Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor,Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.     

Human Recombinant Nerve Growth Factor Replaces Deficient Neurotrophic Support in the Diabetic Rat

Manipulation of neurotrophic support is a developing strategy for new therapy aimed at neurodegenerative diseases. This study demonstrates reduced content and retrograde transport of endogenous nerve growth factor (NGF) in sciatic nerve of diabetic rats. There were also reductions in the diabetic rats in NGF protein and mRNA in skin and muscle of the hindlimb. These deficits correlated with reductions in substance P and calcitonin gene-related peptide–both products of NGF-influenced genes in primary afferents. These manifestations of deficient neurotrophic support were corrected by intensive insulin treatment and surmounted by administration of exogenous human recombinant NGF in a dose-related manner. Impaired neurotrophic support may, therefore, participate in the pathogenesis of diabetic and other peripheral neuropathies.     

Regional Increases in Brain-Derived Neurotrophic Factor and Nerve Growth Factor mRNAs During Amygdaloid Kindling, But Not in Acidic and Basic Fibroblast Growth Factor mRNAs

Summary: We studied mRNA levels for neurotrophic factors using the amygdaloid kindling model of epilepsy. One hour after stage 5 kindled seizures, there were four-to fivefold increases in brain-derived neurotrophic factor (BDNF) mRNA in rat dentate gyrus and perirhinal cortex. Nerve growth factor (NGF) mRNA levels were increased bilaterally in some (but not all) limbic areas. There were no detectable changes in acidic fibroblast GF (aFGF) mRNA or basic fibroblast GF (bFGF) mRNA for 24 h after the kindled seizures. During kindling, levels of BDNF mRNA in the dentate gyrus correlated with projection to generalized seizures, whereas NGF mRNA in the limbic regions continued to increase during seizure development. These results indicate that the induction of mRNAs for neurotrophic factors, especially for BDNF mRNA in the dentate gyrus, corresponds to the increases in metabolic and electrical ictal discharge associated with kindled seizures. The persistent increase observed in NGF mRNA may be related to enhanced synaptic efficacy during kindling, but aFGF and bFGF are presumed to have little relation to the kindling process.     

Positive Feedback between Acetylcholine and the Neurotrophins Nerve Growth Factor and Brain-derived Neurotrophic Factor in the Rat Hippocampus

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.     

Cells that Express Brain-Derived Neurotrophic Factor mRNA in the Developing Postnatal Rat Brain

Brain-derived neurotrophic factor (BDNF) is a member of a family of related neurotrophic proteins which includes nerve growth factor (NGF) and hippocampus-derived neurotrophic factor/neurotrophin-3 (NT-3). To obtain information regarding possible roles for BDNF during postnatal brain development, we have examined the temporal and spatial expression of this trophic factor using in situ hybridization. In specific neocortical regions BDNF mRNA-expressing cells were seen at 2 weeks of age and thereafter. One particular neuronal cell type strikingly labelled was the inverted pyramidal cell population in the deep layers of parietotemporal cortex. In pyriform and cingulate cortices, BDNF mRNA was detected at postnatal day 1 and 1 week of age, respectively, with increasing levels during ontogeny. Several forebrain regions, including the thalamic anterior paraventricular nucleus, hypothalamic ventromedial nucleus as well as the preoptic area, contained moderate levels of BDNF mRNA throughout development. BDNF mRNA was detected transiently in several brainstem structures, notably in the substantia nigra and interpeduncular nucleus. Expression of this trophic factor in hippocampus was relatively low in the early neonatal brain, but attained high levels in the CA3 and CA4 regions as well as in the dentate gyrus by 2 weeks of age. At this early age, which is still during the period of neurogenesis in the dentate gyrus, labelling was restricted to the outer layer, which contained cells with a more mature appearance. However, by 3 weeks of age labelling was distributed throughout the granule cell layer. Our results show both transient and persistent expression of BDNF mRNA in various regions of the developing rat brain and suggest that there is a caudal to rostral gradient of BDNF expression during postnatal brain development, which may be correlated to neuronal maturation.     

Cerebellar Brain-Derived Neurotrophic Factor, Nerve Growth Factor, and Neurotrophin-3 Expression in Male and Female Rats Is Differentially Affected by Hypergravity Exposure During Discrete Developmental Periods

We previously reported that the effects of perinatal exposure to hypergravity on cerebellum and motor functions in rat neonates are strongly dependent on the specific developmental period of exposure. In the present study, we explored the hypothesis that neurodevelopmental changes are associated with altered expression of brain neurotrophins critical for normal brain growth and differentiation. We compared the effects of hypergravity exposure during four developmental periods: period I extended from gestational day (G) 8 through G15; period II from G15 to birth, period III from birth to postnatal day (P) 6; and period IV extended from G8–P12. For comparison we used stationary control (SC) neonates not exposed to hypergravity. Neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin 3 (NT-3) levels were measured in cerebellar homogenates prepared from postnatal day 12 male and female rat neonates using specific ELISAs. Hypergravity exposure affected individual neurotrophins differently and the effect was further determined by the period of hypergravity exposure. ANOVA showed: (1) a significant effect of the period of exposure to hypergravity on cerebellar BDNF (p = 0.009), with maximal decrease of 28.7% in males and 32.1% in females following exposure during period III; (2) a significant effect on NGF (p < 0.0001), with maximal decrease of 35.6% in male and 48.8% in female neonates following exposure during period III; (3) no statistically significant effect on NT-3 expression with a trend towards decreased expression in female rats following exposure during period IV. Although the molecular mechanisms underlying the differential neurotrophins’ response to hypergravity are not clear, an altered pattern of their expression is likely to contribute to neurodevelopmental changes and impaired sensorimotor behavior in exposed neonates.     

Effects of Donor Age and Brain-Derived Neurotrophic Factor on the Survival of Dopaminergic Neurons and Axonal Growth in Postnatal Rat Nigrostriatal Cocultures

Early postnatal rat brain tissue can be grown for several weeks as organotypic slice cultures by the roller-tube method. We have here used this method to study the effects of donor age and brain-derived neurotrophic factor (BDNF) on the survival and growth of tyrosine hydroxylase immunoreactive (TH-i), dopaminergic (DA) neurons during the postnatal period when their nerve fibers normally innervate the striatal target. Tissue slices of ventral mesencephalon (VM) and striatum were prepared from newborn and 7-day-old rats and cocultured for 3–3.5 weeks with different combinations of the two donor ages. After immunocytochemical staining the number of TH-i, ventral mesencephalic neurons were counted, and the growth of TH-i fibers into the striatal part of the cocultures was evaluated. Co-cultures, with both VM and striatal slices prepared from newborn rats, contained a significantly higher number of TH-i neurons and displayed a significantly increased innervation of the striatal slices compared with other combinations of donor ages. Addition of BDNF resulted in both an increased survival of TH-i neurons and an increased growth of TH-i fibers into the cocultured striatal slices. Significant neurotrophic effect of BDNF did, however, require young donor age of both VM and striatal slices. It is suggested that BDNF induces more cells, possibly progenitor cells, to express TH immunoreactivity. Alternatively BDNF may suppress apoptotic cell death documented by others to occur in the postnatal rat substantia nigra pars compacta. Irrespective of the mechanisms, survival of more TH-i neurons was related to an increased innervation of the striatal slices by TH-i nerve fibers. The observed effects of BDNF on both survival and fiber growth of TH-i neurons indicate a potential role of BDNF for treatment of Parkinson's disease or grafts of immature DA neurons transplanted to patients with Parkinson's disease. A significant trophic effect of BDNF did, however, seem to depend on young developmental age of both striatum and VM. Parallel treatment with striatal neurotrophic factors may therefore be a necessary prerequisite to a trophic effect of BDNF under clinical conditions.     

Brain-Derived Neurotrophic Factor Improves Long-Term Potentiation and Cognitive Functions after Transient Forebrain Ischemia in the Rat

We investigated the effect of brain-derived neurotrophic factor (BDNF) on hippocampal long-term potentiation (LTP) and cognitive functions after global cerebral ischemia in the rat. After four-vessel occlusion, BDNF was administered via an osmotic minipump continuously over 14 days intracerebroventricularly. Electrophysiological experiments were performed 14 days after cerebral ischemia. Test stimuli and tetanization were delivered to the Schaffer collaterals of the hippocampus and field excitatory postsynaptic potentials (fEPSP) were recorded in the CA1 region. Cognitive impairment was analyzed repeatedly with a passive avoidance test, a hole-board test, and with an activity center on the same animal. In sham-operated animals, LTP was consistantly induced after delivering a tetanus (increase of initial slope of fEPSP to 173 +/- 12% of baseline; n = 6). After transient forebrain ischemia LTP could not be induced (117 +/- 4% of baseline; n = 7). In ischemic animals treated with BDNF, LTP could be induced (168 +/- 28% of baseline; n = 8). Transient forebrain ischemia resulted in a significant decrease in spatial discrimination performance but not of associative memory. The ratios for working memory (WM) and reference memory (RM) 15 days after ischemia were lower in the ischemic rats (n = 10) than in the sham-operated control animals (n = 10; WM: 22 +/- 6 vs 72 +/- 7; RM: 30 +/- 7 vs 72 +/- 5). Postischemic intracerebroventricular BDNF infusion increased both WM (63 +/- 4; n = 10) and RM (58 +/- 5; n = 10). The spontaneous locomotor activity did not differ significantly in the three groups. These data indicate a protective effect of BDNF for synaptic transmission and cognitive functions after transient forebrain ischemia.     

Differential Effects of MK-801 on Brain-Derived Neurotrophic Factor mRNA Levels in Different Regions of the Rat Brain

We have studied the effects of MK-801, a noncompetitive antagonist of N -methyl- -aspartate-type glutamate receptors, on brain-derived neurotrophic factor (BDNF) mRNA levels in the rat brain. MK-801 decreased BDNF mRNA in the hippocampus and in the superficial layers of the cerebral cortex. However, in single cells of the middle layer of the cerebral cortex and the midline thalamic nuclei BDNF mRNA levels were markedly increased by MK-801. The highest density of these cells was found in the limbic cortex, especially in the retrosplenial and medial entorhinal cortex. Pentobarbital (an enhancer of gabaergic functions) and scopolamine (a muscarinic receptor antagonist) blocked the effects of MK-801 on BDNF mRNA levels in the retrosplenial cortex, but the nicotinic and dopaminergic receptor antagonists mecamylamine and haloperidol, respectively, were ineffective. Pilocarpine, a muscarinic cholinergic agonist increased BDNF mRNA in some, but not all, cortical areas, where MK-801 had elicited an increase in BDNF mRNA. Thus, the observations made with MK-801 demonstrate that depending on the neuronal connections and the transmitter systems involved, a given compound can elicit either a decrease or an increase in BDNF mRNA levels. This may open up pharmacological possibilities to a regionally more refined regulation of the neurotrophin synthesis.     

Critical Periods of Basic Fibroblast Growth Factor and Brain-Derived Neurotrophic Factor in the Development of the Chicken Cochleovestibular Ganglionin Vitro

The temporal roles of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the development of sensory neurons have been studied in a cell culture preparation which models normal embryonic inner ear development (normocytic). Previous studies showed that FGF-2 stimulated migration and differentiation of ganglion cells for the first 2 daysin vitro,but after 5 days led to degeneration, implicating other factors in their later development. To see if BDNF could be such a factor, otocysts were explanted from white leghorn embryos at the time when ganglion cell precursors normally start migrating from the otic epithelium. Cultures were grown in a defined medium, either with or without human recombinant FGF-2 for 2 days or with BDNF. On Day 3, FGF-2 was replaced either with BDNF in defined medium or with defined medium only. Measurements of neuroblast migration and neurite outgrowth were made by time-lapse imaging in living cultures. In cultures receiving BDNF on Day 3, cell migration and neurite outgrowth from the explant increased for more than 3 weeks but not in cultures receiving only defined medium from Day 3. Cultures did not survive more than 3–4 days when receiving either BDNF in defined medium or defined medium alone from the first day. A neutralizing antibody to BDNF inhibited neuronal migration and neurite outgrowth, and it also blocked the effects of exogenous BDNF. BDNF did not enhance the effects of FGF-2 by interacting with it. These experiments defined a temporal sequence in which FGF-2 acts early in development, while BDNF affects a later stage.     

Brain-Derived Neurotrophic Factor Levels in Autism: A Systematic Review and Meta-Analysis

Brain-derived neurotrophic factor (BDNF) plays an important role in activity-dependent synaptic plasticity. Altered blood BDNF levels have been frequently identified in people with autism spectrum disorders (ASD). There are however wide discrepancies in the evidence. Therefore, we performed the present systematic review and meta-analysis aimed at qualitative and quantitative synthesis of studies that measured blood BDNF levels in ASD and control subjects. Observational studies were identified through electronic database searching and also hand-searching of reference lists of relevant articles. A total of 183 papers were initially identified for review and eventually twenty studies were included in the meta-analysis. A meta-analysis of blood BDNF in 887 patients with ASD and 901 control subjects demonstrated significantly higher BDNF levels in ASD compared to controls with the SMD of 0.47 (95% CI 0.07–0.86, p?=?0.02). In addition subgroup meta-analyses were performed based on the BDNF specimen. The present meta-analysis study led to conclusion that BDNF might play role in autism initiation/ propagation and therefore it can be considered as a possible biomarker of ASD.     

The Role of Apolipoprotein E and Ethanol Exposure in Age-Related Changes in Choline Acetyltransferase and Brain-Derived Neurotrophic Factor Expression in the Mouse Hippocampus

Disruption of apolipoprotein E (APOE) is responsible for age-dependent neurodegeneration and cognitive impairment. Elderly individuals are more sensitive than young individuals to the effects of ethanol (EtOH), particularly those affecting cognition. We investigated the role of APOE deficiency and EtOH exposure on age-dependent alterations in choline acetyltransferase (ChAT) and brain-derived neurotrophic factor (BDNF) mRNA and protein expression in the mouse hippocampus. Three-month-old (young) and 12-month-old (aged) ApoE-knockout (ApoE-KO) and wild-type (WT) mice were treated with saline or 2 g/kg EtOH, and the bilateral hippocampus was collected after 60 min for real-time PCR and western blotting analyses. ChAT (P < 0.01) and BDNF (P < 0.01) expression were significantly decreased in both young and aged saline- and EtOH-treated ApoE-KO mice versus young and aged saline- and EtOH-treated WT mice. Aged saline- and EtOH-treated ApoE-KO mice exhibited greater differences in ChAT and BDNF expression (P < 0.01) than young saline- and EtOH-treated ApoE-KO mice. Aged EtOH-treated WT mice also exhibited larger decreases in BDNF expression (P < 0.01)—but not in ChAT expression—than young EtOH-treated WT mice. EtOH decreased ChAT and BDNF expression in both young (P < 0.01) and aged (P < 0.01) ApoE-KO mice versus EtOH-free ApoE-KO mice of the same age. EtOH also decreased BDNF expression in aged (P < 0.01) WT mice versus EtOH-free aged WT mice. In summary, these results suggest that APOE deficiency and EtOH exposure cause age-dependent decreases in ChAT and BDNF in the hippocampus. Importantly, the decreases in ChAT and BDNF were greater in aged EtOH-treated mice, particularly those lacking APOE, raising the possibility that APOE-deficient individuals who consume alcohol may be at greater risk of memory deficit.     

脑源性神经营养因子基因多态性与散发性帕金森病的相关性研究

目的探讨脑源性神经营养因子(Brain-DerivedNeurotrophicFactor,BDNF)基因多态性与散发性帕金森病(SporadicParkinson'sDisease,SPD)的相关性。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析方法对196例健康人和85例SPD患者进行基因分型;利用x2检验统计分析实验数据。结果两组间BDNF基因G196A和C270T多态性位点各基因型和等位基因频率差异均无显著性(P>0.05)。结论BDNF基因多态性与SPD无显著相关性。     

Perinatal Undernutrition Modifies Cell Proliferation and Brain-Derived Neurotrophic Factor Levels During Critical Time-Windows for Hypothalamic and Hippocampal Development in the Male Rat

Maternal perinatal undernutrition (MPU) modifies the activity of the hypothalamic‐pituitary‐adrenal axis and sensitises to the development of metabolic and cognitive adult diseases. Because the hypothalamus and hippocampus are involved in the regulation of neuroendocrine activity, energy metabolism and cognition, we hypothesised that a maternal 50% food restriction (FR50) from day 14 of pregnancy (E14) until postnatal day 21 (P21) would affect the development of these structures in male rat offspring. Protein and mRNA levels of brain‐derived neurotrophic factor (BDNF) and cell proliferation [analysed by 5‐bromodeoxyuridine (BrdU) incorporation] were compared in both control and FR50 rats from E21 to P22. Although the pattern of the evolution of BDNF concentration and cell proliferation throughout development was not strikingly different between groups, several disturbances at specific developmental stages were observed. FR50 rats exhibited a delayed increase of hippocampal BDNF content whereas, in the hypothalamus, BDNF level was augmented from E21 to P14 and associated, at this latter stage, with an increased mRNA expression of TRkB‐T2. In both groups, a correlation between BDNF content and the number of BrdU positive cells was noted in the dentate gyrus, whereas opposite variations were observed in CA1, CA2 and CA3 layers, and in the arcuate and ventromedial nuclei. In the hippocampus, P15‐FR50 rats showed an increased number of BrdU positive cells in all regions, whereas, at P22, a decrease was observed in the CA2. In the hypothalamus, between E21 and P8, MPU increases the number of BrdU positive cells in all regions analysed and, until P15, marked differences were noticed in the median eminence, the paraventricular nucleus and the arcuate nucleus. Taken together, the results obtained in the present study show that MPU changes the time course of production of BDNF and cell proliferation in specific hippocampal and hypothalamic areas during sensitive developmental windows, suggesting that these early perinatal modifications may have long‐lasting consequences.