Differential responses to the application of exogenous NT-3 are observed for subpopulations of motor and sensory neurons depending on the presence of skeletal muscle.

We examined the effects of a single injection of exogenous NT-3, administered at embryonic day (E) 13.5, on the survival of two populations of motor neurons and two populations of sensory neurons. Both wild-type and double knockout, Myf5-/-:MyoD-/-, mutant embryos were examined to determine the effects of the aforementioned neurotrophin on motor and sensory neuron survival in the presence and absence, respectively, of skeletal muscle. We found that, although NT-3 rescues select populations of motor neurons in the absence of muscles, there is a lack of increase in neuron survival when skeletal muscle is present. Additionally, NT-3 was found to rescue a select population of proprioceptive sensory neurons in the absence of target tissue, while, at times, exacerbating neuron cell death when target tissues are present. Lastly, we found that neurons in the spinal cord and brainstem show both a regional and functional specificity in their response to the administration of NT-3 in utero. Our results indicate the possibility that different pathways are involved in the survival of neurons during naturally occurring programmed cell death and during excessively occurring programmed cell death.     

Differential survival response of neurons to exogenous GDNF depends on the presence of skeletal muscle

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent survival factor for neurons in vitro and in vivo. The current study investigated the effects of a single in utero injection with GDNF in both wild-type and Myf5-/-:MyoD-/- embryos. The embryos in the latter group, denoted double mutants (DM), do not contain skeletal muscle and associated neurotrophic factors due to lack of myogenesis and, therefore, neurons of the central and peripheral nervous system undergo excessively occurring programmed cell death (EPCD). We found that treatment with GDNF had no effect on wild type neuronal numbers in any of the anatomic locations investigated. However, GDNF rescued the neurons of the facial motor nucleus, the mesencephalic nucleus and the median motor column in the absence of skeletal muscle. The findings of the current study agree with previous reports that compromised mouse neurons have increased survival response to GDNF.     

The G-protein inhibitor, pertussis toxin, inhibits the secretion of brain-derived neurotrophic factor

Secretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain-derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo-optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain-derived neurotrophic factor between embryonic days 13 and 17 (E13-17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42%, whereas injections at E19 had no effect. Co-injection of brain-derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly owing to diminished competition from endogenously released brain-derived neurotrophic factors for the receptors that mediate retrograde axonal transport.These data suggest that the release of a major fraction of brain-derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins.     

Presence of neurotrophic factors in skeletal muscle correlates with survival of spinal cord motor neurons.

To determine which combination of skeletal muscle-derived neurotrophic factors may be important for the survival of specific subpopulations of developing spinal cord motor neurons, we used Myf5 and MyoD (myogenic regulatory factors) knockouts, containing differentially committed myogenic precursor cells (MPCc) and immunohistochemistry against several muscle-secreted neurotrophic factors. At the peak of motor neuron cell death, skeletal muscle development is delayed in the back and body wall muscles of Myf5-/- embryos and in the limb muscles of MyoD-/- embryos. We hypothesized that, if the skeletal muscle was indeed an important source of survival factors for motor neurons, the back, the abdominal wall, and the forelimb MPCs of Myf5-/- or MyoD-/- embryos should produce at least some neurotrophic factors necessary for the survival of motor neurons. In this report, we demonstrate that (1) different MPCs lacking Myf5, MyoD, or Myf5/MyoD have different capabilities in providing factors potentially required for the survival of motor neurons and intramuscular nerve branching, (2) MPCs in double-mutant embryos do not contain neurotrophic factors in the absence of myogenic specification, and (3) different subpopulations of MPCs contain different combinations of neurotrophic factors potentially required for the survival of the specific subpopulations of innervating motor neurons.     

Transmission blockade during neuronal development

Summary Transmission blockade has been performed with - and -bungarotoxin and with d-tubocurarine in ten to eighteen-day-old chick embryos. Prevention of the naturally occurring nerve cell death in the trochlear nucleus after postsynaptic blockade is reported. Using stereological and biomathematical methods, it was possible to describe an absolute enlargement in volume of the trochlear nucleus despite a significant overall decrease in brain weight after -bungarotoxin and d-tubocurarine treatment. Nuclear and nucleolar diameters and the number of nucleoli per cell nucleus in trochlear neurons show time-dependent changes, but not differences between the controls and curare treated chick embryos. The ultrastructural appearance of trochlear neurons is not different between the controls and curare treated embryos after eight days of transmission blockade, indicating a similar maturation in these two groups. Only a somewhat higher frequency of dense bodies has been found after curare treatment. In contrast to these findings, the electron micrographs of -bungarotoxin treated chick embryos show severe degenerative signs in the trochlear nucleus and other motor brain regions very similar to the morphological changes after axonal lesions (retrograde axonal reaction). The quantitative Golgi-analysis of trochlear neurons demonstrates a significant decrease in dendritic branching after curare treatment. All neurons in the trochlear nucleus are labeled by retrograde transport of horseradish-peroxidase after injection into the upper extraocular muscles both in the controls and curare-treated animals. Therefore, we can assume that the motor neurons have maintained contacts with their peripheral target even after eight days of transmission blockade.Comparing biochemical and pharmacological data published in the literature with our present results, we conclude that an increased number of acetylcholine receptors in the peripheral target represents the cellular basis for the prevention of the naturally occurring neuronal cell death in neuromuscular systems. Therefore, the quantitative relations between neurons and post-synaptic receptor sites may be one of the important factors in the development and maintenance of neuromuscular systems.Supported by Deutsche Forschungsgemeinschaft grant Zi 192/3-4     

Reinnervation of the rat musculocutaneous nerve stump after its direct reconnection with the C5 spinal cord segment by the nerve graft following avulsion of the ventral spinal roots: a comparison of intrathecal administration of brain-derived neurotrophic factor and Cerebrolysin

Experimental model based on the C5 ventral root avulsion was used to evaluate the efficacy of brain-derived neurotrophic factor (BDNF) and Cerebrolysin treatment on motor neuron maintenance and survival resulted in the functional reinnervation of the nerve stump. In contrast to vehicle, BDNF treatment reduced the loss and atrophy of motor neurons and enhanced the regrowth axon sprouts into the distal stump of musculocutaneous nerve. However, the axon diameter of the myelinated fibers was smaller than those of control rats. The morphometric results were related to a low score in behavioral test similar to vehicle-treated rats. Cerebrolysin treatment greatly protected the motor neurons against cell death. Moreover, morphometric features of myelinated axons were better than those of rats treated with vehicle or BDNF. The mean score of grooming test suggested better results of the functional motor reinnervation than after BDNF administration. The majority of rescued motor neurons regenerating their axons through nerve graft in both BDNF- and Cerebrolysin-treated rats expressed choline acetyltransferase immunostaining. The results demonstrate that BDNF has more modest effects in preventing the death of motor neurons and functional recovery of injured motor nerve after root avulsion than Cerebrolysin.     

The use of the optical disector to estimate the total number of neurons in the developing chick lateral motor column: effects of purified growth factors.

Competition between neurons for limited amounts of trophic factors is believed to be the basis for large-scale neuronal death during the normal development of the vertebrate nervous system. In this study, an unbiased stereological counting method, an optical disector/Cavalieri combination, was used to estimate the total number of motor neurons in the lateral motor column of the developing chick and to assess the effects of four growth factors on neuronal numbers. The total number of neurons in lateral motor columns at embryonic day 6 (E6), E8, E10 and E12 were 18,747 +/- 1,369 (mean +/- SD), 15,037 +/- 1,816, 10,245 +/- 940, and 8,802 +/- 797, respectively. Daily exposure from E6 to E9 to three of the growth factors (basic fibroblast growth factor, bFGF; leukemia inhibitory factor, LIF; nerve growth factor, NGF) had no effect on total neuron number at E10. However, exposure to ciliary neurotrophic factor (CNTF) from E6 to E9 significantly increased (P less than 0.05) the number of neurons in the lateral motor column (13,610 +/- 725, compared with 10,058 +/- 204 in normal saline controls). These results are in agreement with previous reports of large scale neuronal death in the developing chick lumbar lateral motor column between E6 and E12 and confirm that exposure to growth factors such as CNTF can mitigate the course of normal ontogenetic cell death. The optical disector/Cavalieri combination is an efficient method for counting neurons: on average, following sectioning and staining, less than 30 min was required to estimate the total number of motor neurons in a lateral motor column with a coefficient of error of approximately 10%.     

Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly

Daily restraint for 3 weeks was shown to atrophy dendrites of hippocampal pyramidal neurons in rats. Brain-derived neurotrophic factor (BDNF), which maintains neuronal survival and morphology, has been shown to decrease in response to acute stress. Plasma glucocorticoid (GC) and serotonergic projections from the raphe nuclei play major roles in reducing BDNF synthesis in the hippocampus. We investigated BDNF mRNA levels there, together with plasma GC levels, GC receptors in the hippocampus/hypothalamus and 5-HT synthesizing enzyme, tryptophan hydroxylase in the raphe nuclei, in animals chronically stressed for 1-3 weeks, using in situ hybridization and immunohistochemistry. In these animals, BDNF mRNA levels were significantly decreased in the hippocampus after 6 h of restraint, but the ability of restraint to reduce BDNF synthesis seemed less robust than that seen in acute stress models. HPA axis response to stress in these animals assessed by plasma GC levels was delayed and sustained, and the GC receptor in the paraventricular hypothalamic nucleus was increased at 1 week. Tryptophan hydroxylase immunoreactivity was increased in the median raphe nucleus at 2-3 weeks. Repetitive stress-induced reduction of BDNF may partly contribute to the neuronal atrophy/death and reduction of hippocampal volume observed both in animals and humans suffering chronic stress and/or depression.     

Brain-derived and glial cell line-derived neurotrophic factors protect a catecholaminergic cell line from dopamine-induced cell death

Brain-derived neurotrophic factor (BDNF) promotes the survival of dopaminergic neurons in primary cultures and protects these neurons from the neurotoxic effects of 6-hydroxydopamine. The protective mechanism of BDNF on neurotoxicity was evaluated using CATH.a cells, a clonal catecholaminergic cell line derived from the central nervous system. Dopamine produced a dose-dependent cell death in CATH.a cells. Treatment of CATH.a cells with BDNF or glia cell line-derived neurotrophic factor (GDNF) reduced dopamine-induced cell death by approximately 60-70%. Nerve growth factor, basic fibroblast growth factor, neurotrophin-4/5 and insulin had no protective effect on dopamine-induced cell death. Dopamine decreased the activity of superoxide dismutase and the levels of glutathione in the CATH.a cells and these decreases were reversed by BDNF. In addition, BDNF treatment alone increased superoxide dismutase activity by 108%. These results suggest that BDNF may safeguard CATH.a cells from dopamine-induced cell death by maintaining or enhancing components of the cell, which protect from oxidative stress.     

脑源性神经营养因子在成年猴脑的分布

采用免疫组织化学方法探讨了 BDNF在成年猴脑的分布。结果显示 ,脑源性神经营养因子阳性反应产物主要分布于下列结构 :大脑皮层 III～ V层的锥体细胞及突起 ;小脑皮质篮状细胞、Purkinje细胞、Golgi细胞及小脑顶核的神经元及其突起 ;海马各区的神经元、纤维和齿状核的颗粒细胞 ;尾状核、豆状核和室旁核部分神经元和纤维 ;脑干脑桥核、舌下神经核、迷走神经背核、前庭神经核、下橄榄核及网状结构的神经元及纤维或膨体。此外 ,在大、小脑的白质也可见到部分脑源性神经营养因子阳性胶质细胞。脑源性神经营养因子阳性反应产物广泛地分布于猴脑的多种区域和细胞 ,提示其功能可能涉及不同类型的神经元及可能的非神经细胞。本研究结果为探讨脑源性神经营养因子在成年猴脑的分布规律及其功能特点提供了有用的形态学依据。     

Statistical coassembly of glycine receptor alpha1 wildtype and the hyperekplexia mutant alpha1(P250T) in HEK 293 cells: impaired channel function is not dominant in the recombinant system

Effects of brain-derived neurotrophic factor (BDNF) on cell migration from the ventricular zone to the cortical plate (CP) in developing mouse cerebral cortex were examined. BDNF (700 ng) was injected into the brain ventricle of 13- or 14-day-old embryos (E13 or E14) after the intraperitoneal administration of 5-bromodeoxyuridine (BrdU) to pregnant mice. BDNF injection at E13 increased the number of BrdU-positive cells migrated into the CP until E15, and caused them to become localized in much deeper layers (V-VI) than expected (IV-V, as in the vehicle-treated mice) by postnatal day 1. However, when the injections were made at E14, BrdU-positive cells predominantly migrated to layers II/III irrespective of BDNF administration. These results demonstrate that BDNF affects particular progenitors at limited stages, and suggest the presence of a Reelin-independent mechanism(s) to regulate cell migration.     

Nineteenth century research on naturally occurring cell death and related phenomena

Research on naturally occurring cell death is older than current opinion gives credit. More than 100 nineteenth century publications deal with it, and we review most of these. Soon after the establishment of the cell theory by Schleiden and Schwann, Carl Vogt (1842) reported cell death in the notochord and adjacent cartilage of metamorphic toads. Subsequent landmark discoveries included the massive cell death that occurs in pupating diptera (Weismann 1864), chondrocyte death during endochondral ossification (Stieda 1872), phagocytosis associated with cell death in the muscles of metamorphic toads (Metschnikoff 1883), chromatolytic (apoptotic) cell death in ovarian follicles (Flemming 1885), the reinterpretation of Sarkoplasten as Sarkolyten in metamorphic amphibia (Mayer 1886), the programmed loss of an entire population of neurons in fish embryos (Beard 1889), the death of scattered myocytes and myofibres in mammalian muscle (Felix 1889), and the death of many motor and sensory neurons in chick embryos (Collin 1906). Other lines of nineteenth century research established concepts important for understanding cell death, notably trophic interactions between neurons and their targets, and intercellular competition.     

The mesencephalic trigeminal nucleus of the duck: development and apoptosis

The normal development of the mesencephalic trigeminal nucleus (MesV) of the white Peking duck (Anas platyrhynchos) was studied from the 9th day of incubation until hatching and during adulthood. In the early days of embryonic development, neurons are present in the posterior commissure and in the mesenchymal tissue outside the leptomeninges in addition to those in the tectal commissure (TC) and in the optic tectum. Following the internucleosomal cleavage of DNA, a massive loss of neurons in the MesV starts in the 11-day embryo and continues until the 15th day of incubation. On the 16th day, the nucleus consists of a numerically larger medial division located in the TC and a smaller lateral division within the stratum griseum periventriculare as is found in the adult animal. The programmed cell death occurring in the MesV is discussed herein and correlated with the analogous apoptotic phenomena observed in the trigeminal motor nucleus.     

In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS

Apoptosis has been recognized to be an essential process during neural development. It is generally assumed that about half of the neurons produced during neurogenesis die before completion of the central nervous system (CNS) maturation, and this process affects nearly all classes of neurons. In this review, we discuss the experimental data in vivo on naturally occurring neuronal death in normal, transgenic and mutant animals, with special attention to the cerebellum as a study model. The emerging picture is that of a dual wave of apoptotic cell death affecting central neurons at different stages of their life. The first wave consists of an early neuronal death of proliferating precursors and young postmitotic neuroblasts, and appears to be closely linked to cell cycle regulation. The second wave affects postmitotic neurons at later stages, and is much better understood in functional terms, mainly on the basis of the neurotrophic concept in its broader definition. The molecular machinery of late apoptotic death of postmitotic neurons more commonly follows the mitochondrial pathway of intracellular signal transduction, but the death receptor pathway may also be involved.Undoubtedly, analysis of naturally occurring neuronal death (NOND) in vivo will offer a basis for parallel and future studies aiming to elucidate the mechanisms of pathologic neuronal loss occurring as the result of conditions such as neurodegenerative disorders, trauma or ischemia.     

Prosaptide exacerbates ischemia-induced behavioral deficits in vivo; an effect that does not involve mitogen-activated protein kinase activation

Prosaposin is a 517 amino acid membrane component and secreted protein(5,7,9) that is proteolytically cleaved to generate the four small glycoproteins; saposins A, B, C and D.(9,13,19) Prosaposin's ability to promote neurite outgrowth(31) and to protect neurons from programmed cell death(28) in vitro, as well as to rescue neurons from ischemia and other damage in vivo(11,12,15,25) implied that prosaposin was neurotrophic/neuroprotectant.(1,7,24,31) The neurotrophic sequence of prosaposin was isolated to smaller peptide fragments termed prosaptides(15,31) within the amino terminal portion of saposin C.(1,6,8,10,17,20,21,28) The proposed use of synthetic prosaptides as peripherally administered neuroprotective and/or neurotrophic therapeutic agents has stemmed from their ability to cross the blood-brain barrier,(27) as well as their reported neurotrophic activity in vitro.(15,23,31) Few studies, however, have attempted to characterize these peptides, presumably due to their reported instability following peripheral administration.(27) With the recent design of a stable 11-mer retro-inverso prosaptide,(15,31) it has become feasible to investigate the pharmacological effects of a stable version of these peptides in the validated rabbit spinal cord ischemia model that has been used extensively in the development of therapeutics to treat ischemic stroke.(4,14,16,18) Our results show not only that prosaptide was not neurotrophic/neuroprotectant in vivo, but rather it worsened ischemia-induced behavioral deficits.     

RNA interference-mediated inhibition of brain-derived neurotrophic factor expression increases cocaine's cytotoxicity in cultured cells

Previous studies showed that cocaine exposure decreased brain-derived neurotrophic factor (BDNF) function and resulted in neuronal cell death. To investigate a role of BDNF in cocaine's cytotoxicity, an RNA interference (RNAi) approach was used. Transfection of neuroblastoma SK-N-AS cells or primary rat hippocampal neurons with the small double-stranded interfering RNA (siRNA) targeting BDNF mRNA, but not the scrambled siRNA, resulted in reductions in levels of BDNF mRNA and proteins by more than 70% in the transfected cells as compared with the control group, suggesting an RNAi-mediated, sequence-specific gene silencing. The results also showed that cocaine-induced cytotoxicity, assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide) assay, was more pronounced in the cells transfected with the siRNA than in the cells transfected with the scrambled siRNA or in the cells treated with Lipofectamine 2000 alone (the control group), suggesting that inhibition of BDNF expression enhances cocaine's cytotoxicity. Together with previous studies showing that cocaine suppresses BDNF expression, the present data suggest that the drug-induced reduction of BDNF productions may make neurons more vulnerable to cocaine's toxic effects and precipitate cocaine-induced central nervous system damages.     

Low brain-derived neurotrophic factor (BDNF) levels in serum of Huntington's disease patients.

Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms and by a progressive degeneration of neurons in basal ganglia and in brain cortex. Brain-derived neurotrophic factor (BDNF) is a pro-survival factor for striatal neurons. Some evidence implicates a brain BDNF deficiency, related to mutated huntingtin expression, in the selective vulnerability of striatal neurons in HD. We compared BDNF serum levels in 42 patients with HD (range 28-72 years, mean age 51.9 +/- 11.5), and 42 age-matched healthy subjects (range 25-68 years, mean age 48.2 +/- 12.5). We evaluated the potential relationship between BDNF serum levels, CAG repeat number (range 40-54, mean 44.8 +/- 3.4) and duration of illness (range 6-228 months, mean 103.6 +/- 62.1). Serum BDNF levels were significantly lower in patients than in age-matched healthy subjects. Lower BDNF levels were associated with a longer CAG repeat length and a longer duration of illness. Severity of the illness, as assessed by the Unified Huntington's Disease Rating Scale (UHDRS) motor and cognitive scores, was negatively related to serum BDNF levels. These results in vivo confirm that the huntingtin mutation causes BDNF production to decline and show that the BDNF deficiency is detectable in HD patients' sera. Further studies on a larger sample size should confirm whether BDNF concentrations in patients' serum could be a useful clinical marker related to the patients' disease phenotype.     

Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3–5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.