Choleragenoid-horseradish Peroxidase Research Articles

Selective C-fiber deafferentation of the spinal dorsal horn prevents lesion-induced transganglionic transport of choleragenoid to the substantia gelatinosa in the rat

The effect of neonatal capsaicin treatment, producing selective elimination of almost all unmyelinated C-fiber sensory axons, was studied on lesion-induced transganglionic labelling of the substantia gelatinosa of the spinal cord by choleragenoid. In both control and capsaicin-pretreated rats, the injection of choleragenoid-horseradish peroxidase conjugate into the intact sciatic nerves resulted in intense labelling only of the deeper layers of the spinal dorsal horn. In the control but not the capsaicin-pretreated rats, the injection of the tracer into sciatic nerves transected 2 weeks previously produced an intense homogeneous labelling of the substantia gelatinosa. It is concluded that the uptake and axonal transport of choleragenoid by capsaicin-sensitive C-fiber afferents may be accounted for by the lesion-induced transganglionic labelling of the substantia gelatinosa, rather than by A-fiber sprouting.

Neurotrophin-3 antisense oligonucleotide attenuates nerve injury-induced Aβ-fibre sprouting

It is proposed that following peripheral nerve injury abnormal sprouting of Aβ-fibre primary afferent neurons in the spinal cord contributes to the allodynia that often occurs with such injury. Allodynia is characterized as pain due to a stimulus which is normally non-noxious. Our recent in vivo experiments show that intrathecal administration of neurotrophin-3 (NT-3), in normal animals, induces allodynia and sprouting of Aβ-fibres. In this study, we examine whether intrathecal administration of NT-3 antisense oligonucleotides (50 μM), via an osmotic pump for 14 days, attenuates nerve injury-induced sprouting and allodynia. The oligonucleotides used in this study were phosphorothioate modified and control experiments, using an ELISA, confirm that intrathecal administration of the antisense induces a significant decrease in NT-3 levels in the spinal cord. All surgery was conducted on anaesthetized Wistar rats (sodium pentobarbitone, i.p. 50 mg/kg). Consistent with previous studies, transganglionic labelling of Aβ-fibres with choleragenoid-horseradish peroxidase (C-HRP) shows that complete transection of the sciatic nerve induces an expansion of C-HRP label into lamina II of the spinal dorsal horn. Using image analysis, we find that intrathecal administration of NT-3 antisense attenuates the density of C-HRP labelling in lamina II in nerve injured animals. A NT-3 sense oligonucleotide (50 μM) has no effect. To test the effect of NT-3 antisense on allodynia, the nociceptive flexion reflex is examined, using an Ugo Basile Analgesymeter, in animals with partial sciatic nerve ligation. Intrathecal administration of 50 μM NT-3 antisense significantly attenuates nerve injury-induced allodynia, whereas the sense oligonucleotide has no effect. These results provide further evidence that endogenous NT-3 contributes to both nerve injury-induced Aβ-fibre sprouting and allodynia and demonstrates the potential of neurotrophin-3 antisense oligonucleotides as therapeutic agents for neuropathic pain.

Neonatal capsaicin treatment prevents the normal postnatal withdrawal of A fibres from lamina II without affecting Fos responses to innocuous peripheral stimulation

The development of spinal cord sensory pathways has been investigated in postnatal day (P) 21 rat pups following neonatal capsaicin treatment. Capsaicin-induced destruction of C fibres was confirmed by 62% loss of Isolectin B4 (IB4)-binding and an 86% loss of calcitonin gene-related peptide (CGRP)-immunoreactive small diameter dorsal root ganglion cells. Neonatal capsaicin treatment prevented the normal withdrawal of choleragenoid–horseradish peroxidase (B-HRP)-labelled A fibres from lamina II (substantia gelatinosa) to deeper laminae postnatally. A fibre terminals projected more dorsally, extending into 43% of lamina II compared to vehicle-treated littermates. A small cell loss in, and/or shrinkage of, substantia gelatinosa cannot account for this. These support the concept of a competitive interaction between A and C fibre afferents to establish final terminal fields. However the continued exuberant A fibre termination in capsaicin-treated rats did not lead to continued c- fos induction in the superficial dorsal horn by innocuous stimulation. In normal development, exuberant A fibre terminals coincide with c- fos activation in lamina II by innocuous skin stimulation [23]. Despite the continued presence of exuberant A fibre terminals, c- fos was not induced by innocuous peripheral stimulation in P21 capsaicin-treated rats implying that these superficial terminals do not activate lamina II neurons in the same way as in the neonate.

Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals.

The role of L-aspartate as an excitatory neurotransmitter in primary afferent synapses in the spinal cord dorsal horn is disputed. To further investigate this issue, we examined the presence of aspartate-like immunoreactivity in primary afferent nerve terminals and other tissue components of the dorsal horn. We also examined the relationship between aspartate and glutamate immunogold labeling density and the density of synaptic vesicles in primary afferent terminals and presumed inhibitory terminals forming symmetric synapses. Weak aspartate immunosignals, similar to or lower than those displayed by presumed inhibitory terminals, were detected in both C-fiber primary afferent terminals in lamina II (dense sinusoid axon terminals, identified by morphological criteria) and in A-fiber primary afferent terminals in laminae III-IV (identified with anterograde transport of choleragenoid-horseradish peroxidase conjugate). The aspartate immunogold signal in primary afferent terminals was only about one-fourth of that in deep dorsal horn neuronal cell bodies. Further, whereas significant positive correlations were evident between synaptic vesicle density and glutamate immunogold labeling density in both A- and C-fiber primary afferent terminals, none of the examined terminal populations displayed a significant correlation between synaptic vesicle density and aspartate immunogold labeling density. Thus, our results indicate relatively low levels and a non-vesicular localization of aspartate in primary afferent terminals. It is therefore suggested that aspartate, rather than being a primary afferent neurotransmitter, serves a role in the intermediary metabolism in primary afferent terminals.

Contribution of neurotrophin-3 to the neuropeptide Y-induced increase in neurite outgrowth of rat dorsal root ganglion cells

Recent studies show that neuropeptide Y acts indirectly, via release of a neurotrophic factor(s) from the spinal cord, to increase the neurite outgrowth of dissociated adult rat dorsal root ganglion cells. This study examines further the neuropeptide Y-induced increase in neurite outgrowth. To characterize the factor(s) mediating the neuropeptide Y-induced increase in neurite outgrowth, we have examined whether antisera to either nerve growth factor or neurotrophin-3 influence the neuropeptide Y-induced increase in neurite outgrowth. Spinal cord slices were incubated with media alone or in combination with 10 nM neuropeptide Y for 2 h at 37°C. The supernatant of spinal cord incubated with neuropeptide Y significantly enhanced the neurite outgrowth of normal dorsal root ganglion cells. Antiserum against nerve growth factor had no effect on the trophic actions of the supernatant. Antiserum against neurotrophin-3, however, significantly attenuated the increase in neurite outgrowth. Consistent with this finding, neurotrophin-3 also increased the percentage of cells with neurites. Transganglionic labelling of A-fibres with choleragenoid–horseradish peroxidase in animals treated intrathecally with neurotrophin-3 for 14 days via an osmotic pump showed that the area of choleragenoid–horseradish peroxidase label expanded into lamina II. In comparison, saline-treated animals had no label in lamina II. In addition, neurotrophin-3-treated animals also had a significant decrease in mechanical nociceptive threshold. The results suggest that neuropeptide Y acts via neurotrophin-3 to mediate an increase in neurite outgrowth of dorsal root ganglion cells. These results have important implications for the mechanisms underlying neuropathic pain.

Central projections of nerves innervating the rabbit maxillary sinus localized using wheat germ agglutinin-horseradish peroxidase or choleragenoid-horseradish peroxidase.

Central projections of nerves innervating the rabbit maxillary sinus were localized by using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or choleragenoid-horseradish peroxidase (B-HRP). Tracer was placed into the left maxillary sinus; rabbits were killed 3 or 5 days later, and histochemical localization of transported WGA-HRP or B-HRP was performed. Labeled cell bodies (437-545/animal) were seen in the ipsilateral trigeminal ganglion. Very few labeled cell bodies (zero to three/animal) were observed in the contralateral ganglion. The area of cell bodies labeled by WGA-HRP appeared similar to the area of cell bodies labeled by B-HRP. Transganglionic projections from either tracer were localized to lamina II of the ipsilateral subnucleus caudalis. In addition, WGA-HRP labeling was occasionally observed in lamina I. No labeling was present in other areas of the brainstem. In contrast to the above results, other studies have demonstrated that B-HRP produces terminal-like labeling in deeper layers of the gray matter. We injected B-HRP into the infraorbital nerve and sciatic nerve, which are known to contain projections to deep layers of the gray matter. Labeling was observed in the deep layers of the medullary or spinal dorsal horn 5 days later, suggesting that nerves innervating the sinus only project to superficial laminae. These results suggest that neurons in superficial laminae of the subnucleus caudalis may be important for the reflex initiation of the increased glandular secretions in the maxillary sinus during sinusitis.

Increased neurite outgrowth of cultured rat dorsal root ganglion cells following transection or inhibition of axonal transport of the sciatic nerve

Dissociated dorsal root ganglion cells (DRGs), taken from rats 2 weeks after sciatic nerve transection, have an increase in the percentage of cells with neurites compared to DRGs taken from normal animals. This study examines the possible factors that may contribute to the nerve injury-induced increase in neuritogenesis. Topical application of the local anaesthetic, bupivicaine, either to the nerve trunk prior to transection or to the proximal nerve stump for 2 weeks had no effect on the increased neurite outgrowth induced by nerve transection. Neurite outgrowth was also not influenced by administration of either nerve growth factor (NGF) via the femoral artery into normal rats or anti-NGF antiserum to the proximal nerve stump. Inhibition of axonal transport by topical application of vinblastine, however, induced a significant increase in neurite outgrowth compared to untreated controls. In addition, vinblastine-treated animals also develop hyperalgesia to mechanical stimulation and transganglionic labelling of sensory neurons with choleragenoid-horseradish peroxidase shows that the area of termination of myelinated sensory neurons in the spinal cord expands into lamina II. The results suggest that nerve injury-induced increase in neurite outgrowth is not dependent on NGF nor nerve impulses generated at the site of injury and supports the view that the absence of an inhibitory factor(s), that in normal animals may regulate neuronal outgrowth.

Evidence that large myelinated primary afferent fibers make synaptic contacts in lamina II of neonatal rats

Choleragenoid horseradish peroxidase (B-HRP) is a retrogradely transported marker that selectively labels large cutaneous myelinated primary afferent fibers. In adults, B-HRP labelled large afferent fibers are seen to enter laminae III–V, and to a lesser extent lamina I, whereas lamina II, which is the major termination site of unmyelinated primary afferents, remains unlabelled. In the neonate, however, there is extensive B-HRP label in lamina II. The present study shows that the B-HRP labelled fibers in the neonate make many synaptic contacts in lamina II. This supports the idea that large primary afferent fibers in neonatal animals make synaptic contact with post-synaptic targets that presumably process nociceptive information. Accordingly to ameliorate pain in neonates it may be more important to block low threshold sensory input whereas in adults it would be more important to block the high threshold inputs.

Organization of hindlimb nerve projections to the rat spinal cord: a choleragenoid horseradish peroxidase study.

The aim of the present study has been to investigate the projections of hindlimb muscle afferent fibers to the spinal cord with particular emphasis on the ventral horn and the column of Clarke. Following transections of the appropriate ventral roots, injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the tibial, peroneal, hamstring, superior gluteal, femoral, and obturator nerves in one group of adult rats. In another group of rats, similar experiments were done with intact ventral roots in order to map the location in the ventral horn of the motoneuron cell columns supplying each investigated nerve. An extensive overlap was found for the different nerve projections to Rexed's laminae V-VII. A somatotopic organization of the nerve projections was seen in the lamina IX cell groups of the ventral horn as well as in the column of Clarke, even though an overlap existed. The densest primary afferent projection from each injected nerve was to its homonymous motoneurons. Only a small to moderate overlap between the projections of the tributary branches of the sciatic nerve was found in the ventral horn, whereas the obturator and femoral nerve projections showed more profound overlap. In the column of Clarke, hindlimb nerves innervating distal muscles projected medially, and nerves innervating proximal muscles projected laterally.

Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy.

We have investigated the time course and extent to which peripheral nerve lesions cause a morphological reorganization of the central terminals of choleragenoid-horseradish peroxidase (B-HRP)-labelled primary afferent fibers in the mammalian dorsal horn. Choleragenoid-horseradish peroxidase is retrogradely transported by myelinated (A) sensory axons to laminae I, III, IV and V of the normal dorsal horn of the spinal cord, leaving lamina II unlabelled. We previously showed that peripheral axotomy results in the sprouting of numerous B-HRP-labelled large myelinated sensory axons into lamina II. We show here that this spread of B-HRP-labelled axons into lamina II is detectable at 1 week, maximal by 2 weeks and persists for over 6 months postlesion. By 9 months, however, B-HRP fibers no longer appear in lamina II. The sprouting into lamina II occurs whether regeneration is allowed (crush) or prevented (section with ligation), and does not reverse at times when peripheral fibers reinnervate the periphery. We also show that 15 times more synaptic terminals in lamina II are labelled by B-HRP 2 weeks after axotomy than in the normal. We interpret this as indicating that the sprouting fibers are making synaptic contacts with postsynaptic targets. This implies that A-fiber terminal reorganization is a prominent and long-lasting but not permanent feature of peripheral axotomy. We also provide evidence that this sprouting is the consequence of a combination of an atrophic loss of central synaptic terminals and the conditioning of the sensory neurons by peripheral axotomy. The sprouting of large sensory fibers into the spinal territory where postsynaptic targets usually receive only small afferent fiber input may bear on the intractable touch-evoked pain that can follow nerve injury.

Morphological relationships of choleragenoid horseradish peroxidase‐labeled spinal primary afferents with myenteric ganglia and mucosal associated lymphoid tissue in the cat esophagogastric junction

The goal of the present study was to gain insight into the environmental factors influencing the activity of primary spinal afferent fibers in the different layers of the esophagogastric junction of the cat and, thus, to analyze the relationships of these afferents with various cellular components. Spinal primary afferent fibers were selectively labeled by anterogradely transported choleragenoid horseradish peroxidase conjugate (B-HRP). B-HRP was injected into the thoracic dorsal root ganglion at the T8-T13 levels. 6-Hydroxydopamine-induced sympathectomy was performed prior to B-HRP injection in order to prevent otherwise unavoidable labeling of sympathetic fibers in the gut wall. Numerous labeled fibers ran between, around, and within the myenteric ganglia. Others crossed the muscle layers directly and entered the mucosa, where some ran near granulocytes and around or through solitary lymphoid follicles. Labeled fibers were observed in the squamous esophageal epithelium but not in the fundic glandular epithelium. The fibers in the myenteric area are probably connected to the muscular tension receptors that have been detected by electrophysiologic techniques. This assumption is based on the observation that only a few fibers appear to terminate in muscle layers and on the fact that the myenteric area is very narrow and subject to powerful forces. Fibers in the myenteric ganglia could be involved in local efferent functions. Fibers in the mucosa could act as nociceptors and might be involved in local immunological responses.

Evidence for vesicular storage of glutamate in primary afferent terminals.

The correlation between the density of gold particles signalling fixed glutamate and the density of synaptic vesicles was examined in primary afferent terminals in the rat dorsal horn. Statistically significant positive correlations between these parameters were found in lamina I and III-V primary afferent terminals, identified through axonal transport of choleragenoid-horseradish peroxidase conjugate, and in lamina II dense sinusoid axon terminals of presumed thin calibre primary afferent origin. Profiles containing pleomorphic synaptic vesicles displayed negative or statistically insignificant correlations between the density of synaptic vesicles and that of gold particles. These observations indicate that primary afferent terminals store glutamate in synaptic vesicles and provide further evidence for a role of glutamate as a primary afferent neurotransmitter.

Enrichment of Glutamate‐like Immunoreactivity in Primary Afferent Terminals Throughout the Spinal Cord Dorsal Horn

Although several lines of evidence indicate that glutamate is a neurotransmitter in primary afferent terminals, controversies exist on the proportion and types of such terminals that release glutamate. In the present study quantitative analysis of immunogold labelling was used to assess the presence of glutamate-like immunoreactivity in primary afferent terminals in laminae I-V of the rat spinal cord dorsal horn. Anterograde transport of choleragenoid-horseradish peroxidase from a spinal ganglion and tetramethyl benzidine histochemistry were used to identify primary afferent terminals in laminae I and III-V. Presumed C-fibre terminals in lamina II were identified on morphological criteria (dense sinusoid axon terminals). Primary afferent terminals in all dorsal horn laminae displayed significantly higher levels of glutamate-like immunoreactivity than pleomorphic vesicle-containing profiles in laminae III-IV and large neuronal cell bodies in laminae III-V. The density of gold particles over primary afferent terminals also significantly exceeded the average density of gold particles over laminae II and III-IV. The highest densities of gold particles were present over dense sinusoid axon terminals in lamina II. These findings suggest that glutamate, alone or in combination with other neuroactive compounds, is involved in the transfer of all sensory modalities from primary afferent fibres to dorsal horn neurons.

Brain stem projections of rat lumbar dorsal root ganglia studied with choleragenoid conjugated horseradish peroxidase.

Brain stem projections from each of the L1-L6 lumbar dorsal root ganglia (DRGs) were investigated in adult rats following DRG injections of choleragenoid-horseradish peroxidase. All these DRGs projected throughout the rostrocaudal extent of the gracile nucleus (Gr). Labeling from L1-L6 DRGs was transported to successively more dorsomedial areas of Gr. Investigation of the Gr projections from the DRGs revealed a somatotopic organization which was most prominent in the middle part of Gr. The cuneate nucleus showed smaller projections from all investigated DRGs. Minor projections to the internal basilar nucleus, external cuneate nucleus, medial vestibular nucleus, ventral cochlear nucleus and trigeminal sensory nuclei were also found from some of the DRGs.

Choleragenoid horseradish peroxidase used for studying projections of some hindlimb cutaneous nerves and plantar foot afferents to the dorsal horn and Clarke's column in the rat

The aim of the present study has been to investigate the spinal projections of cutaneous hindlimb afferents particularly to the deep dorsal horn and to Clarke's column (CC), by using the B-subunit of cholera toxin conjugated to horseradish peroxidase. Injections into three different cutaneous hindlimb nerves in adult rats resulted in dense labeling in the dorsal horn laminae IIi-IV/V, moderate labeling in lamina I and modest labeling in dorsomedial parts of CC. Footpad injections gave similar results, except for a lack of labeling in CC and only weak labeling in laminae I and V. The results suggest that B-HRP should be a useful marker for studying cutaneous myelinated nerve fiber projections to the rat spinal cord.

Distribution of lumbar dorsal root fibers in the lower thoracic and lumbosacral spinal cord of the rat studied with choleragenoid horseradish peroxidase conjugate

Spinal cord projections from lumbar dorsal root ganglia (DRGs) were investigated in adult rats following injections of choleragenoid horseradish peroxidase (B-HRP) into each of the six lumbar DRGs. This method is known to label primarily thick fibers. Labeling was found in all laminae except in the outer part of lamina II. Labeled fibers and terminal-like structures were found 8-13 segments rostral and 1-5 segments caudal to the injected DRGs. A somatotopic organization was revealed in laminae III, where the labeling seemed to be organized in mediolateral zones. Some of these protruded as fingerlike processes through segments rostral and caudal to the root entry level. An interdigitating pattern for these fingerlike processes was seen between some DRGs, while an extensive overlap was found between other DRGs. Many zones were found to correspond to the known central projections of peripheral sensory nerves supplied by the injected ganglion. This suggests that the central projection of a DRG is highly related to the projections of the peripheral nerves included in the DRG. The projections to lamina IV were organized in a similar manner as in lamina III, even though the projections showed a higher degree of overlap than in lamina III. No clear somatotopic organization was found in laminae V-IX. Provided that the topographical relationship between central projections of single peripheral nerves and of DRGs correspond to their peripheral projections, the results of this study, together with results of earlier studies suggest that the outlines of dermatomes are highly related to the territories of peripheral nerves included in the dermatomes.

Dose- and time-dependent selective and non-selective effects of ricin (RCA 120) on rat primary sensory neurons

The distribution of degenerating fibers in the spinal cord was studied in Fink-Heimer-stained sections following treatment of tibial nerve with ricinus communis agglutinin (RCA 120). The ricin was either injected into the nerve or applied in a capsule on the transected nerve. Short survival times and low doses of ricin resulted in degeneration in somatotopically appropriate parts of the medial dorsal horn. Longer survival times and higher doses resulted in degeneration which progressively expanded into inappropriate areas in the central and lateral parts of the dorsal horn and in deeper laminae regardless of the mode of application. Furthermore, the effect of a ricin injection into the tibial nerve on transganglionic transport of choleragenoid horseradish peroxidase (B-HRP) in the peroneal nerve was studied following a simultaneous or delayed B-HRP injection. A simultaneous ricin and a B-HRP injection resulted in primary afferent HRP labeling in the gray matter, regardless of the dose of ricin. Following a delayed B-HRP injection almost no primary afferent labeling was seen in the gray matter, unless a very low dose of ricin was injected. This study shows that treatment of a peripheral nerve with a high dose of ricin and a long survival time may result in a considerable non-selective degeneration of fibers in the spinal cord. A selective degeneration may, however, be obtained by using lower doses or shorter survival times.

A comparison between wheat germ agglutinin- and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat

Horseradish peroxidase conjugates of either the lectin wheat germ agglutinin or choleragenoid, the binding subunit of cholera toxin, were injected into the L5 spinal ganglion of adult rats. This enabled comparison of these two conjugates as anterograde tracers in the primary sensory system. After a postoperative survival of 4 h to 30 days, the rats were perfused and frozen sections from spinal and medullary regions receiving primary afferents were processed for horseradish peroxidase histochemistry with tetramethylbenzidine as the chromogen. Additional observations were made in two adult cats. Following injection of wheat germ agglutinin-horseradish peroxidase the labelling appeared mostly as small-sized granules. The concentration of labelled primary afferents in the grey matter of the spinal cord was greatest in the marginal zone and the substantia gelatinosa and less pronounced in the deep parts of the dorsal horn. Labelling was also found in a region lateral to the central canal and in the ventral horn. Following injection of choleragenoid-horseradish peroxidase the labelling appeared mainly as larger-sized granular profiles. The concentration of labelled primary afferents was greatest in the deep part of the dorsal horn and pronounced in a region lateral to the central canal and in the ventral horn. All these regions are known to receive large calibre fibres. The marginal zone and the substantia gelatinosa, known to receive fine calibre fibres, showed almost no labelling in the rat. In the cat, however, there was somewhat more labelling in the substantia gelatinosa. Labelling of neuronal cell bodies indicating transneuronal transport was seen after injection of wheat germ agglutinin-horseradish peroxidase. Transneuronal labelling did not seem to occur after injection of choleragenoid-horseradish peroxidase. The present findings show that wheat germ agglutinin- and choleragenoid-horseradish peroxidase give rise to markedly different labelling patterns. A possible explanation for the different labelling in the marginal zone and substantia gelatinosa could be that certain primary sensory neurones lack either receptors for choleragenoid on their neuronal plasma membrane or the ability to transport the choleragenoid-horseradish peroxidase complex.

NEURON PATTERNS CONTROLLING TRANSMISSION OF IPSILATERAL HIND LIMB REFLEXES IN CAT

NEURON PATTERNS CONTROLLING TRANSMISSION OF IPSILATERAL HIND LIMB REFLEXES IN CAT