Influence of Temocapril on Cultured Ventral Spinal Cord Neurons

Temocapril, a angiotensin-converting enzyme (ACE) inhibitor, was tested for neurotrophic activity in primary explant cultures of ventral spinal cord of fetal rats (VSCC). Temocapril had a remarkable effect on neurite outgrowth with a 4.2- to 5.1-fold increased over that of control VSCC at their effective concentrations. In temocapril-treated VSCC, choline acetyltransferase (ChAT) activity was also increased 2.4–3.2 times over that of control at 10^–9 and 10^–8 M, respectively. Our data suggest that temocapril is a candidate for neurotrophic factors on spinal motor neurons in vitro. A possible therapeutic role for temocapril in damaged motor neurons, such as in motor neuropathy and amyotrophic lateral sclerosis, remains to be defined.     

Thyrotropin-Releasing Hormone Enhances Choline Acetyltransferase and Creatine Kinase in Cultured Spinal Ventral Horn Neurons

The effect of 0.1 mM thyrotropin-releasing hormone (TRH) on ventral horn neurons was investigated in eight experimental sets of tissue cultures established from ventral and dorsal portions of spinal cords of 13-15-day rat embryos. Cultures were treated with TRH from day 1 for 2-5 weeks. TRH-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had more numerous and more healthy-appearing neurons and thicker bundles of long cell processes. In TRH-treated VSCC, choline acetyltransferase (ChAT) activity was greater than 16 times (p less than 0.005) and creatine kinase greater than 3 times (p less than 0.005) that of control VSCC. Morphologic and biochemical parameters of dorsal spinal cord cultures remained unchanged by TRH treatment. Since lower motor neurons are numerous in the ventral spinal cord (and not present in the dorsal cord) and since lower motor neurons are the major ChAT-containing spinal cord cells, our data demonstrating a beneficial effect of TRH on VSCC suggest a tropic effect of TRH on lower motor neurons.     

T-588 Protects Motor Neuron Death Against Glutamate-Induced Neurotoxicity

To examine the possible neuroprotective effect of T-588 against glutamate-induced neurotoxicity, we analyzed the pharmacological utility of T-588 in a postnatal organotypic culture model of motor neuron degeneration. Treatment with 10^–5 M of glutamate resulted a motor neuron loss and decreased activity of choline acetyltransferase (ChAT). Cotreatment of 10^–5 M of glutamate and T-588 revealed a protective effect against motor neuron death and decreased ChAT activity. We concluded that T-588 may play important roles in the survival and maintenance of spinal motor neurons in its neuroprotection against glutamate-induced neurotoxicity. Our data may provide a rationale for designing a therapeutic strategy for protection against pathologically induced motor neuron damage or cell death such as amyotrophic lateral sclerosis and motor neuropathy.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.     

胍丁胺对体外培养脊髓神经元的影响及在谷氨酸诱导损伤条件下的作用

研究胍丁胺(agmatine,AG)对培养脊髓神经元的作用及谷氨酸(glutamate,Glu)损伤后的影响,探讨其对神经元的毒性作用及其可能的机理.采用原代细胞培养法,分离培养胚胎大鼠脊髓神经元,3d后加入不同浓度的AG(0.5～80mg/L)继续培养12、24、36h,接着采用四甲基氮唑蓝法和中性红法分别测定胍丁胺对细胞存活率以及毒性的影响.在加入AG的同时给予2mmol/LGlu对细胞进行损伤,建立体外的损伤模型.然后进行细胞形态学观察、NeuN免疫细胞化学染色、四甲基氮唑蓝法测定细胞存活率、Hoechst33342和PI染色检测细胞凋亡和坏死率,同时对神经元中丙二醛(malondialdehyde,MDA)含量的变化检测,最后对各组进行比较和统计分析.结果发现,AG作用浓度低于40mg/L对正常培养的脊髓神经元的存活没有明显的影响,而80mg/L的AG对神经元生长有毒副作用.在给予Glu损伤后,细胞生长状态较差,细胞出现退化,而且细胞存活率显著下降,细胞坏死和凋亡率显著增高(P<0.05或0.01),而同时给予AG干预(AG-Glu组),细胞生长较正常培养的细胞生长状态略差,但明显优于Glu组,而且细胞凋亡和坏死率降低,细胞存活率增高,MDA含量减少(P<0.05).结果提示AG作为一种新发现的神经递质或调质,在正常生理情况下对神经元的生长没有毒副作用,而在Glu诱导损伤条件下,能够抑制Glu诱导的损伤.其发生机制可能与AG通过拮抗N-甲基-M-天冬氨酸(N-methyl-D-aspartate,NMDA)受体,阻断或抑制Glu的氧化毒性的级链反应有关.     

Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed-volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15-50 mM) produced a dose-related increase in basal NA release that was calcium dependent. The potassium-evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective alpha 2-adrenoceptor antagonist idazoxan (10(-6)-10(-4) M) or (b) the thyrotrophin-releasing hormone (TRH) analogue RX 77368 (pGlu-His-3,3'-dimethyl ProNH2; 10(-5) and 10(-4) M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 microM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10(-7) M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10(-4) M) significantly attenuated the potassium- or RX 77368-induced release of NA, whereas substance P (3 X 10(-5) and 1 X 10(-4) M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.     

Effects of GnRH on Neurite Outgrowth,Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos

It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.     

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.     

Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µ M ) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca²⁺-free medium and was inhibited by an antagonist of NMDA receptors or a voltage-sensitive Ca²⁺ channel blocker. Preincubation with W7 also inhibited the glutamate-evoked cyclic AMP increase. NMDA, aspartate, and high-K⁺ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)-glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca²⁺ influx via both NMDA receptors and voltage-sensitive Ca²⁺ channels followed by an activation of the Ca²⁺/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.     

Enhancement of Neurite Outgrowth and Aspartate-Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8-day-old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine-coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8-day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6- to 1.8-fold and for γ-aminobutyric acid to a lesser degree when examined at a concentration for high-affinity uptake (10^-6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of K^m, but to an increase of V^max. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate-glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.     

向蟾蜍脊髓灰质腹角注射谷氨酸钠对腓肠肌收缩的影响

首先绘制了蟾蜍脊髓灰质腹角运动神经元池的精确定位图谱,然后分别将浓度为1、0.5、0.1、0.01 mol/L的谷氨酸钠溶液及生理盐水（0.65% NaCl,对照组）微量（0.1 µL）注射到蟾蜍脊髓灰质腹角运动神经元池,采用BL-420F 生物机能实验系统记录腓肠肌收缩曲线,以收缩波的上升相持续时间、最大张力、张力变化速率、下降相持续时间为指标对各组腓肠肌的收缩曲线进行比较。结果：4组实验组腓肠肌的收缩形式是不同程度的强直收缩;各组收缩波的上升相持续时间、最大张力、张力变化速率在一定程度上存在谷氨酸钠量效依赖关系,且1 mol/L组腓肠肌强直收缩的张力、张力变化速率显著大于其他各组,这可能是由于谷氨酸钠与运动神经元上谷氨酸受体结合的数量不同造成的。     

Neurotrophic Effects of GnRH on Neurite Outgrowth and Neurofilament Protein Expression in Cultured Cerebral Cortical Neurons of Rat Embryos

The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.     

Cell Survival Characteristics and Choline Acetyltransferase Activity in Motor Neurone-Enriched Cultures from Chick Embryo Spinal Cord

The effect of muscle extract on cell survival and choline acetyltransferase (ChAT) activity in cultures of enriched cholinergic neurones from 7-day chick embryo spinal cord was examined. When neurones were grown on hydrated collagen gels, considerable cell survival and ChAT activity were obtained even in the absence of tissue extract. These parameters were stimulated twofold in the presence of skeletal muscle extract but not liver or skin extracts. The cholinergic neurotrophic activity was found to be heat- and trypsin-sensitive, nondialysable, and to act in the virtual absence of glial cells. These data are consistent with a retrogradely acting motor neurone trophic activity.     

Nuclear Organization in the Spinal Cord Depends on Motor Neuron Lamination Orchestrated by Catenin and Afadin Function

1. Download high-res image (89KB)
2. Download full-size image

     

Differential Release of Endogenous 5-Hydroxytryptamine, Substance P., and Neurokinin A from Rat Ventral Spinal Cord in Response to Electrical Stimulation

Abstract: The release of endogenous 5-hydroxytryptamine (5-HT), substance P (SP), and neurokinin A (NKA) from superfused tissue slices of rat ventral lumbar spinal cord, where SP and NKA coexist with 5-HT in terminals of descending bulbospinal neurons, was investigated. Electrical field stimulation was performed using square-wave pulses of 2-ms duration and 30 mA stimulus intensity. The following four different patterns of stimulation were used: 2 Hz continuous, 20 Hz continuous, 20 Hz intermittent, and 50 Hz intermittent. 5-HT was measured in the slice superfusates by HPLC with electrochemical detection. SP and NKA were measured by radioimmunoassay. The release of 5-HT was significantly enhanced using all stimulation paradigms and the evoked release of 5-HT per pulse was independent of the stimulation frequency. The release was found to be calcium dependent and there was no increase in the efflux of 5-hydroxyindoleacetic acid in response to stimulation. At 2 Hz (continuous), no significant increase in the release of SP was observed. Stimulation at higher frequencies yielded a significant increase in the release of SP per pulse. At 20 Hz, the release was increased by 73% (continuous) and 74% (intermittent), and at 50 Hz (intermittent) by 175% of basal efflux. The evoked release of NKA was also frequency dependent. At 2 Hz (continuous), no significant increase in the release of NKA was observed. At 20 Hz (intermittent), the evoked release per pulse was increased by 33% and at 50 Hz (intermittent) by 53% compared with the basal efflux of NKA. The results suggest that coexisting neurotransmitters/neuromodulators in the spinal cord may be released in different proportions depending on the stimulation frequency and that only 5-HT is released when the nerve terminal is activated by low-frequency stimulation.     

Hydroxyl Radicals Generated In Vivo Kill Neurons in the Rat Spinal Cord: Electrophysiological, Histological, and Neurochemical Results

Abstract: We have used microdialysis to establish an experimental model to characterize mechanisms whereby released substances cause secondary damage in spinal cord injury. We use this model here to characterize damaging effects of the hydroxyl radical (OH') in vivo in the spinal cord. OH'was generatad in vivo by pumping H₂O₂ and FeCI₂/EDTA through parallel microdialysis fibers inserted into the spinal cord. These agents mixed in the tissue to produce OH'by Fenton's reaction. Two types of control experiments were also conducted, one administering only 5 m M H₂O₂ and the other only 0.5 m M FeCI₂/0.82 m M EDTA. During administration of these chemicals, electrical conduction was recorded as one test for deterioration. OH'blocked conduction completely in 2.5-5 h and Fe²⁺/EDTA partly blocked conduction, but H₂O₂ alone did not cause detectable blockage. Histological examination supported the hypothesis that neurons were killed by OH', as Fe²⁺/EDTA and H₂O₂ alone did not destroy significant numbers of neurons. OH', H₂O₂, and Fe²⁺ all caused gradual increases in extracellular amino acid levels. These results are consistent with Fe²⁺-catalyzed free radical generation playing a role in tissue damage upon spinal cord injury.     

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.     

Spinal Cord Transcriptome Analysis Using Suppression Subtractive Hybridization and Mirror Orientation Selection

Comparison of cDNA libraries derived from the spinal cord with those derived from the visual cortex by means of forward and reverse subtractive hybridization resulted in the cataloguing of 60 genes differentially expressed in the spinal cord. 1. The differentially expressed genes represent a mixture of novel and known sequences with known and unknown protein products. 2. The possibility that the subtraction process was simply overwhelmed by background sequences was significantly reduced by several observations including comparisons between suppression subtractive hybridization (SSH) and mirror orientation selection (MOS). 3. Nearly half of all genes up-regulated in the spinal cord are of myelin origin. 4. Twenty-five percent of all up-regulated clones in the spinal cord versus the visual cortex are for proteolipid protein. 5. Ten percent of all up-regulated clones in spinal cord versus visual cortex are for ferretin heavy chain, which is known to be produced in oligodendroglial cells in the CNS. 6. Two of the up-regulated sequences, proteolipid protein and N-myc down-regulated gene 4, are identified with genes known to directly affect neuron survival. 7. Two of the up-regulated genes, ferritin and transferrin, are indirectly associated with apoptosis through their ability to sequester iron and reduce free radical formation.