Effect of axotomy on cholinergic enzyme activities in the spinal cord and sciatic nerve of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity measured in the ventral and dorsal part of the dog spinal cord (L6-S2) and in the stumps of the sciatic nerve 5, 10, 15 and 21 days after its transection were compared with the corresponding activities in the intact contralateral nerve and in sham-operated animals. AChE was also examined histochemically. Changes in the enzyme activities in the central nerve stump were correlated with activity changes in the spinal cord. In the central nerve stump, a marked (25%) increase in AChE activity was found on the fifth day after transection, but by the 21st day it fell below control value levels; up to the 15th day it showed good correlation with AChE activity in the ventral spinal cord. Histochemically, pronounced reduction of enzymatic activity was found in the ipsilateral part of the spinal cord. On the 15th day, ChAT activity in the ventral spinal cord was also significantly decreased and the accumulation of the enzyme in the central nerve stump was negligible. On the contrary, at the last 21-day interval examined, a significant increase in ChAT activity and a nonsignificant increase in AChE activity was found in the spinal cord, but their activities in the central nerve stump were decreased. In the degenerated peripheral nerve stump ChAT activity dropped by an average of 99% and AChE activity by 48% during the first 15 days after transection but, on the 21st day, AChE activity was 22% higher than at the preceding interval.     

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: Effect of axotomy

Although Ca²⁺/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIα and IIβ are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIβ antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIα, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIα immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIα is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIα as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIβ down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIα appears to support axonal regrowth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 796–810, 1997     

Localization of cholinergic neurons in the cat lower brain stem

The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.     

Effects of axotomy on the activity of the motor neurons of the external ocular motor nucleus during saccadic movements

The instantaneous firing frequency of cat abducens nucleus motoneurons (Mns) during spontaneous saccadic eye movements has been analyzed. Recordings were carried out from both control and axotomized Mns. Firing frequency of control Mns increased gradually during the first four to five interspike intervals, at which point maximum firing frequency was reached. Axotomized Mns showed an increase in firing frequency only up to the second or third interval, decreasing rapidly then. Linear relationships, with high correlation coefficients, were established between the first five intervals versus maximum frequency or peak eye velocity during saccades, in both control and axotomized Mns. However, the latter showed a decrease in the linear correlation from the third interval because of the decrease in the slope of the relationship. Functional implications of these results are discussed according to the present hypothesis on the effects of axotomy upon oculomotor neurons.     

Cholinergic enzyme activity during partial brain ischemia in the dog

Activity of cholinacetyltransferase (ChAT. EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was monitored during occlusion of arteria cerebri media dx. (MCA) in five areas of the brain cortex, in nucleus caudatus and in the thalamus of the ipsilateral and contralateral hemisphere. After 1 hour of MCA occlusion ChAT and AChE activity was reduced in the ischemised region of the hemisphere, i. e. in gyrus ectosylvius anterior and gyrus sylvius anterior, whereas after 4 hours of occlusion the differences were not significant. In nc. caudatus and thalamus the activity of enzymes during ischemia did not change much.     

低温对沙土鼠脑缺血后微管运动蛋白和结构蛋白活性的影响

目的：研究低温对脑缺血后沙土鼠微管运动蛋白(Kinesin）微管结构蛋白（microtubule associated protein 2,MAP2）活性的影响,并探讨二者活性变化与延迟性神经元死亡（delayed neuronal death,DND）的关系。方法：Kinesin和MAP2的活性应用免疫组织化学染色结合计算机图象分析的方法测定,DND应用病理检查方法判断。结果：低温明显减少脑缺血后的DND。前脑缺血再灌注后MAP2和kinesin活性随再灌注时间延长而进行性下降,且kinesin活性下降程度大于MAP2。低温明显减少脑缺血后MAP2和kinesin活性的下降程度。Kinesin活性下降的严重程度与脑缺血后DND的严重程度相一致。结论：低温可明显减少脑缺血后的DND,其机制与其减少脑缺血后运动蛋白kine-sin活性的下降有关。     

miR-126、miR-31在运动神经元与神经干细胞分化所得胆碱能神经元间表达情况的比较

目的了解运动神经元和神经干细胞诱导分化所得胆碱能神经元间miR-126和miR-31的差异表达情况,并以此来探讨两种细胞之间的差异。方法应用ABI公司的TaqMan MicroRNA Assays real-time PCR技术,观察miR-126和miR-31在运动神经元与神经干细胞分化所得胆碱能神经元中的表达情况。结果 miR-126在神经干细胞分化所得胆碱能神经元中的表达是在运动神经元中的0.002倍(P<0.05)。miR-31在神经干细胞分化所得胆碱能神经元中的表达是在运动神经元中的56.444倍(P<0.05)。结论 miR-126和miR-31在运动神经元与神经干细胞分化所得胆碱能神经元中的表达存在差异,对二者预测靶基因参与的生物学过程分析,暗示两种细胞可能在信号传导和发育上存在有差别。     

Neuropeptide messenger plasticity in the CNS neurons following axotomy

Neuronal peptides exert neurohormonal and neurotransmitter (neuromodulator) functions in the central nervous system (CNS). Besides these functions, a group of neuropeptides may have a capacity to create cell proliferation, growth, and survival. Axotomy induces transient (1–21 d) upregulation of synthesis and gene expression of neuropeptides, such as galanin, corticotropin releasing factor, dynorphin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, cholecystokinin, angiotensin II, and neuropeptide Y. These neuropeptides are colocalized with “classic” neurotransmitters (acetylcholine, aspartate, glutamate) or neurohormones (vasopressin, oxytocin) that are downregulated by axotomy in the same neuronal cells. It is more likely that neuronal cells, in response to axotomy, increase expression of neuropeptides that promote their survival and regeneration, and may downregulate substances related to their transmitter or secretory activities.     

Effect of luliberin on the activities of mitochondrial respiratory enzymes]

Luliberin, a luteinizing hormone-releasing hormone, was shown to inhibit the respiratory enzymes of rat liver mitochondria and submitochondrial particles prepared from beef heart mitochondria. At the hormone concentration of 8.10(-6) M the NADH-oxidase activity of the submitochondrial particles was inhibited by 50%. The fragments of the hormone and its analogs and pyroglutamic acid, oxytocin and bradikinin possessed practically no inhibiting effects. In the case of submitochondrial particles the inhibition was only observed in the presence of Ca2+ and was significantly decreased after addition of bovine serum albumin and phospholipase inhibitors -- butacaine and dicaine. It is assumed that the effect of luliberin on the respiratory chain is mediated through mitochondrial phospholipase.     

Effects of partial unfused contractions of the gastrocnemius medialis muscle on homonymous and synergist motor neurons in cats

Autogenetic inhibition of homonymous and synergist motoneurones can be elicited by very weak partial twitches of gastrocnemius medialis muscle, but during sustained contractions the amplitude of inhibitory post-synaptic potentials decreases quickly. A similar decrease also occurs during stronger contractions. The mechanism responsible for this decrease is still active in low spinal preparations. Pre-synaptic inhibition of Ib afferent fibres might contribute to this reduction of efficiency in the transmission of Ib afferent inputs to motoneurones.     

The arrangement of endoplasmic reticulum and proteosynthesis in spinal ganglia neurons of dog after ischemia

The authors studied ultrastructural and biochemical changes in spinal ganglia neurons of dogs after ischemia. Partial spinal cord ischemia was induced by occlusion of the abdominal aorta just below the renal arteries and the arterial blood pressure was registered above and below the occlusion. - In the course of 2-4 hours' ischemia the amount of free ribosomes increased. In some cases the formation of filamentous material or tubular structures inside the cisterns of endoplasmic reticulum was apparent. The proteosynthesis declined. Incorporation of 14C - leucine into the spinal ganglion was 50% as compared to the control animals. The morphological and biochemical changes were more pronounced after 4 h ischemia.     

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.     

Effect of elastin peptides on the activities of antioxidant enzymes in fibroblasts.

The effect of elastin peptides (kappa-elastin) was investigated on murine fibroblasts. The data indicate that elastin peptides increase the activities of antioxidant enzymes detoxifying free radicals and increase the lipid peroxide concentration within the cell. These results suggest that the influence of elastin peptides on oxygen metabolism may be related to their activities in vivo following elastin degradation and can contribute to their role in the pathogenesis of atherosclerosis.     

Effect of pH on properties of the cholinergic receptor membrane of Limnaea stagnalis neurons

The effect of pH on responses of completely isolated neurons ofLimnaea stagnalis during perfusion and microapplication of acetylcholine was investigated by a microelectrode voltage clamp method. A decrease in pH led to a decrease in the sensitivity of the cholinergic receptor membrane. At pH 5.8–6.0 responses to acetylcholine disappeared completely. Increasing the pH to 10.6 did not affect the response to this agent. The ionization constant of the group responsible for the change in sensitivity averaged 6.7. The effect of pH was shown to be unconnected with its influence on the ion channels of the cholinergic receptor membrane and it is evidently due to protonation of the functionally important group in the combining site of the cholinergic receptor.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 8, No. 6, pp. 640–644, November–December, 1976.     

Effect of static magnetic field on some enzymes activities in rats

The magnetic field of 0.008 T and 0.15 T inductions influence lasting 7 weeks (7 days a week), 1 h daily determines the increase of the activity of cytoplasmatic enzymes (glutamic pyruvic transaminase, glutamic oxalacetic transaminase, lactic dehydrogenase), the decrease of cholinesterase activity and the growth of alkaline phosphatase activity in the plasma of the examined animals. The observed changes were reversible. 2 months after the exposure had been stopped, the tested parameters were back to normal.     

Direct interactions between immunocytes and neurons after axotomy in Aplysia

Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.