, ‐β2,3 and ‐γ2 in the majority of neurons. This effect was detectable after 15 min and lasted at least 12 h. Neurotrophin‐4 (NT‐4), but not neurotrophin‐3 (NT‐3), also reduced GABAA receptor immunoreactivity, supporting the proposal that this effect is mediated by trkB. Altogether the results suggest that exposure to BDNF induces a rapid reduction in postsynaptic GABAA receptor number that is responsible for the decline in GABAergic mIPSC amplitudes.

Neurotrophins and activity-dependent plasticity of cortical interneurons

Neocortical and hippocampal GABA-containing interneurons are susceptible to activity-dependent modulation, such as regulation of soma size, numbers of synaptic contacts, and levels of GABA or neuropeptide expression. In vitro, the effects of neuronal activity on morphology and gene expression of GABA-containing neurons are mimicked, in part, by members of the neurotrophin gene family, such as brain-derived neurotrophic factor (BDNF). In the neocortex and hippocampus, BDNF is synthesized and secreted in an activity-dependent manner by pyramidal neurons, the target cells of GABA-containing neurons, suggesting that BDNF is an activity-dependent, target-derived trophic factor for these interneurons. In support of this, neuronal activity fails to upregulate the expression of neuropeptide Y in hippocampal cultures from BDNF-deficient mice. We, therefore, hypothesize that neurotrophins might mediate some of the actions of neuronal activity on GABA-containing neurons.

Fast actions of neurotrophic factors

A diversity of neurotrophic factors are required for the differentiation and survival of neurons and for maintaining their phenotype. By virtue of the rapid time scale of signal transduction in the cytosol, many of these factors also acutely regulate neuronal functions as diverse as synaptic transmission and nerve growth. These fast actions greatly expand the regulatory role of neurotrophic factors, particularly in the synaptic plasticity of developing nervous systems.

Postsynaptic target specificity of neurotrophin-induced presynaptic potentiation.

The role of the target cell in neurotrophin-induced modifications of glutamatergic synaptic transmission was examined in cultured hippocampal neurons. Brain-derived neurotrophic factor (BDNF) induced rapid and persistent potentiation of evoked glutamate release when the postsynaptic neuron was glutamatergic, or excitatory (E-->E), but not when it was GABAergic, or inhibitory (E-->1). This target-specific action of BDNF was also found at divergent outputs of a single presynaptic neuron innervating both glutamatergic and GABAergic neurons, suggesting that individual terminals can be independently modified. Surprisingly, BDNF increased the frequency of miniature postsynaptic currents at both E-->E and E-->I, although it had no effect on evoked currents at E-->I. Finally, potentiation by neurotrophin-3 (NT-3) was also target specific. The selective effect at E-->E suggests that retrograde signaling by the postsynaptic target cell endows a localized presynaptic action of neurotrophins.

Differential Regulation of Nerve Growth Factor (NGF) Synthesis in Neurons and Astrocytes by Glucocorticoid Hormones

Glucocorticoid hormones are important regulators of brain development and ageing. Here we show that dexamethasone, a synthetic glucocorticoid, differentially affects the expression of nerve growth factor (NGF) in cultured neurons and astrocytes. Dexamethasone increased the levels of NGF mRNA in cultured hippocampal neurons in a time‐ and concentration‐dependent manner, whereas it down‐regulated the NGF mRNA levels in astrocytes. However, dexamethasone had no effect on the mRNA levels of brain‐derived neurotrophic factor in the hippocampal neurons. Aldosterone, a mineralocorticoid, in higher concentrations also up‐regulated NGF mRNA levels in the hippocampal neurons. Dexamethasone increased the levels of NGF mRNA in the rat hippocampus in vivo, but not to the same extent as observed with kainic acid, a glutamate receptor agonist. There is no apparent diurnal rhythm in the hippocampal NGF protein levels corresponding to circadian variations in the levels of glucocorticoid hormones in serum. The increase in NGF mRNA in the hippocampus in vivo following dexamethasone treatments may reflect the physiological response of hippocampal neurons to high glucocorticoid levels reached under conditions of stress.

Regulation of brain-derived neurotrophic factor mRNA levels in hippocampus by neuronal activity.

Neuronal activity increases synthesis of brain-derived neurotrophic factor (BDNF) mRNA in vivo and in vitro. We have investigated the pathways through which neuronal activity stimulated by kainic acid regulates BDNF mRNA levels in cultured hippocampal neurons and transgenic mice. Kainic acid induced the transcription of BDNF mRNA without influencing the mRNA stability. Interestingly, the half-life of the 4.2 kb BDNF transcript was much shorter than that of the 1.6 kb transcript (23 +/- 4 min. vs. 132 +/- 30 min). Increase in the BDNF mRNA levels by kainic acid was not blocked by the protein synthesis inhibitor cycloheximide demonstrating that BDNF is regulated as an immediate early gene in hippocampal neurons. Although calmodulin antagonists are known to abolish the effect of kainic acid on BDNF mRNA, this effect was very similar in Ca(+2)-calmodulin-dependent protein kinase II alpha knock-out mice and in wild-type mice. Surprisingly, even high doses of kainic acid failed to increase nerve growth factor (NGF) mRNA in mouse hippocampus although elevation in rat brain has been consistently observed.

Neurotrophins as Mediators of Neuronal Plasticity

Publisher Summary Neurotrophic molecules — in particular, the members of the nerve growth factor (NGF) gene family — have so far predominantly been considered under the aspect of their function in regulating neuronal survival and differentiation of specific populations of neurons during embryonic development and in maintaining characteristic structural and functional properties of these neurons in adulthood. Apart from the trophic support of specific populations of neurons during embryonic development and in adulthood, there is increasing evidence that neurotrophins also play a role in synaptic plasticity. This can be deduced from the following observations — (1) the regionally differential rapid regulation of synthesis of NGF and brain-derived neurotrophic factor (BDNF) by neuronal activity in response to subtle physiological stimuli; (2) the activity-dependent release of neurotrophins from all neuronal processes demonstrated so far only for NGF. This, together with the enhancement of transmitter release by neurotrophins from nerve terminals, expressing the corresponding Trk receptors, is compatible with the hypothesis that the neurotrophins may stabilize and/or rearrange specific synapses in an activity-dependent manner. This interpretation is also supported by recent observations that long-term potentiation is strongly impaired in hippocampal slices of homo- and heterozygote BDNF knock-out mice.