化学剂引起的豚鼠“踏步自动作用”的研究

传统的“踏步自动作用”(stepping automatism)用电刺激和踏车(treadmill)相结合的方法在中脑或脊髓猫上引起。本文发现4-R-2,2,5,5-四(三氟甲基)-氢化咪唑化合物能在整体豚鼠引起规律的“踏步自动作用”。用上述化合物引起的豚鼠踏步运动仅见“对角肢”(diago-hal limbs)和“同侧肢”(homolateral limb-)两种踏步模式,并且前肢较后肢规律,前肢的踏步幅度铰后肢高数倍。     

诱发踏步物质和中枢运动控制

1980年发现若干4-R-咪唑啉衍生物能在豚鼠等四足动物诱发规律的“踏步自动作用”(stepping automatism);1983年在上海将这些物质命名为“诱发踏步物质”(substances capable of inducing stepping,SIS)。X光电影的分析表明,SIS诱发的“踏步周期”相似于四足动物的正常踏步运动。SIS仅强抑制α-运动神经元的兴奋性突触后电位,对抑制性突触后电位无影响。SIS还能在麻雀等两足动物诱发拍翅飞行运动。     

冷冻或切除小脑蚓部山顶(Culmen)对豚鼠“踏步自动作用”(Stepping automatism)的影响

本研究探讨部分冷冻或切除小脑蚓部(vermis)对整体豚鼠“踏步自动作用”(steppingautomatism)的影响。“踏步自动作用”由我们近年来发现的诱发踏步物质(SIS)(4-R-2,2,5,5-四(三氟甲基)-咪唑啉)所引起。结果表明部分冷冻或切除小脑蚓部的山顶(culmen,Ⅴ和Ⅳ叶)和中央叶(Centralis,Ⅲa,b)明显增强豚鼠的“踏步自动作用”。冷冻小脑不能触发,但仅能调控“踏步自动作用”。这种调控作用对自动化程度差的弱“踏步自动作用”特别显著。蚓部山顶(Ⅴ叶为主)同时调控左右前肢踏步,而一侧蚓部山顶及其半球则主要调控同侧前肢踏步。此外,本研究的结果表明当介面温度(冷冻头和小脑幕间)致冷至5℃—0℃左右,冷冻小脑便可基本模拟部分切除小脑效应。     

电刺激家鸽中脑诱导运动的初步研究

用电刺激方法研究浅麻醉状态下家鸽中脑的不同区域诱发的行为反应,认识家鸽中脑运动相关脑区的空间编码规律。在中脑的半环隆枕(TOS)、丘间核等核团区域诱导出扇动翅膀、呼吸加快、羽毛倒伏等运动行为。     

胆碱能和多巴胺能化合物对豚鼠“踏步自动作用”的影响

中枢M-胆碱受体阻滞剂东莨菪碱(0.2—0.5mg/kg)和多巴胺前体L-多巴[特别是″L-多巴 RO-4-4602″(300mg/kg 50 mg/kg)],能增强由4-氨基-2,2,5,5-四(三氟甲基)-氢化咪唑(6—8mg/kg)引起的豚鼠“踏步自动作用”。胆碱酯酶可逆性抑制剂毒扁豆碱(0.5mg/kg)和多巴胺受体阻断剂氟哌啶醇(0.1—0.2mg/kg)则减弱上述“踏步自动作用”,从而表明豚鼠的“踏步自动作用”不仅和胆碱能及多巴胺能系统的活动有关,而且可能和两者间的交互影响有关。此外,豚鼠规律的强踏步运动常见伴随θ节律的呈现。     

电刺激大壁虎中脑诱发相反方向脊柱侧弯的研究

深入探索大壁虎Gekko gecko中脑运动调控功能,通过对头部固定的大壁虎(n=10)中脑脑区采用不锈钢微电极施加电刺激,发现对中脑不同深度脑区的电刺激能诱发出脊柱侧弯、四肢动等运动反应.在实验过程中,一些刺激针道在中脑围脑室灰质诱发出脊柱同侧弯曲反应,而在中脑腹侧被盖诱发出脊柱对侧弯曲反应.通过对刺激脑区的组织学检验,确定了大壁虎中脑内控制相反方向脊柱侧弯运动核团分布的大致区域.     

刺激大鼠“涌泉”穴诱发的脊髓背表面电位

刺激大鼠“涌泉”穴诱发的节段性背表面电位(Y-sCDP),在远节段(L3-T7)逐渐消失,而在更远节段(C6)又记录到一个背表面电位(Y-dCDP),该电位为一慢电位正波(P波),高位横断脊髓后Y-dCDP完全消失,说明它来源于脊髓上结构,当电解损毁中脑导水管周围灰质(PAG)后,P波幅值显著降低(P<0.05),说明刺激“涌泉”穴产生的Y-dCDP为激活PAG等痛觉调制核团下行诱发的脊髓背表面电位。     

豚鼠离体腹腔神经节神经元自发f-EPSP和AP

应用细胞内生物电记录技术观察豚鼠腹腔神经节(CG)神经元自发快兴奋性突触后电位(f-EPSP)和动作电位(AP)的特征,分析其形成的可能机制。发现在豚鼠离体CG上存在自发f-EPSP和AP,发放频率不同。自发f-EPSP的幅度(5.67±2.66)mV(n=26),明显低于刺激内脏大神经诱发f-EPSP的幅度(13.26±6.74)mV(n=34,P<0.01),而自发的AP后超极化幅度(AHPA)(13.86±4.24)mV(n=30),明显高于刺激内脏大神经诱发的AHPA(8.99±2.79)mV(n=54,P<0.01)。六烃季铵或低Ca2 /高Mg2 Krebs液能完全阻断自发的AP,但自发的f-EPSP则不被完全阻断。结果提示豚鼠离体CG神经元有自发性电活动,这除与突触前膜ACh的随机释放有关以外,可能还有对Ca2 不敏感的其他递质介导。     

电针颧髎穴和尾部电流致痛后c—fos在大鼠中枢脑桥延髓内DBH阳性神经元中的表达

本研究在大鼠上分别用电针大鼠面部颧髎穴和尾部电流刺激致痛后,报道DBH阳性神经元与c—fos蛋白在中枢神经系统中的共存。实验在雄性SD大鼠上进行。刺激后2小时,经心脏用4％多聚甲醛磷酸缓冲液灌注固定,脑组织作冰冻冠状切片。在同一切片上顺序进行Fos抗体—ABC—DABni和DBH抗体—PAP—DAB双免疫细胞化学方法染色,并以不刺激动物作对照,结果表明:针组和痛组动物均可在脑干若干核团内观察到Fos样免疫阳性(FLI)显示神经元在细胞核上,星紫黑色。光镜下在A_1、A_2、A_3蓝斑与蓝斑下核处有DBH样免疫阳性(DLI)在细胞体、突起和终末上,呈棕黄色,但胞核不染。光镜下出现三种形式神经元:1.仅有FLI胞核染色。2.仅有DLI胞质、突起和终末染色。3.FLI胞核存在DLI胞体中(即共存)。统计分析表明。在脑干部位,FLI在网状外侧核(RL)、孤束核(nts)、延髓头端腹侧区(RVM)和导水管周围灰质的腹侧、腹外侧和外侧部位(PAG)针组显著多于痛组。共存神经元仅在A_1部位针组显著地多于痛组,A_2、A_5及蓝斑部位。针组与痛组的共存数差异不显著。     

刺激大鼠中脑导水管周围灰质诱发的脊髓背根电位及其传出途径的分析

电刺激大鼠中脑导水管周围灰质(PAG),在腰5(L_5)背根可记录到—稳定的负性背根电位(DRP),简称 PAG-DRP。PAG-DRP 具有空间和时间总和性质,沿背根作电紧张性扩布,且能被 GABA 能拮抗剂印防己毒素(Picrotoxin)所抑制。电解损毁中缝大核(NRM)对刺激背侧 PAG 诱发的 PAG-DRP 无明显影响,而可使刺激腹侧 PAG 诱发的 PAG-DRP电位幅值降低40%左右。结果表明,PAG 下行抑制作用中有突触前抑制参与;NRM 参与腹侧 PAG-DRP 的产生,背侧 PAG-DRP 则可能由 NRM 以外的其他核团中继。     

Significance of peripheral feedback in stepping movement generation under epideral spinal cord stimulation

In acute experiments on decerebrated and spinalized cats, the role of peripheral afferent input from hindlimbs in stepping patterns formation under epidural spinal cord stimulation (ESCS), was investigated. The hindlimb muscles' electromyographic activity and kinematic parameters of evoked stepping were analyzed. It has been shown that epidural stimulation (20-100 microA, 5 Hz) of L4-L5 spine segments induced coordinated stepping on the treadmill belt. In conditions of weight-bearing support (stopped treadmill, hindlimbs lifted above the treadmill), the stepping rhythmic was unstable, stepping cycle period and its internal structure having changed as well. With increased speed of locomotion the stepping frequency increased due to the duration of the support phase decreasing. Forward stepping could be reversed to backward stepping by changing the direction of the treadmill belt movement. In 2-4 hours after complete spinal transection (T8-T9), the epidural stimulation elicited stepping movements on a moving treadmill only. It was found that the influence of peripheral feedback on initiation of the stepping after spinalization increased. Peripheral feedback seems to play a major role in determining the fundamental features of motor output during the ESCS.     

Role of afferent activity in the generation of stepping movements

Efferent fibers of the hind limbs were divided and electrical activity of a filament from the ventral root of S1 was recorded in experiments on mesencephalic cats capable of locomotion in response to stimulation of the "locomotor region" of the midbrain. In response to weak stimulation of the locomotor region, when the forelimbs were not performing stepping movements, regular waves of activity appeared in the filament with a period close to the duration of the step during walking (0.5–1.0 sec). This periodic process was largely dependent on the tonic afferent inflow: various extero- and interoceptive stimuli applied to the hind limb could change the period of generation or abolish it. Active stepping movements of the forelimbs as well as passive movements of the hind limbs led to synchronization of activity in the filament with these movements. After division of the afferent fibers to the hind limbs the animals performed one or two steps in response to stimulation of the dorsal root of S1 by a short series of pulses. They could also perform independent stepping movements of the hind limb if 15–30% of the fibers in the dorsal root of L7 remained intact.M. V. Lomonosov Moscow State University. Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 4, No. 4, pp. 401–409, 1972.     

Morphological conservation of limb natural pendular period in the domestic dog (Canis familiaris): Implications for locomotor energetics

For better understanding of the links between limb morphology and the metabolic cost of locomotion, we have characterized the relationships between limb length and shape and other functionally important variables in the straightened forelimbs and hindlimbs of a sample of 12 domestic dogs (Canis familiaris). Intra-animal comparisons show that forelimbs and hindlimbs are very similar (not significantly different) in natural pendular period (NPP), center-of-mass, and radius of gyration, even though they differ distinctly in mass, length, moment-of-inertia, and other limb proportions. The conservation of limb NPP, despite pronounced dissimilarity in other limb characteristics, appears to be the result of systematic differences in shape, forelimbs tending to be cylindrical and hindlimbs conical. Estimating limb NPP for other species from data in the literature on segment inertia and total limb length, we present evidence that the similarity between forelimbs and hindlimbs in NPP is generally true for mammals across a large size range. Limbs swinging with or near their natural pendular periods will maximize within-limb pendular exchange of potential and kinetic energy. As all four limbs of moderate- and large-size animals swing with the same period during walking, maximal advantage can be derived from the pendular exchange of energy only if forelimbs and hindlimbs are very similar in NPP. We hypothesize that an important constraint in the evolution of limb length and shape is the locomotor economy derived from forelimbs and hindlimbs of similar natural pendular period. J. Morphol. 234:183–196, 1997. © 1997 Wiley-Liss, Inc.     

Quadrupedal locomotion in squirrel monkeys (Cebidae: Saimiri sciureus): a cineradiographic study of limb kinematics and related substrate reaction forces

Quadrupedal locomotion of squirrel monkeys on small-diameter support was analyzed kinematically and kinetically to specify the timing between limb movements and substrate reaction forces. Limb kinematics was studied cineradiographically, and substrate reaction forces were synchronously recorded. Squirrel monkeys resemble most other quadrupedal primates in that they utilize a diagonal sequence/diagonal couplets gait when walking on small branches. This gait pattern and the ratio between limb length and trunk length influence limb kinematics. Proximal pivots of forelimbs and hindlimbs are on the same horizontal plane, thus giving both limbs the same functional length. However, the hindlimbs are anatomically longer than the forelimbs. Therefore, hindlimb joints must be more strongly flexed during the step cycle compared to the forelimb joints. Because the timing of ipsilateral limb movements prevents an increasing amount of forelimb retraction, the forelimb must be more protracted during the initial stance phase. At this posture, gravity acts with long moment arms at proximal forelimb joints. Squirrel monkeys support most of their weight on their hindlimbs. The timing of limb movements and substrate reaction forces shows that the shift of support to the hindlimbs is mainly done to reduce the compressive load on the forelimb. The hypothesis of the posterior weight shift as a dynamic strategy to reduce load on forelimbs, proposed by Reynolds ([1985]) Am. J. Phys. Anthropol. 67:335-349; [1985] Am. J. Phys. Anthropol. 67:351-362), is supported by the high correlation of ratios between forelimb and hindlimb peak vertical forces and the range of motion of shoulder joint and scapula in primates.     

A radiographic study of the fetus in late pregnancy and during foaling.

The position, posture and presentation of the fetus were studied by serial radiography of the abdomen in 18 crossbred Pony mares near term and during first- and second-stage labour. In 3 mares the fetal position was assessed before and after induction of parturition with the synthetic prostaglandin, fluprostenol. In late gestation and up to the time of first-stage labour the fetus lay in ventral position with the forelimbs and poll flexed or partly flexed. At this time fetal movements were confined to flexion and extension of neck and forelimbs, but at parturition the head and limbs gradually extended the the forelimbs, head and neck rotated so that dorsal position and cranial extension were achieved. From this position, with the forelimbs and muzzle engaged in the cervical canal, delivery was quickly effected. The trunk and hindlimbs came into dorsal position during second-stage labour. The mechanics of these fetal movements and their relation to causes of dystocia are discussed.     

Vitamin A mediated limb deformities in the common Indian toad, Bufo melanostictus (Schneider)

Several types of limb deformities were induced by vitamin A in B. melanostictus. These ranged from total suppression of all the limbs (ectromelia) to partial development of either the forelimb or the hindlimb or both (mesomelia) to reduction or absence of digits in either the forelimbs or hindlimbs or both and absence of long bones in either the forelimbs or hindlimbs or both (phocomelia) or duplication of the hindlimbs (polymelia). All the limb abnormalities were induced in the developing limbs of the tail amputated tadpoles of B. melanostictus following vitamin A treatment, which is all the more interesting. The results suggest that vitamin A induces the above mentioned abnormalities by either switching on or over-expressing or disrupting the limb-specific hox genes by yet unknown mechanisms.     

A model for nonexercising hindlimb muscles in exercising animals

Nonexercising muscles appear to be metabolically active during exercise. Animal models for this purpose have not been established. However, we have been able to teach animals to run on their forelimbs while their hindlimbs are suspended above the treadmill with no visible limb movement. To document that indeed this mode of exercise does not provoke additional muscle activity, we have compared the levels of neural activation of the soleus and plantaris muscles using a computer analysis of the electromyographic interference pattern, recorded from bipolar fine wire electrodes implanted across each muscle. Via computer analyses of the electromyographic interference patterns the frequencies and amplitudes of motor unit action potentials were obtained. The data were sampled during 20 s of every minute of observation. Comparisons were made in four conditions: (i) resting on the treadmill while bearing weight on the hindlimbs (normal rest), (ii) running on the treadmill (15 m/min, 8% grade) on all four limbs (normal exercise), (iii) resting while the hindlimbs were suspended in a harness above the treadmill (suspended rest), and (iv) exercising with the forelimbs (15 m/min, 8% grade) while the hindlimbs were suspended above the treadmill (suspended exercise). All four experimental conditions were carried out for 90 min each and were performed by each animal. The results clearly show that muscle activities (frequencies and amplitudes), when the hindlimbs are suspended above the treadmill, at rest or during exercise, are lower than the activities in these same muscles when the animals are at rest, supporting only their body weight. Activities in the same muscles during exercise were from 300 to 2000% greater than during hindlimb suspension.(ABSTRACT TRUNCATED AT 250 WORDS)     

MLR-induced inhibition of barosensory cells in the NTS

A central motor command arising from the mesencephalic locomotor region (MLR) is widely believed to be one of the neural mechanisms that reset the baroreceptor reflex upward during exercise. The nucleus tractus solitarius (NTS), a dorsal medullary site that receives input from baroreceptors, may be the site where central command inhibits baroreceptor input during exercise. We, therefore, examined the effect of electrical stimulation of the MLR on the impulse activity of cells in the NTS in decerebrate paralyzed cats. Of 129 NTS cells tested for baroreceptor input by injection of phenylephrine (7-25 microg/kg iv) or inflation of a balloon in the carotid sinus, 58 were stimulated and 19 were inhibited. MLR stimulation (80-150 microA) inhibited the discharge of 48 of the 58 cells stimulated by baroreceptor input. MLR stimulation had no effect on the discharge of the remaining 10 cells, each of which displayed no spontaneous activity. In contrast to the 77 NTS cells responsive to baroreceptor input, there was no change in activity of 52 cells when arterial pressure was increased by phenylephrine injection or balloon inflation. MLR stimulation activated each of the 52 NTS cells. For 23 of the cells, the onset latency to MLR stimulation was clearly discernable, averaging 6.4 +/- 0.4 ms. Our findings provide electrophysiological evidence for the hypothesis that the MLR inhibits the baroreceptor reflex by activating NTS interneurons unresponsive to baroreceptor input. In turn, these interneurons may release an inhibitory neurotransmitter onto NTS cells receiving baroreceptor input.     

Mutations of the mouse Twist and sy (fibrillin 2) genes induced by chemical mutagenesis of ES cells

A prior phenotype-based screen of mice derived from ethylmethanesulfonate-mutagenized embryonic stem cells yielded two mouse limb defect mutants. Animals heterozygous for the polydactyly ems (Pde) mutation display preaxial polydactyly of the hindlimbs, and homozygous syndactyly ems (sne) animals are characterized by a fusion of the middle digits of their hindlimbs and sometimes forelimbs. We now report that Pde is a new allele of the basic helix-loop-helix protein gene Twist. Sequencing the full-length cDNA and several hundred basepairs of genomic DNA upstream of the coding region failed to reveal a mutation, suggesting that the lesion may be in a regulatory element of the gene. sne is a new fused phalanges (fp) allele of the shaker-with-syndactylism deletion complex (sy), and we show that the genomic lesion is a small deletion removing an entire exon, coincident with the insertion of the 3' end of a LINE element belonging to the TF subfamily.     

Islet1 regulates establishment of the posterior hindlimb field upstream of the Hand2-Shh morphoregulatory gene network in mouse embryos

How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1(-/-) hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.