Metabotropic Purinoreceptors in Rat Dorsal Horn Neurons: Predominantly Dendritic Location

Elevations of the cytosolic free Ca²⁺ concentration ([Ca²⁺] _i ) in rat dorsal horn neurons induced by addition of ATP to the medium were compared in spinal cord slices and after isolation of the neurons. In slices, application of ATP results in an increase in the [Ca²⁺] _i by 201 ± 12 nM, on the average; in a Ca²⁺ -free external solution the respective rise was 156 ± 14 nM (n = 45 of 76 examined cells), which indicate the presence of active purinergic metabotropic receptors in about 59% of the neurons. In freshly isolated neurons with absent dendrites, we found no metabotropic responses. Thus, the results confirm the conclusion on the localization of metabotropic postsynaptic purinoreceptors mostly on the dendritic tree of dorsal horn neurons.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

脊髓背角痛觉传递和调制的一些化学解剖学观察

本实验研究了脊髓背角内Ｃ纤维末梢的分布和突触学特征及其一些神经递质化学构筑;定量观察了急性痛引起背角的递质变化;显示了初级传入Ｃ纤维,抑制性中间神经元和背角伤害性感受神经元三者之间的突触关系,并探讨它们在痛觉信息传递和调制中的作用。     

Cyclic AMP Regulates the Expression of Neurokinin1 Receptors by Neonatal Rat Spinal Neurons in Culture

Neurokinin1 (NK1) receptors are up-regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)-responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl-cAMP (dbcAMP) resulted in a time-dependent increase in 125I-Bolton-Hunter-substance P (BHSP) binding in the cultures; treatment with dibutyryl-cyclic GMP did not. Treatment with forskolin plus 3-isobutyl-1-methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP-induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP-induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)-induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.     

不同频率电针刺激对神经痛模型大鼠脊髓背角P物质的影响

目的:观察P物质(Substance P,SP)在慢性坐骨神经压迫损伤(chronic constriction injury,CCI)模型脊髓中表达的变化,探讨电针镇痛的机制是否与脊髓背角中SP表达的变化有关。方法:选择32只雄性、体重180-200 g的SD大鼠,并将其随机均分为4组(n=8)。空白组(Con组)为正常痛阈值大鼠;假电针组(CCI+A组)在损伤的坐骨神经旁置入电针,但无电流刺激;2 Hz组和100Hz组分别给予相应频率电流刺激30 min。在实验开始前和术后1、4、7、14、20、22天记录大鼠的热缩足反射潜伏期(Paw Withdrawal Latency,PWL)和机械刺激缩足反射阈值(Paw Withdrawal Threshold,PWT)。免疫组化方法检测脊髓背角SP的表达。结果:术后20天,电针治疗后,100 Hz组和2 Hz组PWT分别为(7.33±1.42)g和(7.80±1.42)g,均显著高于假电针组(2.60±1.46)g,差异有统计学意义(P0.05)。100 Hz组在术后20天后和2 Hz组在术后14天后PWL值均显著高于假电针组,差异有统计学意义(P0.05)。免疫组化显示:2 Hz组和100 Hz组大鼠脊髓背角中P物质阳性细胞显著低于假电针组(P0.05)。结论:坐骨神经旁电针刺激能够显著减轻CCI模型大鼠热痛觉及机械痛觉过敏,其机制可能与抑制脊髓背角SP的表达有关。     

Effects of Gabapentin on Calcium Transients in Neurons of the Rat Dorsal Root Ganglia

In our experiments on rat dorsal root ganglia (DRG) neurons, we studied the effects of an antiepileptic agent, gabapentin, on calcium transients evoked by depolarization of the membrane using the fluorescence calciumsensitive dye Fura-2/AM. Application of gabapentin to neurons with large-diameter somata practically did not change the characteristics of calcium transients. In mid-sized neurons, the amplitude of transients decreased, on average, by 27% with respect to the control, while in small-sized neurons the transients changed insignificantly (on average, less than by 7%). The mid-sized neurons were additionally subjected to the capsaicin test, which allowed us to differentiate primary nociceptive neurons of this group where TRPV1-type channels are expressed. In capsaicin-sensitive neurons, application of gabapentin led to a decrease in the amplitude of calcium transients, on average, by 37%, while such a decrease was only 16% in capsaicininsensitive neurons. Based on our own data and findings of other researchers on the ability of gabapentin to demonstrate affine binding with the accessory α₂δ subunit of voltage-dependent calcium channels and also on the peculiarities of expression of these channels in somatosensory neurons of the corresponding types, we discuss the probable pattern of expression of subunits of the α₂δ-1 subtype in DRG cells of different sizes. We demonstrated that the effects of gabapentin on calcium transients in nociceptive and hypothetically nonnociceptive mid-sized DRG neurons are selective (the effects in neurons involved in the sensation of acute pain are probably more intense). Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 281–287, July–August, 2008.     

鸡脊髓背角胶状质中calbindin-D28k阳性终末的超微结构及与substance P阳性终末之间的联系

目的 观察鸡脊髓背角胶状质中calbindin-D28k（CB）阳性终末的超微结构及其与含有substance P（SP）中央末梢之间的联系.方法 应用免疫电镜技术观察鸡脊髓背角胶状质中CB阳性终末的超微结构,并应用激光共聚焦显微镜观察鸡脊髓背角胶状质中CB和SP阳性突触小球中央末梢之间的关系.结果 电镜下观察：1）突触小球中含有心小泡的中央末梢呈CB免疫阳性;2）突触小球内或外的部分含小泡的树突呈CB免疫阳性;以及3）突触小球外的部分轴突呈CB免疫阳性.在突触结构内,CB免疫阳性反应物主要分布于突触后膜上.免疫荧光双标记法显示,SP阳性的含有心小泡的中央末梢呈CB阳性.结论 突触小球的中央末梢中CB与SP共存,提示CB可能通过其钙离子缓冲作用,参与脊髓的痛觉调制.     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Lead Reduces Depolarization-Induced Calcium Entry in Cultured DRG Neurons Without Crossing the Cell Membrane: Fura-2 Measurements

1. Cultured dorsal root ganglion neurons of rat pups were depolarized by exposure to 50 mM K⁺ and the rise of [Ca²⁺]_i was measured using fura-2 as an indicator.2. Lead in the extracellular solution reduced the rise of [Ca²⁺]_i in a concentration-dependent manner, with a threshold concentration of 0.25 M. More than 80% of the calcium entry was prevented by 5 M lead. The IC₅₀ and the Hill coefficient were 1.3 M and 1, respectively.3. This effect was considered to be due to a reduction of VACCCs, since applications of NMDA did not result in any rise of [Ca²⁺]_i.4. Since Pb²⁺ itself changes the fura-2 signal in a typical and characteristic manner, fura-2 is also an indicator for Pb²⁺. No changes in fura-2 signals were detected when lead (5 M) was applied for several minutes in the absence of calcium, indicating that Pb²⁺ did not enter the cells.5. Thus it is concluded that lead prevents calcium entry by reducing VACCCs but does not cross the cell membrane itself.     

Calcium Mobilization by Ryanodine Promotes the Phosphorylation of Initiation Factor 2α Subunit and Inhibits Protein Synthesis in Cultured Neurons

Abstract: Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca²⁺ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2α subunit, and double-stranded RNA-dependent eIF-2α kinase (PKR) activity were analyzed. Glutamate (100 µ M ) and K⁺ (60 m M ), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 m M ), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 n M ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2α subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

The Cholinergic Regulation of Intracellular Calcium in the Human Neuroblastoma, SH-SY5Y

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 +/- 14 nM (mean +/- SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 microM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 +/- 52 (mean +/- SEM, n = 12) and 422 +/- 48 nM (mean +/- SEM, n = 7), respectively, in less than 4 s. This initial calcium transient declined to a plateau of 268 +/- 36 and 240 +/- 27 nM for carbachol and acetylcholine, respectively, in approximately 40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 +/- 0.05, 8.3 +/- 1.6, 411 +/- 36, and 240 +/- 46 nM (mean +/- SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 +/- 0.18, 13.5 +/- 8.5, 192 +/- 32, and 414 +/- 25 nM (mean +/- SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects.(ABSTRACT TRUNCATED AT 250 WORDS)