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H Thoenen

Publications and source records attributed to H Thoenen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Localization of cellular storage compartments and sites of constitutive and activity-dependent release of nerve growth factor (NGF) in primary cultures of hippocampal neurons.

The characteristics of the constitutive and activity-mediated secretion of NGF from native hippocampal slices are the same as those from hippocampal cultures transfected with an NGF-overexpressing plasmid (Blöchl and Thoenen, 1995). In these cultures, the distribution of intracellular NGF immunoreactivity-including the co-localization with endoplasmic reticulum (ER) and Golgi markers-in soma, dendrites, and axons, as visualized by confocal microscopy, is compatible with a localization of NGF in an ER-like compartment. Since the positively charged NGF molecule is bound, at the sites of its release, to the negatively charged neuronal surface, at low salt buffer concentrations, it was possible to attribute the different release mechanisms to specific neuronal surface sites. Constitutive secretion of NGF is confined to the neuronal soma and the very proximal parts of dendrites. In contrast, the activity-dependent secretion, initiated by high potassium or glutamate also occurs all along the neuronal processes, in particular dendrites. This release is independent of extracellular calcium, but depends on calcium released from intracellular calcium stores and is mediated by sodium influx via voltagegated sodium channels and non-NMDA glutamate receptors. The confocal intensity analysis of the NGF surface staining permitted quantitative assessment of the different release mechanisms to different neuronal domains.

Animals↗
PubMed1996

Ciliary neurotrophic factor enhances the rate of oligodendrocyte generation.

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

Animals↗
PubMed1996

Cell density increases Bcl-2 and Bcl-x expression in addition to survival of cultured cerebellar granule neurons.

The proto-oncogene bcl-2 and its family members, bcl-x and bax are recognized as major regulators of cell death and survival. Although Bcl-2 and Bcl-x are expressed in brain, little is known how they are regulated in neurons. Here we have studied the expression of bcl-2, bcl-xL and bax mRNA in rat cerebellar granule neurons cultured under conditions which influence neuron survival. Insulin-like growth factor-1 and brain-derived neurotrophic factor supported the survival of these neurons, but affected neither the expression of bcl-2, bcl-xL nor bax mRNA. In contrast, bcl-2 and bcl-xL mRNAs were up-regulated in cerebellar granule neurons plated at high density exhibiting an increased neuronal survival. Western blots showed that cell density also increased Bcl-2 protein level. However, conditioned medium from dense cultures did not affect the level of bcl-2 mRNA nor survival of the neurons. This suggests that cell density promotes survival and regulates Bcl-2 expression in cerebellar granule neurons through a signaling pathway different from known neurotrophic factors.

Animals↗
PubMed1996

Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation.

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.

Afferent Pathways↗
PubMed1996

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.

Animals↗
PubMed1996

Inactivation of bcl-2 results in progressive degeneration of motoneurons, sympathetic and sensory neurons during early postnatal development.

Bcl-2 is a major regulator of programmed cell death, a critical process in shaping the developing nervous system. To assess whether Bcl-2 is involved in regulating neuronal survival and in mediating the neuroprotective action of neurotrophic factors, we generated Bcl-2-deficient mice. At birth, the number of facial motoneurons, sensory, and sympathetic neurons was not significantly changed, and axotomy-induced degeneration of facial motoneurons could still be prevented by brain-derived neurotrophic factor (BDNF) or ciliary neurotrophic factor (CNTF). Interestingly, substantial degeneration of motoneurons, sensory, and sympathetic neurons occurred after the physiological cell death period. Accordingly, Bcl-2 is not a permissive factor for the action of neurotrophic factors, and although it does not influence prenatal neuronal survival, it is crucial for the maintenance of specific populations of neurons during the early postnatal period.

Animals↗
PubMed1996

The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments.

Brain-derived neurotrophic factor (BDNF) is a member of the NGF gene family, which has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes like long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF-gene. We then used hippocampal slices from these mice to investigate whether LTP is affected by this mutation. Mutant mice showed significantly weaker LTP in the CA1 region. The magnitude of the potentiation as well as the percentage of cases in which LTP could be induced successfully was clearly reduced whereas important pharmacological and morphological control parameters in the hippocampus of these animals were unaffected. Adenoviral vectors were used to re-express BDNF in acute slices of BDNF-knock-out mice. In most cases LTP could be rescued with this approach. These results suggest that BDNF has an important functional role in the expression of LTP in the hippocampus.

Animals↗
PubMed1996

Autocrine-paracrine regulation of hippocampal neuron survival by IGF-1 and the neurotrophins BDNF, NT-3 and NT-4.

In contrast to sympathetic and sensory neurons in the peripheral nervous system, the neurotrophic requirements for neurons in the central nervous system (CNS) have not been clearly identified. The inactivation of specific neurotrophic factors and their receptors by gene targeting has shown that there are no major changes in neuron numbers in the CNS. This suggests an overlap between the action of different neurotrophic factors in the brain during development. Here we have studied the survival of hippocampal neurons prepared from embryonic rats using different culture conditions. Whereas the hippocampal neurons survive well in culture when plated at high density, they die at lower cell densities in the absence of appropriate neurotrophic factors. Under the latter conditions, both insulin-like growth factor-1 (IGF-1) and neurotrophins - brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) - rescued a large proportion of cultured neurons. In addition, hippocampal neurons from BDNF knockout mice exhibited enhanced cell death compared with cells from wild-type animals. BDNF and IGF-1 both increased the survival of the hippocampal neurons lacking BDNF, showing complementary action for these factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neuron survival at low cell densities, showing autocrine or paracrine action of the factors. At higher cell densities, however, the antibodies had no effect, demonstrating that there is a sufficient amount of endogenous factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neurons depend for survival on local neurotrophic factors such as IGF-1, BDNF and NT-3, which act in an autocrine/paracrine manner. The multifactorial support of hippocampal neurons ensures a maximal degree of neuron survival even in the absence of an individual factor

Animals↗
PubMed1996

Brain-derived neurotrophic factor reversed experience-dependent synaptic modifications in kitten visual cortex.

During a critical period of early postnatal development the functional architecture of the visual cortex is shaped by experience-dependent circuit selection following a Hebbian mechanism. One consequence is that monocular deprivation (MD) leads to competitive repression of the input from the deprived eye. Recently it has been proposed that this process might involve activity-dependent competition for neurotrophic substances because the synthesis of brain-derived neurotrophic factor (BDNF) is regulated by visual input. Here we investigate the effects of intracortical infusion of BDNF and nerve growth factor (NGF) on MD effects in the visual cortex. Neuronal responses were monitored with optical and single unit recording techniques in the visual cortex of kittens that had been infused intracortically either with BDNF, NGF or cytochrome C while subjected to MD for 1 week during the peak of the critical period. NGF or cytochrome C had no effect on the consequences of MD. After BDNF treatment, by contrast, ocular dominance (OD) shifted towards the deprived eye in a zone extending 2.5-3.5 mm from the infusion cannula, and neurons lost their orientation selectivity. At intermediate distances both eyes activated the cortex equally well and responses were again tuned for orientation; at still larger distances OD was shifted towards the normal eye. Thus, BDNF antagonizes the functional effects of MD and at high concentrations causes paradoxical disconnection of non-deprived afferents and a loss of orientation selectivity.

Animals↗
PubMed1995

Neurotrophins and neuronal plasticity.

There is increasing evidence that neurotrophins (NTs) are involved in processes of neuronal plasticity besides their well-established actions in regulating the survival, differentiation, and maintenance of functions of specific populations of neurons. Nerve growth factor, brain-derived neurotrophic factor, NT-4/5, and corresponding antibodies dramatically modify the development of the visual cortex. Although the neuronal elements involved have not yet been identified, complementary studies of other systems have demonstrated that NT synthesis is rapidly regulated by neuronal activity and that NTs are released in an activity-dependent manner from neuronal dendrites. These data, together with the observation that NTs enhance transmitter release from neurons that express the corresponding signal-transducing Trk receptors, suggest a role for NTs as selective retrograde messengers that regulate synaptic efficacy.

Animals↗
PubMed1995

Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo- and heterozygous mutant mice showed significantly reduced LTP in the CA1 region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.

Animals↗
PubMed1995

Expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function.

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT-3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT-4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT-3 mRNA essentially remained unchanged, whereas NT-4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT-4, whereas BDNF expression seems to play a role mainly after nerve lesion.

Animals↗
PubMed1995

TrkA tyrosine residues involved in NGF-induced neurite outgrowth of PC12 cells.

The proto-oncogene product gp140prototrk (TrkA) is the receptor tyrosine kinase that mediates nerve growth factor-induced neuronal survival and differentiation. In receptor tyrosine kinases, specific intracellular tyrosine residues become phosphorylated after ligand binding and the phosphorylated tyrosines induce the cascade of signal transduction. Here we have identified intracellular tyrosine residues of TrkA involved in nerve growth factor-induced neurite outgrowth of PC12 cells, using site-directed mutagenesis and a PC12 cell line expressing very low levels of endogenous TrkA (PC12nnr5 cells). We analysed eight conserved intracellular tyrosine residues of TrkA while the three putative autophosphorylation sites conferring tyrosine kinase activity were left intact. Five tyrosine residues, Y499, Y643, Y704, Y760 and Y794, in rat TrkA were involved in nerve growth factor-induced neurite outgrowth. None of these tyrosines mediated the full activity of wild-type TrkA, and a pair of these tyrosines, Y760 and Y794, promoted neurite outgrowth in an additive manner. These data indicate that no single tyrosine is sufficient to induce complete neurite outgrowth but the five tyrosine residues Y499, Y643, Y704, Y760 and Y794 cooperate to exhibit the full activity of wild-type TrkA.

Animals↗
PubMed1995

Characterization of nerve growth factor (NGF) release from hippocampal neurons: evidence for a constitutive and an unconventional sodium-dependent regulated pathway.

We investigated the mechanism of neuronal nerve growth factor (NGF) release with regard to the potential function of NGF as a mediator of neuronal plasticity in the CNS. The analysis was performed in hippocampal slices and in primary cultures of hippocampal neurons, transiently transfected with an NGF cDNA construct to increase the level of NGF expression. In both systems there was activity-dependent NGF release initiated by high potassium (KCl), veratridine, glutamate or carbachol. Replacement of 90% of sodium in the medium with N-methyl-glucamine strongly reduced this release. The KCl- and veratridine-initiated NGF release was suppressed by tetrodotoxin; release by glutamate was less sensitive to tetrodotoxin but was sodium-dependent. The glutamate effect could be inhibited by GYKI52644, an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, but not by MK-801, an antagonist of NMDA receptors. The activity-dependent release of NGF did not depend on extracellular Ca2+, but was sensitive to the intracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, and to depletion of intracellular calcium stores. Conversely, mobilization of Ca2+ from intracellular stores with caffeine and thapsigargin mimicked the effect of depolarization. Basal NGF release could be reduced by either temperature block (15 degrees C) or tetrodotoxin to approximately 50%. The combination of both treatments reduced NGF release to below the detection limit, suggesting that basal release has constitutive and regulated components, the latter presumably resulting from spontaneous activity of interconnected neurons.

Animals↗
PubMed1995

Brain-derived neurotrophic factor messenger RNA is expressed in the septum, hypothalamus and in adrenergic brain stem nuclei of adult rat brain and is increased by osmotic stimulation in the paraventricular nucleus.

We have detected scattered brain-derived neurotrophic factor mRNA-producing neurons in the medial septal nucleus, which contains cholinergic neurons that are responsive to brain-derived neurotrophic factor and nerve growth factor. In the brainstem, many adrenergic neurons showed a positive signal for brain-derived neurotrophic factor messenger RNA. Several hypothalamic nuclei contain brain-derived neurotrophic factor messenger RNA-positive neurons, among them paraventricular, median preoptic, vetromedial and dorsomedial nuclei. Osmotic stimulus, which activates vasopressin-producing neurons increased brain-derived neurotrophic factor messenger RNA levels in the paraventricular nucleus demonstrating that this factor is regulated by neuronal activity not only in the hippocampus and cortex but also in the hypothalamus.

Adrenergic Fibers↗
PubMed1995

Gene transfer through the blood-nerve barrier: NGF-engineered neuritogenic T lymphocytes attenuate experimental autoimmune neuritis.

Nerve-specific autoimmune T lymphocytes were used as vehicles to deliver therapeutically useful neurotrophic factors across the endothelial blood-nerve barrier. P2 protein-reactive T-lymphocyte lines from Lewis rats were transduced with a recombinant retrovirus containing the mouse nerve growth factor (NGF) gene. The engineered T cells released high amounts of NGF dependent on antigenic stimulation in vitro. After intravenous injection, the T cells infiltrated the rat peripheral nervous system and persisted there for at least two weeks. Local release of NGF from engineered T cells was demonstrable by immunocytochemistry and by an anti-inflammatory effect on infiltrating macrophages.

Animals↗
PubMed1995

Characterization of TrkB receptor-mediated signaling pathways in rat cerebellar granule neurons: involvement of protein kinase C in neuronal survival.

TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-gamma (PLC-gamma) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-gamma, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c-ras in a concentration-dependent manner. These results suggest that two different pathways, the c-ras and the PLC-gamma pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.

Animals↗