Axoplasmic transport of somatostatin and substance P in the Vagus nerve of the rat, guinea pig and cat

The axoplasmic transport of somatostatin (SS) and substance P (SP) in the cervical vagus nerve was studied in the rat, guinea pig and cat. In preliminary studies, neuropeptide immunoreactivity (IR-SS and IR-SP) was evaluated in extracts of nodose ganglion and vagus nerve using gel and reverse-phase high-performance liquid chromatography (HPLC). In each species, a single immunoreactive form of SP co-eluted with the synthetic undecapeptide on a Bio-Gel P-10 column. More than 95% of transported vagal IR-SS co-eluted with synthetic SS-14. A small percentage in each species co-eluted with SS-28. No larger form, corresponding to a prosomatostatin, was identified in any of the 3 species. On HPLC, IR-SP and IR-SS co-eluted with their synthetic forms. To quantify neuropeptide transport, the vagus nerve was ligated distal to the nodose ganglion. 24 h later in each species, the content of IR-SS and IR-SP was more than 6 times greater in a 3-mm segment of nerve proximal to the ligature than in equal length segments distal to ligature or in the unligated contralateral nerve. In the proximal segment, the net content of IR-SP (pg/3-mm segment, mean +/- S.E.M.) was 366 +/- 45 in the rat, 2038 +/- 184 in the guinea pig, and 912 +/- 108 in the cat. The content of IR-SS in the same segment was 36 +/- 4, 66 +/- 13, and 575 +/- 59 pg/3-mm, respectively. The apparent transport velocities were similar for each peptide and among species. The contribution of the nodose ganglion to transported neuropeptide was estimated by crushing the vagus above the nodose ganglion and simultaneously ligating the nerve distal to the ganglion. The percent contribution of the ganglion to transported IR-SS following this procedure was 50% in the rat, 73% in the guinea pig, and 16% in the cat. Nodose ganglion contribution to IR-SP transport was 31%, 50% and 74%, respectively. Estimated turnover of IR-SS and IR-SP within the ganglion ranged from 4.1 to 6.8 times per 24 h in each species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retrograde axonal transport of receptor-bound opiate in the vagus and delayed accumulation in the nodose ganglion

Opiate receptors measured in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature. The time-course of accumulation was completely different in the proximal and the distal segments; the labelling was maximal 4 h after injection of [3H]lofentanil above the ligature but 16-24 h below the ligature. In unligated rats, a peak of radioactivity appeared in the nodose ganglion 16 h after injection; vagotomy, vinblastine or chronic treatment with capsaicin prevented the appearance of this delayed accumulation in the ganglion. These foregoing experiments suggest that opiate may act in the cell body of sensory neurones after being internalized at the nerve terminals and then transported retrogradely through fast axoplasmic mechanisms.     

Capsaicin applied to peripheral nerve inhibits axoplasmic transport of substance P and somatostatin

Capsaicin was applied locally to the sciatic or saphenous nerve, and the effects on axoplasmic transport, neurogenic plasma extravasation, and thermal pain were studied. Capsaicin (10 mg/ml) led to a complete block of axoplasmic transport of immunoreactive substance P (I-SP) and somatostatin (I-SRIF) in rat sciatic nerve without affecting the transport of noradrenaline or acetylcholinesterase. Inhibition of I-SP transport was also found in sciatic nerves of guinea-pig, cat and rabbit. In contrast, one or two weeks after systemic capsaicin treatment (125 mg/kg s.c.), orthograde transport of I-SP was the same in control and capsaicin-treated rats. After local capsaicin application to the sciatic nerve, a decrease of I-SP was found not only in skin and sciatic nerve distal to the site of application, but also in dorsal root ganglia, dorsal roots and the dorsal half of the spinal cord segments L 4-5. This was accompanied by a loss of acid phosphatase activity in the substantia gelatinosa supplied by sciatic nerve afferents. Plasma extravasation by mustard oil was reduced in the skin of the hind paw with a time course identical to the I-SP depletion. The response to noxious heat (hot plate test) was, however, abolished earlier. These results indicate that capsaicin applied to a peripheral nerve inhibits axoplasmic transport in sensory but not in adrenergic or cholinergic neurons, which leads to long-term biochemical and functional changes of the entire sensory neuron. In addition, capsaicin appears to inhibit impulse propagation in certain populations of sensory neurons.     

Novel pathway for LPS-induced afferent vagus nerve activation: possible role of nodose ganglion

The afferent vagus nerve has been suggested to be an important component for transmitting peripheral immune signals to the brain. However, there is inconsistent evidence showing that subdiaphragmatic vagotomy did not inhibit the brain mediated behavioral and neural effects induced by the peripheral application of lipopolysaccharide (LPS). LPS triggers innate immune cells through Toll-like receptor 4 (TLR4). In the present study, we found that TLR4 mRNA and protein was expressed in the rat nodose ganglion. Thus, it is suggested that LPS could activate afferent vagus nerve at the level of nodose ganglion, which exists centrally from the subdiaphragmatic level of vagus nerve. The results could provide evidence for the novel pathway of LPS-induced afferent vagus nerve activation.     

Axonal transport of substance P in the vagus and sciatic nerves of the guinea pig

The axonal transport and apparent subcellular distribution of substance P-like immunoreactive material (SPLI) were examined in nerves of guinea pigs by means of a sensitive radioimmunoassay and by immunohistofluorescence. Crushes or ligations were made at various levels above and below the nodose ganglion of the vagus, on the sciatic nerve, and on the central process of the S1 spinal ganglion. From the relative rates of accumulation of SPLI in the adjacent segments, it was concluded that the bulk of the substance P produced in the sensory ganglion cells was being exported toward the terminal regions of their peripheral branches. The average velocity of transport of SPLI in the peripheral direction was calculated to be 1 mm/h in the sciatic nerve and 1.25 mm/h in the vagus. The removal of SPLI from regions of nerve distal to a ligature indicated that only 26% of the peptide in vagus nerve and 17% of the peptide in sciatic nerve was available for rapid transport. It was therefore estimated that the mean velocity of the moving fraction was 5-6 mm/h. Stop-flow experiments with local cooling and rewarming in vivo suggested that some SPLI may have been transported as rapidly as 10 mm/h. The behavior of SPLI during ultracentrifugation of nerve and ganglion extracts indicated that this peptide was normally present both in a soluble form and in association with particles but was transported primarily in the latter form.     

An electron microscopic study on substance P-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs

Substance P-immunoreactive nerve fibers in the celiac ganglion of guinea pigs were revealed with the PAP procedures to contain abundant small clear vesicles mixed with a few large granular vesicles. The immunoreactive materials were localized around cytoplasmic components including vesicles and on the inside of the plasma membrane. The immunoreactive fibers directly apposed to unlabelled dendrites of postganglionic neurons and also to preganglionic axons. Morphological features of synapses could be identified at sites of apposition to unlabelled dendrites: clusters of vesicles in the immunoreactive fibers, intercellular spaces of about 20 nm, and an intermediate density on the postjunctional membrane of unlabelled dendrites. On the other hand, no distinct electron density together with accumulations of vesicles was seen underneath the apposed membrane of unlabelled axons. These findings indicate at the ultrastructural level that substance P-fibers form axo-dendritic synapses on the postganglionic neurons and also suggest the presence of the presynaptic interaction between substance P-fibers and some preganglionic axons in this ganglion.     

Substance P depolarizes nerve terminals in an autonomic ganglion

The effect of substance P on presynaptic nerve terminals was examined by intracellular impalements of calyciform terminals within the chick ciliary ganglion. Substance P produced a slow depolarization of the nerve terminals which was associated with an increase in input resistance. The postsynaptic ciliary neurons were unaffected by exposure to substance P, indicating that at this synapse the physiological effects of substance P may be largely presynaptic in nature.     

An electron microscopic study on enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs

Enkephalin-like immunoreactive nerve fibers in the celiac ganglion of guinea pigs were characterized by a high population of large granular vesicles mixed with small clear vesicles. The immunoreactive material is confined to the large granular vesicles. The immunoreactive nerve fibers formed many axo-dendritic as well as axo-somatic synapses and also formed a few synapses with presumed preganglionic axons containing numerous vesicles. The immunoreactive fibers were regarded as presynaptic at these synapses. These findings suggest that enkephalin might play a role as a neurotransmitter or neuromodulator in the ganglionic transmission of this prevertebral ganglion.     

Afferent projections of the cervical vagus and nodose ganglion in the dog

The distribution within the brain stem of the afferent projections of the cervical vagus and the nodose ganglion was studied with horseradish peroxidase (HRP) and HRP-wheat germ agglutinin conjugate. Two to eight days after application of tracer into the cervical vagosympathetic trunk or the nodose ganglion the brain stems and ganglia were perfused and processed by the tetramethyl benzidine method. Vagal afferent fibers entered the lateral medulla as a distinct bundle spatially separate from the vagal efferent rootlets which were caudal and ventral to the afferents. Labeled axons in the solitary tract began to enter the nucleus tractus solitarii (nTS) 4.5 mm anterior to obex and were seen throughout the ipsilateral nTS as far as 3.5 mm caudal to obex. Label density varied within the nTS, with heaviest labeling in the dorsal and dorsolateral portions. Label was also seen in the ipsilateral area postrema (ap) and dorsal motor nucleus of the vagus. Labeled fibers crossed in the commissural portions of ap and nTS to enter the contralateral ap and nTS.     

Axonal transport of opiate receptors in capsaicin-sensitive neurones

Opiate receptors measured in vitro or in vivo with [³H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature, thus indicating a bidirectional axoplasmic transport of these receptors. When rats were treated with capsaicin, the accumulation of opiate receptors was tremendously reduced in the vagus whereas muscarinic receptors in ligated sciatic nerves were unaffected. Since capsaicin is known to affect sensory neurones, mostly those containing substance P, the present results support the idea that the opiate receptors in the vagus are associated with substance P neurones.     

Peripheral but not central axotomy promotes axonal outgrowth and induces alterations in neuropeptide synthesis in the nodose ganglion of the rat

We investigated the effects of central and peripheral axotomy of the sensory neurons in the nodose ganglion on neurite outgrowth and neuropeptide expression. Axonal outgrowth was studied in ganglia subjected to a conditioning lesion of the vagus nerve 6 days prior to in vitro explantation. In such cultures, a conditioning effect, i. e. a shorter initial delay and faster axonal outgrowth, was observed after peripheral axotomy, while central axotomy had no effect. Neuropeptide expression was measured by immunocytochemistry 3 days after axotomy. Peripheral axotomy induced an increase in the number of neurons expressing the C-terminal flanking peptide of neuropeptide Y (C-PON), galanin (GAL) and vasoactive intestinal peptide (VIP). In contrast, central axotomy did not affect neuropeptide expression. These results suggest that both axonal outgrowth and expression of neuropeptides in the sensory neurons of the nodose ganglion could be regulated by the contact of the cells with their peripheral, but not their central targets.     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用

目的观察神经生长因子（nerve growth factor, NGF）对原代培养的背根神经节（dorsal root ganglion, DRG）神经元中P物质（substance P, SP）的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体（vanilloid receptor 1, VR1）mRNA的表达,用放射免疫分析（radioimmunoassay,RIA）法检测SP的基础释放量和辣椒素（100 nmol/L）刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用(英文)

目的观察神经生长因子(nerve growth factor, NGF)对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中P物质(substance P, SP)的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体(vanilloid receptor 1, VR1)mRNA的表达,用放射免疫分析(radioimmunoassay,RIA)法检测SP的基础释放量和辣椒素(100 nmol/L)刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

In vivo synthesis of substance P in the corpus striatum of the rat and its transport to the substantia nigra

Incorporation of [³⁵S]methionine into substance P in the striatum of the rat and the subsequent transport of the labelled peptide to the substantia nigra has been demonstrated in vivo. After a 4-h infusion of [³⁵S]methionine into the corpus striatum and an additional interval of 4 h radiolabelled substance P was found in the striatum and the substantia nigra of the animals. The criteria for concluding that the labelled product was substance P were: (a) gel chromatography and subsequent ion exchange chromatography of an acetic acid extract of the infused striatum or the ipsilateral substantia nigra yielded a peak of radioactivity co-eluting with endogenous immunoreactive substance P or a sample of synthetic substance P; (b) the radioactive material from this peak also co-chromatographed with synthetic substance P on high-voltage paper electrophoresis or high-pressure liquid chromatography; and (c) bound specifically to the substance P antibody. Intracisternal injection of colchicine (70 μg, i.c.) completely suppressed the appearance of radiolabelled substance P immunoreactive material in the substantia nigra.The data indicate that synthesis of substance P occurs in nerve cell bodies located in the corpus striatum and that substance P is transported to the substantia nigra by a colchicine sensitive mechanism.     

Axonal transport of [35S]methionine labeled proteins in Xenopus optic nerve: phases of transport and the effects of nerve crush on protein patterns

Axonal transport of proteins in the Xenopus optic nerve was examined by labeling proteins in the eye with [35S]methionine injected intraocularly and then analyzing the labeled proteins in the eye, nerve, and tectum on linear gradient SDS polyacrylamide gels at different times after the injection. Because the optic nerve in Xenopus is short, in order to distinguish transported proteins from locally synthesized proteins, the optic nerve on one side of the animal was crushed at the orbit (to stop axonal transport) 5-30 min prior to injection and the crushed and normal nerve segments were compared. Proteins in the intact nerve which were absent in the crushed nerve were identified as axonally transported proteins. By such criteria several waves corresponding to transported material moving at greater than or equal to 6 mm/day, 1.6-2.8 mm/day, and approximately 0.2 mm/day were detected in the nerve. The most rapid phases of transport could be further resolved in the optic tectum into 3 additional components at 60-96 mm/day, 30-48 mm/day, and 6-11 mm/day. Analysis of labeled proteins in the crushed nerves distal to the crush, near the injury site, revealed several locally synthesized proteins (mol. wt. 54,000, 48,000, 43,000 daltons) which were not present in normal, uninjured nerves. Such proteins are probably synthesized by glia in response to injury.     

Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of l-2,3-[³H]proline into newly-synthetized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2–36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of l-2,3-[³H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.     

Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P

This study examined the sciatic nerve axonal transport of substance P-like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8- and 12-week alloxan-diabetic rats, respectively. One group of diabetic rats received acetyl-l-carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced body growth. Axonal transport of SPLI was studied by measurement of 24-hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8-week diabetic rats. In the sciatic nerve of ALCAR-treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12-week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerves showed the typical sorbitol increase and myo-inositol loss that were significantly counteracted by ALCAR. This study suggests that ALCAR treatment prevents diabetes-induced sensory neuropathy by improving altered metabolic pathways such as polyol activity and myo-inositol synthesis, and by preventing the reduction of synthesis and axonal transport of substance P. © 1995 Wiley-Liss, Inc.     

Cholecystokinin-like immunoreactivity occurs in ocular sensory neurons and partially co-localized with substance P

Based on immunohistochemical analysis of the trigeminal, superior cervical, ciliary and sphenopalatine ganglia and of the eye after sensory denervation and sympathectomy, cholecystokinin (CCK)-like immunoreactive nerves in the guinea pig eye derive from the trigeminal ganglion. Substance P (SP) also occurs in some ocular sensory neurons, suggesting the possible co-localization in this system of CCK- and SP-immunoreactivities. A double-labeling immunofluorescence technique stained 3 types of trigeminal cells and ocular nerve fibers: some immunoreactive for both peptides, some immunoreactive only for CCK and some immunoreactive only for SP.     

Synthesis, and central and peripheral axonal transport of substance P in a dorsal root ganglion-nerve preparation in vitro

A preparation of the rat L5 dorsal root ganglion with 6 mm lengths of dorsal root and peripheral branch attached was incubated in vitro over a 9 h period. The substance P-like immunoreactivity (SPLI) of the preparation increased linearly with time and SPLI was transported down both branches. The turnover-time of ganglion SPLI was 3.6 h. Four times as much SPLI accumulated in the peripheral branch as in the dorsal root. When axonal transport was inhibited by demecolcine, SPLI was formed at the same rate but accumulated in the ganglion. Anisomycin inhibited SPLI synthesis after a delay of 2 h. It was apparent that the SPLI of the preparation was contained in two pools, only one of which underwent rapid axonal transport. The mobile pool of axonal SPLI comprised 30% of the total and moved with a velocity of 4.9 mm . h-1.     

Streptozotocin-induced diabetes reduces retrograde axonal transport in the afferent and efferent vagus nerve

Diabetes-induced alterations in nerve function include reductions in the retrograde axonal transport of neurotrophins. A decreased axonal accumulation of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of streptozotocin (STZ)-induced diabetic rats was previously shown. In the current study, no changes in the NGF and NT-3 protein or mRNA levels in the stomach or atrium, two vagally innervated organs, were noted after 16 or 24 weeks of diabetes. Moreover, the amounts of neurotrophin receptor (p75, TrkA, TrkC) mRNAs in the vagus nerve and vagal afferent nodose ganglion were not reduced in diabetic rats. These data suggest that neither diminished access to target-derived neurotrophins nor the loss of relevant neurotrophin receptors accounts for the diabetes-induced alteration in the retrograde axonal transport of neurotrophins. To assess whether diabetes causes a defect in axonal transport that may not be specific to neurotrophin transport, we studied the ability of a neuronal tracer (FluoroGold, FG) to be retrogradely transported by vagal neurons of control and diabetic rats. After vagal target tissue (stomach) injections of FG, the numbers of FG-labeled afferent and efferent vagal neurons were counted in the nodose ganglion and in the dorsal motor nucleus of the vagus, respectively. After 24 weeks of diabetes, FG was retrogradely transported to more than 50% fewer afferent and efferent vagal neurons in the STZ-diabetic compared to control rats. The diabetes-induced deficit in retrograde axonal transport of FG is likely to reflect alterations in basic axonal transport mechanisms in both the afferent and efferent vagus nerve that contribute to the previously observed reductions in neurotrophin transport.