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.     

Regulation of Ciliary Neurotrophic Factor in Cultured Rat Hippocampal Astrocytes

Ciliary neurotrophic factor (CNTF) is a pleiotrophic cytokine which is detectable only at very low levels in the intact adult rat CNS, but following an aspirative lesion to the dorsal hippocampus and overlying cortex, CNTF mRNA levels are dramatically up-regulated in reactive astrocytes. In cultured rat hippocampal astrocytes, CNTF mRNA levels are high, similar to the levels in reactive astrocytes in vivo , but are strongly suppressed after administration of isoproterenol and forskolin, which stimulate the production of intracellular cyclic AMP, induce marked morphological change in the astrocytes and up-regulate glial fibrillary acidic protein mRNA and nerve growth factor mRNA in these cells. Following a single administration of forskolin to cultured astrocytes, suppression of CNTF mRNA was sustained for up to 7 days. A similar down-regulation was observed with the endogenous adrenergic agonists noradrenaline and adrenaline as well as, to a lesser extent, dopamine and adenosine. Down-regulation of CNTF mRNA resulted in a gradual reduction in the level of CNTF protein within the astrocytes. A single addition of forskolin or isoproterenol resulted in a drop in CNTF protein levels to 29 and 52% of control levels respectively after 9 days in vitro , although the rate of turnover of CNTF remained the same. Down-regulation of CNTF mRNA in cultured hippocampal astrocytes by adenylyl cyclase activation was quite specific, as a wide range of growth factors, cytokines and neurotransmitters had little or no effect upon CNTF mRNA levels.     

Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg²⁺ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation - spontaneous and agonist induced.     

Epidermal Growth Factor Prevents Oxygen-triggered Apoptosis and Induces Sustained Signalling in Cultured Rat Cerebral Cortical Neurons

Epidermal growth factor (EGF), a conventional mitogenic factor, acts as a neurotrophic factor on several types of neurons in the central nervous system. We found that EGF prevented the death of rat cerebral cortical neurons cultured in a 50% oxygen atmosphere. This high-oxygen-triggered cell death showed features of apoptotic cell death, which was blocked by inhibitors of RNA or protein synthesis. EGF prevented the oxygen-induced death of the cultured cortical neurons in a dose-dependent manner. Basic fibroblast growth factor (bFGF) also prevented this cell death, although there was no apparent additive effect of EGF and bFGF. Among the cultured cortical neurons, we observed neurons possessing the EGF receptor and cells expressing c-Fos protein in response to EGF. The cortical neurons were cultured in the presence of cytosine arabinoside, and the number of glial fibrillary acidic protein-positive astroglial cells was >0.5% of that of the corresponding microtubule-associated protein 2-positive neurons. Therefore, the effect of EGF on the cultured cortical neurons is thought to be due to a direct action. We also examined EGF-induced signalling in the cultured cortical neurons. We found that EGF induced the sustained tyrosine phosphorylation of the EGF receptor and sustained the activation of mitogen-activated protein kinase in the cultured cortical neurons. We suggest that EGF may exert the survival effect through the prolonged activation of the EGF signalling.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

Muscarinic Modulation of Intrinsic Burst Firing in Rat Hippocampal Neurons

Intracellular recordings in rat hippocampal slices were used to examine how exogenous and endogenous cholinergic agonists modulate the firing pattern of intrinsically burst-firing pyramidal cells. About 24% of CA1 pyramidal cells generated all-or-none, high-frequency bursts of fast action potentials in response to intracellular injection of long positive current pulses. Application of carbachol (5 μM) converted burst firing in these neurons into regular trains of independent spikes. Acetylcholine (5 μM) exerted a similar effect, provided acetylcholine esterase activity was blocked with neostigmine (2 μM). Atropine (1 μM) reversed this cholinergic effect, indicating its mediation by muscarinic receptors. Cholinergic agonists also caused mild neuronal depolarization but the block of intrinsic burst firing was independent of this effect. Repetitive stimulation of cholinergic fibres in the presence of neostigmine (2 μM) evoked a slow cholinergic excitatory postsynaptic potential (EPSP) lasting about a minute. During the slow EPSP, burst firing could not be evoked by depolarizing pulses and the neurons fired in regular mode. These effects were prevented by pretreatment with atropine (1 μM). Exogenously applied cholinergic agonists and endogenously released acetylcholine also reduced spike frequency accommodation and suppressed the long-duration afterhyperpolarization in burst-firing pyramidal cells in an atropine-sensitive manner. A membrane-permeable cAMP analogue (8-bromo-cAMP; 1 mM) also reduced frequency accommodation and blocked the long-duration afterhyperpolarization, but did not affect intrinsic burst firing at all. Taken together, the data show that muscarinic receptor stimulation transforms the stereotyped, phasic response of burst-firing neurons into stimulus-graded, tonic discharge.     

Effect of Acute Soman Exposure on GABAA Receptors in Rat Hippocampal Slices and Cultured Hippocampal Neurons

Exposure of the central nervous system to organophosphorus (OP) nerve agents causes seizures and neuronal cell death. Benzodiazepines are commonly used to treat seizures induced by OPs. However, it is known that soman-induced seizures are particularly resistant to benzodiazepine treatment, as compared with other OPs. This study investigated the effect of soman on γ-aminobutyric acid (GABA) neurotransmission in acute rat hippocampal slices and the surface expression of GABA(A) receptors in cultured rat hippocampal neurons. Results showed that GABA-mediated inhibitory post synaptic currents (IPSCs) are significantly reduced by soman in a concentration-dependent manner in acute rat hippocampal slices. Furthermore, confocal microscopic and cell-based ELISA assays revealed that soman caused rapid internalization of GABA(A) receptors in cultured rat hippocampal neurons. The effect of soman on GABA(A)R endocytosis was not due to inhibition of acetylcholinesterase (AChE) because (1) the acetylcholine muscarinic receptor antagonist atropine did not block soman-induced GABA(A)R endocytosis; and (2) physostigmine, at concentrations that completely inhibit AChE activity, did not cause GABA(A)R endocytosis. Moreover, blocking of the N-methyl-D-aspartate (NMDA) receptors by 2-amino-5-phosphonovalerate (APV) had no effect on soman-induced GABA(A)R endocytosis, suggesting that the soman effect was not secondary to glutamate receptor over activation. Regardless of the exact mechanism, the observation that soman induces rapid GABA(A)R endocytosis may have significant implications in the development of effective countermeasures against soman-induced seizures.     

G-Protein Modulation of Glycine-resistant NMDA Receptor Desensitization in Rat Cultured Hippocampal Neurons

Activation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors increases the excitability of most neurons within the CNS. A common feature of ionotropic glutamate receptors is their ability to undergo desensitization. In the present experiments we have examined the role of guanine nucleotide-binding proteins(G-proteins) in the regulation of NMDA receptor desensitization. Repeated NMDA receptor activation with 2 mM extracellular Ca²+ increased the degree of glycine-resistant NMDA receptor desensitization of subsequent responses to NMDA recorded in the presence of 0.2 mM Ca²+. The recovery of glycine-resistant NMDA receptor desensitization after repeated NMDA receptor activation in the presence of 2 mM Ca²+ was significantly reduced in neurons intracellularly dialysed with guanosine-5'-0-(3-thiotriphosphate), guanosine-5'-triphosphate or AIC1₃ and CsF, compounds known to activate G-proteins. lntracellular dialysis with guanosine-5'-0(2thiodiphosphate), adenosine triphosphate, or adenosine-5'-G(3-thiotriphosphate) was ineffective. The calcium permeability of NMDA receptor-channels was not altered by intracellular dialysis with GTPyS. This suggests that modulation of NMDA receptor desensitization by G-proteins represents a novel mechanism forregulation of glutamate-gated ion channel activity.     

低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响

目的 观察低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响。方法 取培养12d的两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12h,分别观察它们的形态变化和神经元存活数,并用抗rhIL-6单克隆抗体进行免疫组化染色,观察缺氧对大鼠海马培养神经元IL-6免疫反应的影响。结果 低氧预处理可增强海马神经元对rhIL-6的免疫反应,经低氧预处理的海马神经元缺氧后神经元存活数和对rhIL-6的免疫反应均明显高于对照组。结论 低氧预处理氧预处理的海马神经元缺氧后神经元存活数和对thIL-6的免疫反应均明显高于对照组。结论 低氧预处理可使体外培养的海马神经元对缺氧产生耐受,其中rhIL-6的免疫反应增加可能是海马神经元对缺氧的一种适应性变化,提示IL-6可能参与脑缺氧耐受性的形成,并在海马神经元缺氧损伤的调控中起重要作用。     

Widespread and Developmentally Regulated Expression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues

The neurotrophin gene family includes four structurally related proteins with neurotrophic activities. Two of them, nerve growth factor and brain-derived neurotrophic factor (BDNF), have been studied in detail and information has recently emerged on the expression and function of the third member, neurotrophin-3. In contrast, little information is available on neurotrophin-4 (NT-4), the most recently isolated member of this family. In this report we have used a sensitive RNAase protection assay to analyse the developmental expression of NT-4 mRNA in the rat brain and in 12 different rat peripheral organs. In heart, liver and muscle plus skin NT-4 mRNA levels were maximal at embryonic day (E) E13 (the earliest time point tested), with reduced levels at later times of development. In lung, kidney and thymus similar levels were seen from E13 to postnatal day (P) 1, with reduced levels in the adult. In testis, ovary and salivary gland NT-4 mRNA was detected at E16 with a peak shortly after birth. During brain development, NT-4 mRNA was maximal at E13 followed by a decrease around birth, after which the level increased. The postnatal increase of NT-4 mRNA was also seen in cerebral cortex and brain stem analysed separately, while in the hippocampus similar levels were found from P1 to adulthood. NT-4 mRNA was detected in all ten adult rat brain regions analysed with only small regional variations, being highest in pons–medulla, hypothalamus, thalamus and cerebellum. Adult rat thymus, thyroid, muscle, lung and ovary contained higher levels of NT-4 mRNA than brain, while all other adult tissues showed similar or lower levels than in the brain. The highest level of NT-4 mRNA overall was found in P1 testis. For comparison, BDNF mRNA was analysed in the same tissues. The expression of BDNF mRNA was in many cases different from that of NT-4 mRNA. The peak of NT-4 mRNA expression in several of the peripheral tissues coincided with the peak of naturally occurring neuronal cell death in peripheral ganglia. This is consistent with the possibility that NT-4 acts as a target-derived trophic factor in vivo. The widespread and increased expression of NT-4 mRNA during postnatal brain development could reflect a trophic role of NT-4 for central nervous system neurons. However, non-neuronal functions of NT-4 are also possible, particularly in male and female reproductive tissues, where the NT-4 protein could function as a growth factor for immature germ cells.     

Differential Regulation of Preprotachykinin-A mRNA Expression in Striatum by Excitation of Hippocampal Neurons

In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innvervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.     

Distinct Effects of Clostridial Toxins on Activity-dependent Modulation of Autaptic Responses in Cultured Hippocampal Neurons

Clostridial neurotoxins proteolyse specific proteins implicated in synaptic vesicle exocytosis, but their actions on the release machinery in functional synapses is not well understood. Here we examine the effects of botulinum toxin A (BoNT/A) and tetanus toxin (TeTx) on autaptic transmission in cultured rat hippocampal neurons using whole-cell voltage clamp recordings. The proportion of cells responding to stimulation with an excitatory postsynaptic current (EPSC) and the magnitude of the remaining responses decreased gradually with increasing concentration of either toxin. However, the activity-dependent modulation (5 Hz repetitive stimulation) of EPSCs remaining after toxin inhibition differed markedly between the two toxins. The TeTx inhibition was associated with a persistent activity-dependent depression similar to that in control cells. In contrast, the BoNT/A inhibition was accompanied by a reversal of the modulation into facilitation, resembling that induced by lowering of the calcium concentration. These results demonstrate a difference between BoNT/A and TeTx in their mode of inhibition of synaptic vesicle exocytosis, which suggests that they exert their preferential actions at distinct steps of the release process.     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Survival-promoting and Protein Kinase C-regulating Roles of Basic FGF for Hippocampal Neurons Exposed to Phorbol Ester, Glutamate and Ischaemia-like Conditions

Recent evidence suggests that protein kinase C (PKC) is involved in the pathophysiology of neurodegenerative diseases. We examined the effect of basic fibroblast growth factor (bFGF) on the survival of cultured rat hippocampal neurons exposed to conditions in which PKC is likely to play a role. bFGF reduced neuron damage caused by the PKC-activating phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA), glutamate and ischaemia-like culture conditions. bFGF was able to counteract the excessive activation of PKC caused by these treatments. Moreover, bFGF prevented the loss of PKC occurring after prolonged exposure to TPA or ischaemia-like conditions. These results indicate that both the overactivation and the abnormal degradation of PKC can lead to neuron degeneration, and that the neurotrophic competence of bFGF may reside in its ability to regulate and normalize the PKC phosphorylating system.     

缺氧-复氧对大鼠海马培养神经元Bcl-2、Bax表达和神经元凋亡的影响

目的 观察缺氧-复氧对体外培养海马神经元Bcl-2和Bax表达和神经元凋亡的影响。方法 取培养12d的海马神经元，置于恒温(36℃)密闭容器内，连续充以无氧气体[90％(体积分数)N2、10％(体积分数)CO2]，在缺氧条件下继续培养4h后，再于常氧培养箱内复氧培养24h和72h。于不同时间观察神经元存活数，并分别用抗Bcl-2和Bax抗血清进行免疫组织化学染色，观察缺氧-复氧后大鼠海马培养神经元Bcl-2和Bax表达。并用原位末端标记(TUNEL)法和流式细胞术分别检测缺氧-复氧对体外培养海马神经元凋亡的影响。结果 缺氧-复氧后24～72h,海马神经元对Bcl-2的表达逐渐减弱，对Bax的表达逐渐增强，对Bax／Bcl-2比值逐渐增大，凋亡神经元百分率逐渐增多。结论 缺氧-复氧后24～72h神经元凋亡的发生与神经元Bcl-2表达逐渐减弱，Bax表达逐渐增强，Bax／Bcl-2比值逐渐增大有关。