FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.     

Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells

Biochemical and biophysical studies on Aplysia sensory neurons indicate that inhibitory responses to the molluscan peptide FMRFamide are mediated by lipoxygenase metabolites of arachidonic acid. These compounds are a new class of second messengers in neurons.     

Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K+ channels of Aplysia sensory neurones in cell-free membrane patches

Selected actions of neurotransmitters and hormones on ion channels in nerve and muscle cells are now thought to be mediated by cyclic AMP-dependent protein phosphorylation. Although the cyclic AMP-dependent protein kinase (cAMP-PK) affects the cellular properties of several neurones, its mode of action at the single-channel level has not been characterized. In addition, little is known about the identity or subcellular localization of the phosphoproteins that control channel activity and, in particular, whether the critical substrate proteins are cytoplasmic or membrane-associated. In Aplysia sensory neurones, serotonin produces a slow modulatory synaptic potential mediated by cAMP-PK that contributes to presynaptic facilitation and behavioural sensitization. Previously, we have found that serotonin acts on cell-attached membrane patches to produce prolonged all-or-none closures of a specific class of K+ channels (S channels) whose gating is weakly dependent on voltage and independent of intracellular calcium. We demonstrate here that in cell-free membrane patches from Aplysia sensory neurones, the purified catalytic subunit of cAMP-PK produces all-or-none closures of the S channel, simulating most (but not all) aspects of the action of serotonin on cell-attached patches. This result suggests that protein kinase acts on the internal surface of the membrane to phosphorylate either the channel itself or a membrane-associated protein that regulates channel activity.     

Postsynaptic hyperpolarization during conditioning reversibly blocks induction of long-term potentiation

Activity-induced changes in the efficacy of synaptic transmission between neurones are central to several prominent theories of learning. In both in vivo and in vitro preparations of the hippocampus, a conditioning high-frequency stimulus delivered to afferent fibres results in a long-term potentiation of synaptic transmission at those inputs. Evidence has been provided supporting both presynaptic and postsynaptic sites as loci where critical events occur in the development of potentiation. In this study we report that long-term potentiation is reversibly blocked by intracellular injection of hyperpolarizing current in the postsynaptic cell during the conditioning high-frequency stimulus, suggesting the involvement of a voltage-dependent postsynaptic mechanism.     

Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.     

高喷灌浆技术存在问题的探讨

从高喷灌浆工程的实践经验出发，重点分析了高喷灌浆技术存在的水泥材料消耗大、施工工艺复杂、劳动强度大和质量难以控制等问题，并提出了解决方法。     

野木瓜注射液对小鼠皮层神经细胞的抗氧化保护作用

研究了野木瓜注射液(IS)对小鼠大脑皮层神经细胞氧化损伤的保护作用.在细胞培养液中加入100μmol/L H2O2和不同浓度IS,培养数日后通过观察细胞的形态变化,检测细胞外液LDH的活力,胞内超氧化物歧化酶(SOD)的活性,测定细胞活性来观测IS对氧化受损神经细胞的保护作用,并通过脱氧核糖法对IS的羟自由基(.OH)清除率进行测定.结果表明:浓度在25～300 mg/L,IS均能抑制神经细胞的氧化损伤,神经细胞SOD酶活力上升,细胞外液LDH活力降低,浓度为100 mg/L时效果最显著.与阳性对照甘露醇相比,IS有较高的.OH清除率.IS有较高的抗氧化能力,能有效抑制H2O2导致的皮层神经细胞的损伤.     

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries. I     

大鼠丘脑中央下核神经元对不同刺激的反应

目的：观察丘脑中央下核（Sm)神经元对伤害性机械刺激,手针和电针刺激的反应特性。方法：在麻醉大鼠,用玻璃微电极细胞外记录的方法,观察Sm神经元对上述刺激的反应。结果：大多数对手针刺激反应的神经元一般地也对伤害性皮肤或肌肉机械刺激发生反应,这些神经元也对电针刺激穴位发生反应,其反应随刺激强度、串长或重复次数的增加而加强,揭示针刺与伤害性机械刺激具有相同的属性。结论：Sm可能在针感形成及针刺兴奋细胞纤维产生的镇痛中起重要作用。     

Induction of c-fos-like protein in spinal cord neurons following sensory stimulation

It has been suggested that the proto-oncogenes c-fos and c-myc participate in the control of genetic events which lead to the establishment of prolonged functional changes in neurons. Expression of c-fos and c-myc are among the earliest genetic events induced in cultured fibroblast and phaeochromocytoma cell lines by various stimuli including growth factors, peptides and the intracellular second messengers diacylglycerol, cAMP and Ca2+. We report here that physiological stimulation of rat primary sensory neurons causes the expression of c-fos-protein-like immunoreactivity in nuclei of postsynaptic neurons of the dorsal horn of the spinal cord. Activation of small-diameter cutaneous sensory afferents by noxious heat or chemical stimuli results in the rapid appearance of c-fos-protein-like immunoreactivity in the superficial layers of the dorsal horn. However, activation of low-threshold cutaneous afferents results in fewer labelled cells with a different laminar distribution. No c-fos induction was seen in the dorsal root ganglia, gracile nucleus or ventral horn. Thus, synaptic transmission may induce rapid changes in gene expression in certain postsynaptic neurons.     

Direct measurement of the transfer rate of chloroplast DNA into the nucleus

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.     

Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (gamma-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.     

Time to collision is signalled by neurons in the nucleus rotundus of pigeons.

Throughout the animal kingdom, the sight of a rapidly approaching object usually signals danger and elicits an escape response. Gibson suggested that the symmetrical expansion of an object's image (looming) is the critical variable determining that the object is on a collision course with the observer. Similarly, large expanding flow-fields like those produced by locomotion may precipitate manoeuvres such as turning or landing. From such observations it has been shown that the optic flow parameter, tau, which specifies time to contact with the approaching object best fits the behavioural data. We describe a subpopulation of neurons in the nucleus rotundus of the pigeon brain that respond selectively to objects moving on a collision course towards the bird.     

Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons

Nerve growth factor (NGF) is a trophic molecule essential for the survival of sympathetic and sensory neurons during ontogeny. The extent to which NGF is involved in the maintenance or regulation of the differentiated phenotypes of mature peripheral neurons is much less clear, however. Biochemical analysis of the actions of NGF upon peripheral neurons has been hampered by the lack of a preparation of neuronal cells that are responsive to NGF but do not require it for survival. We report here that in adult dorsal root ganglion neurons, which can be isolated, enriched and maintained in culture in the absence of neuronal growth factors, the expression of mRNAs encoding the precursors of two neuropeptides, substance P and calcitonin gene-related peptide is regulated by NGF. Our results provide the first direct evidence of a continuous dynamic role for NGF in regulation of peptide neurotransmitter/neuromodulator levels in mature sensory neurons.