Biomedical subjects

H Thoenen

Publications and source records attributed to H Thoenen.

At least 19 recordsLinked to original sources

PubMed2000

Ultrastructural identification of storage compartments and localization of activity-dependent secretion of neurotrophin 6 in hippocampal neurons.

A modulatory role of neurotrophins (NTs) in activity-dependent neuronal plasticity by pre- and postsynaptic mechanisms is now well established. In this context, it is important to identify the storage compartments and to localize the precise site(s) and mechanism of NT secretion in order to deduce the spatial and temporal availability of NTs. We approached these questions at the ultrastructural level, exploiting the unique property of NT6 to bind tightly to heparan sulfate proteoglycans at the neuronal surface (R. Götz et al., 1994, Nature 372, 266-269), permitting the localization of secretion sites excluding diffusion artifacts. The myc tagging of NT6 permitted glutaraldehyde fixation and hence good preservation of the membrane structure, permitting immunogold labeling of NT6myc at the neuronal surface. NT6myc is preferentially secreted from neurites compared to neuronal cell bodies. In agreement with light-microscopic observations, the ultrastructural localization of NT6myc by postembedding procedures showed a predominant localization in ER-like membrane-confined compartments, partially associated with microtubules.

Amino Acid Sequence↗

PubMed1999

Neurotrophin-evoked rapid excitation through TrkB receptors.

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Action Potentials↗

PubMed1999

Retinal ganglion cell loss after the period of naturally occurring cell death in bcl-2-/- mice.

Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.

Aging↗

PubMed1998

Calbindin-D28k fails to protect hippocampal neurons against ischemia in spite of its cytoplasmic calcium buffering properties: evidence from calbindin-D28k knockout mice.

Cytoplasmic calcium-binding proteins are thought to shield neurons against damage induced by excessive Ca2+ elevations. Yet, in theory, a mobile cellular Ca2+ buffer could just as well promote neuronal injury by facilitating the rapid dispersion of Ca2+ throughout the cytoplasm. In sharp contrast to controls, in mice lacking the gene for calbindin-D28k, synaptic responses of hippocampal CA1 pyramidal neurons which are normally extremely vulnerable to ischemia, recovered significantly faster and more completely after a transient oxygen-glucose deprivation in vitro, and sustained less cellular damage following a 12 min carotid artery occlusion in vivo. Other cellular and synaptic properties such as the altered adaptation of action potential firing, and altered paired-pulse and frequency potentiation at affected synapses in calbindin-D28k-deficient mice were consistent with a missing intraneuronal Ca2+ buffer. Our findings provide direct experimental evidence against a neuroprotective role for calbindin-D28k.

Animals↗

PubMed1998

A role for BDNF in mechanosensation.

Brain-derived neurotrophic factor (BDNF) is a survival factor for certain sensory neurons during development. Using electrophysiology in BDNF-deficient mice, we show here that slowly adapting mechanoreceptors (SAM), but not other types of cutaneous afferents, require BDNF in postnatal life for normal mechanotransduction. Neurons lacking BDNF did not die, but instead showed a profound and specific reduction in their mechanical sensitivity, which was quantitatively the same in BDNF -/- and BDNF +/- animals. Postnatal treatment of BDNF +/- mice with recombinant BDNF completely rescued the mechanosensitivity deficit. Therefore BDNF is important for regulating SAM mechanosensitivity, independent of any survival-promoting function.

Adaptation, Physiological↗

PubMed1997

Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity.

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.

Animals↗

PubMed1997

Endogenous ciliary neurotrophic factor is a lesion factor for axotomized motoneurons in adult mice.

Ciliary neurotrophic factor (CNTF) is an abundant cytosolic molecule in myelinating Schwann cells of adult rodents. In newborn animals in which CNTF is not yet expressed, exogenous CNTF that is locally administered very effectively protects motoneurons from degeneration by axotomy. To evaluate whether endogenous CNTF, released after nerve injury from the cytosol of Schwann cells, supports motoneuron survival, we transected the facial nerve in 4-week-old pmn mice. In this mouse mutant a rapidly progressing degenerative disease of motoneurons starts by the third postnatal week at the hindlimbs and progresses to the anterior parts of the body, leading to death by the seventh to eighth week. Apoptotic death of motoneurons can be observed during this period, as revealed by TUNEL staining. In 6-week-old unlesioned pmn mice approximately 40% of facial motoneurons have degenerated. Facial nerve lesion dramatically increased the number of surviving motoneurons in pmn mice. This protective effect was absent in pmn mice lacking endogenous CNTF. Quantitative analysis of leukemia inhibitory factor (LIF) mRNA expression revealed that the dramatic upregulation seen in wild-type mice after peripheral nerve lesion did not occur in pmn mice. Therefore, endogenous LIF cannot compensate for the lack of CNTF in pmn crossbred with CNTF knock-out mice. Thus, endogenous CNTF released from lesioned Schwann cells supports the survival of axotomized motoneurons under conditions in which motoneurons are in the process of rapid degeneration.

Animals↗

PubMed1997

Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene.

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin) in vivo, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.

Animals↗

PubMed1997

Reduced size of retinal ganglion cell axons and hypomyelination in mice lacking brain-derived neurotrophic factor.

While brain-derived neurotrophic factor (BDNF) delays the death of axotomized retinal ganglion cells in rodents, it is unclear if it affects any aspect of the normal development of these cells. Here we examined the optic nerve of bdnf-/- mice. Axonal numbers were normal, but their diameter, as well as the proportion of myelinated axons, was reduced at postnatal day 20 (P20). In contrast, the facial nerve was not hypomyelinated. Expression levels of mRNAs coding for the myelin proteins PLP and MBP were substantially reduced in the hippocampus and cortex at P20, but not in the sciatic nerve. Intraventricular injections of BDNF into the ventricles of wild-type mice at P10 and P12 up-regulated expression of PLP in the hippocampus at P14. These results indicate a role of BDNF, discussed as indirect, in the control of myelination in the central nervous system.

Animals↗

PubMed1997

Post-occlusion treatment with BDNF reduces infarct size in a model of permanent occlusion of the middle cerebral artery in rat.

In view of the protective effect of brain derived neurotrophic factor (BDNF) against metabolic/excitotoxic insults in vitro, we investigated whether BDNF could limit infarct size after permanent occlusion of the middle cerebral artery in rat (MCAO). BDNF was delivered into the territory of the middle cerebral artery via an osmotic mini-pump (1 microg/h). Infusion of BDNF was started shortly after MCAO, and 24 h later brains were removed for infarct volume determination. Intraoperative and postoperative measurements of physiological variables revealed no differences among vehicle-treated and BDNF-infused animals. However, we found a 33% reduction in total infarct volume in BDNF-treated animals (p<0.05), and a 37% reduction in cortical infarct volume (p<0.05).

Animals↗

PubMed1997

Rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices using adenoviral vectors.

Primary cultures of hippocampal neurons were infected with an adenovirus coding for beta-galactosidase. Expression could be detected as early as 4 h after infection and steadily increased to high levels at 24 h without evidence for a functional impairment of the infected neurons. Similarly, adenovirus-mediated gene transfer into acute hippocampal slices was detectable 4 h after infection and could be localized to discrete areas of the CA1 region by microinjection of the virus stock solution. Infected slices were still suitable for electrophysiological experiments.

Adenoviridae↗

PubMed1997

Vulnerability of midbrain dopaminergic neurons in calbindin-D28k-deficient mice: lack of evidence for a neuroprotective role of endogenous calbindin in MPTP-treated and weaver mice.

Calbindin-D28k (calbindin) is an intracellular calcium binding protein of unknown in vivo function. It is abundantly expressed in many populations of neurons, and it can, presumably by buffering calcium overload, protect cells against excitotoxic damage. In the midbrain, calbindin is preferentially expressed in those dopamine neurons which are spared from degeneration in Parkinson's disease and its animal models. Whether calbindin itself determines neuronal vulnerability is questioned in other lesion models where calbindin expression is not positively correlated with neuronal resistance. To study the possible neuroprotective role of calbindin in vivo, we generated calbindin-deficient mice by gene targeting and assessed the viability of midbrain dopamine neurons in both a chemical and a genetic lesion paradigm. Tyrosine hydroxylase-immunoreactive neurons were counted in calbindin null-mutant mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in a calbindin-deficient weaver strain (homozygous for weaver and the calbindin null mutation). The extent and pattern of neuron loss observed in MPTP-treated wild-type and homozygous weaver mice were as previously described. Surprisingly, no significant differences were observed between MPTP-treated calbindin null mutants and their wild-type littermates, or between calbindin-weaver double mutant mice and weaver mice. Thus, in all four groups the same subpopulation of tyrosine hydroxylase-positive midbrain neurons (i.e. those normally containing calbindin) were preferentially spared. Calretinin, a closely related calcium-binding protein, which is also expressed in some midbrain dopamine neurons, was not up-regulated in these surviving neurons. These findings indicate that the resistance of calbindin-containing neurons in the MPTP and weaver models is not causally related to the expression of calbindin, and that endogenous calbindin is not required for protection of these neurons.

Animals↗

PubMed1996

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Adenoviridae↗

PubMed1996

Introduction of the negative selection marker into replacement vectors by a single ligation step.

Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.

Animals↗

PubMed1996

Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF.

BACKGROUND: The survival and differentiation of motoneurons during embryonic development, and the maintenance of their function in the postnatal phase, are regulated by a great variety of neurotrophic molecules which mediate their effects through different receptor systems. The multifactorial support of motoneurons represents a system of high security, because the inactivation of individual ligands has either no detectable, or relatively small, atrophic or degenerative effect on motoneurons. RESULTS: Leukaemia inhibitory factor (LIF) has been demonstrated to support motoneuron survival in vitro and in vivo under different experimental conditions. However, when LIF was inactivated by gene targeting, there were no apparent changes in the number and structure of motoneurons and no impairment of their function. The slowly appearing, relatively mild degenerating effects in motoneurons that resulted from ciliary neurotrophic factor (CNTF) gene targeting were substantially potentiated by simultaneous inactivation of the LIF gene, however. Thus, in mice deficient in LIF and CNTF, the degenerative changes in motoneurons were more extensive and appeared earlier. These changes were also functionally reflected by a marked reduction in grip strength. CONCLUSIONS: Degenerative disorders of the nervous system, in particular those of motoneurons, may be based on multifactorial inherited and/or acquired defects which individually do not result in degenerative disorders, but which become apparent when additional (cryptic) inherited disturbances or sub-threshold concentrations of noxious factors come into play. Accordingly, the inherited inactivation of the CNTF gene in a high proportion of the Japanese population may represent a predisposing factor for degenerative disorders of motoneurons.

Animals↗

PubMed1996

Brain-derived neurotrophic factor promotes the differentiation of various hippocampal nonpyramidal neurons, including Cajal-Retzius cells, in organotypic slice cultures.

Brain-derived neurotrophic factor (BDNF) is widely expressed in the central nervous system, where its function is poorly understood. The aim of this study was to investigate the effects of BDNF on the differentiation of hippocampal nonpyramidal neurons using organotypic slice cultures prepared from postnatal rats. The application of BDNF induced an increase in immunostaining for the microtubule-associated protein (MAP)-2 in non-pyramidal neurons of the stratum oriens. BDNF promotes the elongation of the dendrites of these neurons, as demonstrated by analysis after biocytin labeling. Calbindin-D- and calretinin-containing subgroups of nonpyramidal cells in the stratum oriens were responsive to BDNF but not to nerve growth factor, as shown by an increase in the number of neurons immunostained for these proteins. BDNF also induced an increase in neuropeptide Y immunostaining of stratum oriens neurons. In contrast, BDNF had no effect on parvalbumin immunostaining, despite the fact that these cells express the BDNF receptor trkB. In addition, BDNF increased calretinin immunoreactivity in Cajal-Retzius cells situated around the hippocampal fissure. The Cajal-Retzius neurons persisted in slices beyond the time at which they degenerate in vivo. However, BDNF is not required for the survival of these cells, because they also persisted in slices from BDNF knock-out mice. The present results indicate that BDNF exerts an effect on the morphology of stratum oriens nonpyramidal cells and their calcium-binding protein levels. BDNF also regulates the calretinin content of Cajal-Retzius cells but is not necessary for their survival.

Animals↗

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↗