Cholera Toxin-horseradish Peroxidase Research Articles

The central organization of the vagus nerve innervating the colon of the rat

Background: The extent to which the vagus nerve innervates the colon remains controversial. Methods: In 29 rats the tracer cholera toxin-horseradish peroxidase was injected into the cecum, the ascending, transverse, or descending colon or the rectum. For comparison, control injections were made into the stomach. Results: For all areas of colon except the rectum, brainstem motoneuronal labeling was limited to the lateral third of the dorsal motor nucleus of the vagus nerve bilaterally. In contrast, gastric injections resulted in motoneuronal labeling limited to the medial portions of the nucleus. The number of labeled motoneurons was greatest following injection of the cecum, and it significantly decreased for the more distal areas of the colon. Colonic motoneuron dendrites projected into the nucleus of the solitary tract and within the dorsal motor nucleus of the vagus nerve. Sensory afferent terminal labeling was limited to the commissural and medial subnuclei of the nucleus of the solitary tract. For the rectum, sensory and motor labeling was limited to the spinal cord. Conclusions: The distribution of labeling within the vagal complex indicates that all regions of the colon, except the rectum, are innervated by the celiac and accessory celiac branches of the vagus nerve.

Primary sensory afferents in the thymus of the guinea pig demonstrated with anterogradely transported horseradish peroxidase conjugates

After injections of wheat germ agglutinin- or B-subunit of cholera toxin-horseradish peroxidase (HRP) conjugates into the C 2–C 4 dorsal root ganglia of the guinea pig and anterograde axonal transport of the conjugates, labeled nerve fibers were found in the thymus mainly on the ipsilateral side. The distribution of the HRP-labeled fibers was similar to that of nerves immunoreactive to substance P and calcitonin gene-related peptide, which were partly colocalized. The results show that the thymus is innervated by primary sensory afferents which have their cell bodies in dorsal root ganglia.

Organizational and morphological features of cat sacrocaudal motoneurons.

We have investigated the organizational and morphological features of motoneurons from cat sacrocaudal spinal cord, the portion of the neuraxis that innervates the tail. This information is pertinent for development of a new model of spinal cord injury. An understanding of sacrocaudal circuitry is essential for physiological and behavioral assessment of the effects of sacrocaudal lesions. Observations from Nissl-stained sections corroborated Rexed's cytoarchitectural scheme. Putative motoneurons were located within two regions of the ventral horn: the ventromedial nucleus (lamina IX) and the nucleus commissuralis. To map motoneuron pools, cholera toxin-horseradish peroxidase conjugate was injected into each dorsal tail muscle. The dorsomedial muscle was innervated by ipsilateral nucleus commissuralis motoneurons. The dorsolateral and intertransversarius muscles were innervated by ipsilateral lamina IX and nucleus commissuralis motoneurons. Cell bodies of retrogradely labeled sacrocaudal motoneurons ranged from 22 to 82 microns in diameter; the unimodal distributions peaked between 45 and 50 microns. Dendritic trees of motoneurons, revealed by retrograde labeling or by intracellular injection with horseradish peroxidase, were extensive. Five to eight primary dendrites originated from the cell body. Dendritic branches extended throughout the ipsilateral ventral gray matter, with processes spreading into the surrounding white matter and the base of the dorsal horn. Dendrites from motoneurons with their soma in the lateral portion of lamina IX formed a longitudinal plexus at the gray/white border. Medial dendrites from motoneurons in the nucleus commissuralis formed bundles in the ventral gray commissure and spread throughout the contralateral ventral horn. It is speculated that contralateral dendrites subserve synchronized co-contraction of medial muscles from both sides of the tail.

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: A cholera toxin‐HRP study

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.

The Role of N‐Methyl‐D‐Aspartate‐Type Glutamatergic Neurotransmission in the Photic Induction of Immediate‐Early Gene Expression in the Suprachiasmatic Nuclei of the Syrian Hamster

Abstract This study investigated the role of N-methyl-D-aspartate (NMDA)-type glutamatergic neurotransmission in mediating the photic induction of immediate-early gene expression in the Suprachiasmatic nucleus (SCN) of the Syrian hamster. Activation of c-fos, c-jun and egr-1 was assessed by immunocytochemical detection of their protein products. To characterize the circadian basis to the inductive effects of light, hamsters were allowed to free-run in constant dim red light and received a 1 h light pulse at different circadian phases relative to activity onset (defined as CT 12). In control animals which did not receive light pulses, c-fos and egr-1 expression was absent or restricted to a small area of the dorsolateral region of the SCN, and expression of c-jun could not be detected in the SCN. In hamsters killed after presentation of a light pulse at either CT 14 or CT 20, there was a marked increase in c-fos and egr-1 immunoreactivities throughout the ventrolateral division of the SCN. In contrast, light pulses given at CT4 or CT 8 failed to activate immediate-early gene expression. Light pulses did not induce c-jun immunoreactivity at any circadian phase tested. Staining for c-fos was maximal 1 h after the start of the light pulse and had started to decline by 2 h. At this later time, c-jun expression was still undetectable. To compare the distribution of retinal afferents with that of c-fos induction, hamsters held on a light schedule of 16 h light: 8 h dark received an intraocular injection of cholera toxin-horseradish peroxidase conjugate 3 days before exposure to a 1 h light pulse given 2 h after lights off. Comparison of adjacent sections processed for c-fos immunoreactivity or for cholera toxin-horseradish peroxidase revealed that light-induced c-fos expression was precisely restricted to retinal terminal fields in the SCN. Light pulses also induced c-fos expression in the retinoreceptive ventral lateral geniculate nucleus and intergeniculate leaflet but not in the retinal fields of the dorsal lateral geniculate nucleus, indicating that the expression of cfos in response to light is spatially specific. The aim of the subsequent experiments was to investigate the role of NMDA-type glutamatergic neurotransmission in mediating the effects of light on c-fos expression in the SCN. To determine whether NMDA had the potential to activate c-fos expression in the SCN, hamsters were infused with 2.5 nmol NMDA or vehicle via an intracerebroventricular (icv) cannula positioned adjacent to the nuclei. In contrast to the effects of light, icv NMDA activated c-fos expression at both CT8 and CT 14. The distribution of immunoreactivity was more widespread than that observed after light, extending throughout the SCN and adjacent hypothalamus. To test whether NMDA receptors had a physiological role in the photic response, hamsters were treated systemically with the non-competitive NMDA antagonist MK801 (dose range 0.6 to 6.0 mg/kg body wt, ip) or vehicle prior to exposure to a 1 h light pulse given at CT 14 or CT 20. Expression of c-fos was still detectable in the dorsolateral SCN but MK801 blocked expression in the ventral portion of the retinoreceptive zone of the SCN. MK801 (10 or 100 nmol) delivered centrally (icv) also prevented light-induced c-fos expression in the ventral region of the SCN bordering the optic chiasm, though staining again persisted in the dorsolateral region. The induction of c-fos by icv NMDA, and the partial blockade of light-induced c-fos expression by the antagonist MK801, are consistent with the hypothesis that glutamate mediates the effects of light on SCN activity. However, the persistent photic induction of c-fos expression in a subfield of retinal afferents following treatment with MK801 suggests that other, non-NMDA-type mechanisms may contribute to photic entrainment.

Access to gastric tissue promotes the survival of axotomized neurons in the dorsal motor nucleus of the vagus in neonatal rats

Lesioning the vagus nerve in the neck (cervical vagotomy) results in a rapid and virtually complete loss of motoneurons in the dorsal motor nucleus of the vagus in neonatal rats. The present study sought to determine whether access to gastric target tissue will promote the survival of these motoneurons after axotomy. Quantitative analysis demonstrates that subdiaphragmatic vagotomy, which leaves the cut vagal axons in close proximity to their normal gastric targets, results in significantly less motoneuron loss than cervical vagotomy. Furthermore, the loss of motoneurons after cervical vagotomy can be significantly reduced by transplanting embryonic gastric tissue to the neck of vagotomized neonatal host rats, in the vicinity of the cut axons. The survival effect of transplanted gastric tissue appears specific because control transplants of embryonic bladder tissue fail to reduce motoneuron death after cervical vagotomy. Injections of the neural tracers Fluoro-Gold and cholera toxin-horseradish peroxidase into gastric transplants labeled surviving motoneurons in cervically vagotomized rats, whereas tracer injections into bladder transplants or into host cervical tissues did not. These results indicate that neonatal vagal motoneurons are capable of making the adjustments necessary to survive axotomy if they have access to gastric target cells. The apparent dependence of injured neonatal vagal motoneurons on gastric tissue offers a new system in which to examine in vivo the trophic interactions between neurons and their targets.

Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat

The motor innervation for palatal, pharyngeal, laryngeal, and esophageal muscles originates within the nucleus ambiguus. Although the viscerotopic organization of the upper alimentary tract in the nucleus ambiguus has been extensively studied, little information concerning the dendritic arborization of nucleus ambiguus motoneurons is available. The neural tracer cholera toxin-horseradish peroxidase, which is particularly effective at revealing dendrites of retrogradely labeled neurons, was used to determine the dendritic architecture and organization of nucleus ambiguus motoneurons. In 72 rats, cholera toxin-horseradish peroxidase in volumes of 1.0-18 microliters was directly applied under pressure to the musculature of various sites along the upper alimentary tract. Motoneurons innervating the soft palate, pharynx, cricothyroid muscle, and cervical esophagus were all found to have extensive dendrites that extended into the adjacent reticular formation with a distinct pattern for each muscle group. In contrast, the dendrites of motoneurons innervating the thoracic and subdiaphragmatic esophagus were confined to the compact formation of the nucleus ambiguus. Dendritic bundling within the confines of the nucleus ambiguus was prominent following injection of tracer into the soft palate, pharynx, and esophagus. The bundles were primarily oriented in a rostrocaudal direction. These data suggest that the extensive extranuclear dendritic arborization of motoneurons innervating the soft palate, pharynx, larynx, and cervical esophagus provide a wide ranging target for multiple central afferents that may be involved in the differential control of muscles that participate in multiple complex motor functions. The lack of extensive extranuclear dendrites of motoneurons innervating the distal esophagus suggest that they receive focused central afferents. The prominent bundling of dendrites within the nucleus ambiguus may provide for synchronization of motoneurons innervating a specific muscle and perhaps for synchronization of motoneurons innervating different muscles acting in sequence.

Representation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in rat

Motor fibers of the accessory celiac and celiac vagal branches are derived from the lateral columns of the dorsal motor nucleus of the vagus nerve. These branches also contain sensory fibers that terminate within the nucleus of the tractus solitarii. This study traces the innervation of the intestines by using the tracer cholera toxin-horseradish peroxidase. In 53 rats, the tracer was injected into either the stomach, duodenum, jejunum, terminal ileum, cecum, or ascending colon. With all cecal injections, prominent retrograde labeling of cell bodies occurred bilaterally in the lateral columns of the dorsal motor nucleus of the vagus nerve above, at, and below the level of the area postrema. Dendrites of laterally positioned neurons projected medially and rostrocaudally within the dorsal motor nucleus of the vagus nerve and dorsomedially into both the medial subnucleus and parts of the commissural subnucleus of the nucleus of the tractus solitarii. Sensory terminal labeling occurred in the dorsolateral commissural subnucleus at the level of the rostral area postrema and the medial commissural subnucleus caudal to the area postrema. Additionally, there was sensory terminal labeling within a small confined area of the dorsomedial zone of the nucleus of the tractus solitarii immediately adjacent to the fourth ventricle at a level just anterior to the area postrema. Stomach injections labeled motoneurons of the medial column of the entire rostrocaudal extent of the dorsal motor nucleus of the vagus nerve and a sensory terminal field primarily in the subnucleus gelatinosus, with less intense labeling extending caudally into the medial and ventral commissural subnuclei. Dendrites of gastric motoneurons project rostrocaudally and mediolaterally within the dorsal motor nucleus of the vagus nerve and dorsolaterally within the nucleus of the tractus solitarii. They are most pronounced at the level of the rostral area postrema where many dendrites course dorsolaterally terminating primarily within the subnucleus gelatinosus. Injections of the duodenum labeled a small number of the cells within the medial aspects of the dorsal motor nucleus of the vagus nerve. Jejunal, ileal, and ascending colon injections labeled cells sparsely within the lateral aspects of the dorsal motor nucleus of the vagus nerve bilaterally. No afferent terminal labeling was evident after injection of these areas of the bowel.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen regulates synaptic input to motoneurons of the adult rat spinal cord

Adult male rats (Sprague-Dawley) were castrated and implanted subcutaneously with Silastic capsules containing testosterone or nothing. Sham-castrated males served as controls. Four weeks following castration, cholera toxin-horseradish peroxidase (CT-HRP) was injected bilaterally into the bulbocavernosus muscles and animals were sacrificed 2 d later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine (TMB) method for visualization of retrogradely transported CT-HRP, and examined at the ultrastructural level. Neuronal structures apposing the membranes of 150 TMB-labeled SNB neurons were analyzed by measuring the percentage of somatic and proximal dendritic membranes covered by synaptic contacts, synaptoid contacts, and neuron-neuron contacts. Most of the neuronal structures in the control and experimental SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic and proximal dendritic membranes covered by synapses 4 weeks after castration was reduced to approximately 30% of those in control animals. However, treatment with testosterone for 4 weeks after castration prevented this decline. Castration and testosterone treatment also influenced the size and number of synaptic contacts per unit length of somatic and proximal dendritic membranes, and the incidence of neuron-neuron contacts and double synapses onto SNB motoneurons. These results indicate that androgen is critical for maintaining the organization of synaptic inputs to these spinal motoneurons in adult male rats.

The organization of vagal innervation of rat pancreas using cholera toxin—horseradish peroxidase conjugate

The present study was initiated to address the current controversy concerning the parasympathetic innervation of the pancreas, using a more sensitive tracer. The location of retrogradely labeled neurons within the dorsal motor nucleus of the vagus (DMV) was examined 48 h following injections of cholera toxin - horseradish peroxidase (CT-HRP) into designated areas of the rat pancreas. The brainstem and spinal cord were searched for any additional labeled neurons located outside of the DMV. Separate groups of animals were used for control injections into the adipose tissue of the greater omentum, the spleen, abdominal musculature, and the diaphragm. In addition, CT-HRP was dripped over the surfaces of the abdominal viscera in another group of animals. These control cases were designed to indicate whether diffusion of the neural tracer away from injection sites had occurred and had resulted in labeling of neurons which did not innervate the injected areas. Following injection of CT-HRP into the right lobe of the pancreas, labeled neurons were found primarily within the medial region of the left DMV. Injection of CT-HRP into the left lobe of the pancreas resulted in retrogradely labeled neurons predominantly within the medial region of the right DMV. Following injections into the entire pancreas, neural labeling was seen bilaterally within the DMV and was concentrated within the medial regions, with a slightly higher degree of labeling within the right DMV. No labeled neurons were seen within the nucleus ambiguus or other areas of the brainstem or spinal cord following pancreatic injections. Furthermore, no afferent labeling within the nucleus of the solitary tract (NTS) was observed, although a very small number of neurons within the nodose ganglia were labeled. The dendrites of backfilled DMV neurons could be seen extending across the midline to the contralateral DMV as well as dorsally into certain subnuclei of the NTS, and to the borders of the area postrema and the fourth ventricle. These results indicate that both the motor and sensory innervation of the rat pancreas are more restricted than has been previously suggested.

The vagal and spinal innervation of the gastro-duodenal junction of sheep.

This paper describes the distribution of vagal afferent, vagal preganglionic and thoraco-lumbar spinal afferent neurones to the gastro-duodenal junction of sheep which may form part of the structural basis for duodeno-gastric reflex mechanisms. After retrograde transport of cholera toxin-horseradish peroxidase (CT-HRP) from subserosal micro-injections into the gastro-duodenal junction, light-microscopic examination revealed CT-HRP-labelled cell bodies projecting from the medulla oblongata and the dorsal root ganglia. Medullary cell bodies were distributed bilaterally in the dorsal motor nucleus of the vagus (d.v.m.n.) 4.50 mm rostral and 2.52 mm caudal to a transverse plane through the obex. The sizes of brain-stem cell bodies, the extent of their dendritic arborization, axonal structure and orientation are described. Spinal sensory neurones were distributed to the thoraco-lumbar dorsal root ganglia T6-L3. Labelled cells and axons were found in the left and right vagal nodose ganglion. No labelled material was present in the lateral columns of the thoraco-lumbar spinal cord. A brain-stem map of medullary neurones was constructed from coronal sections of cresyl fast violet material. This enabled comparison between preparations and confirmed that all CT-HRP-labelled cell bodies and dendritic projections in the brain stem were confined to the d.v.m.n.

The central neural connections of the area postrema of the rat

We applied the neuroanatomical tracers cholera toxin-horseradish peroxidase and wheat germ agglutinin-horseradish peroxidase to investigate the neural connections of the area postrema (AP) in the rat. We find that the AP projects to the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus bilaterally both rostral and caudal to obex; the nucleus ambiguus; the dorsal aspect of the spinal trigeminal tract and nucelus and the paratrigeminal nucleus; the region of the ventrolateral medullary catecholaminergic column; the cerebellar vermis; and a cluster of structures in the dorsolateral pons which prominently include a discrete set of subnuclei in the lateral parabrachial nucleus. The major central afferent input to the area postrema is provided by a group of neurons in the paraventricular and dorsomedial hypothalamic nuclei whose collective dendrites describe a horizontally oriented plexus which encircles the parvocellular nucleus of the hypothalamus bilaterally. In addition, the caudal NTS may project lightly to the AP. The lateral parabrachial nucleus provides a very light input as well. These connections, when considered in the context of the known vagal afferent input and reduced blood-brain barrier of AP, place this structure in a unique position to receive and modulate ascending interoceptive information and to influence autonomic outflow as well.

Electrophysiological properties of neurons in the A1 area projecting to the paraventricular nucleus of the hypothalamus in rats

The lumbar spinal cord of the rat contains two sexually dimorphic motor nuclei, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN). Postnatally, SNB and DLN motoneurons grow substantially and reach their adult morphology by 7 weeks of age. The masculinization of SNB and DLN motoneuron dendrites depends upon steroid hormones. After early castration, the growth of SNB and DLN dendrites is markedly attenuated, but testosterone replacement restores this growth. In the SNB, initial dendritic growth is also supported in castrates treated with estrogen. By using castration and hormone replacement techniques, we examined the development of DLN motoneuron morphology in estrogen-treated castrated rats to determine if estrogen also supports the growth of DLN motoneurons. In addition, given that dorsal root ganglia may be a site of estrogen action, we tested the hypothesis that estrogen acts at primary afferents to support DLN dendritic growth. Thus, we attempted to block the potential trophic effect of estrogen by performing unilateral dorsal rhizotomies in estrogen-treated castrates. DLN motoneuron morphology was analyzed at 4 and 7 weeks of age by using cholera toxin horseradish peroxidase (BHRP) histochemistry. As found for SNB motoneurons, estrogen treatment transiently supported development. DLN motoneurons in estrogen-treated castrates developed normally through 4 weeks of age, but by 7 weeks, DLN motoneuron morphology in estrogen-treated castrates was no longer different from that in oil-treated castrates. Moreover, deafferentation via unilateral dorsal rhizotomy did not inhibit estrogen's ability to masculinize the early development of DLN motoneurons. Thus, the trophic effect of estrogen did not appear to act via the dorsal root ganglia to support the early postnatal development of DLN motoneurons.

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.

Cytoarchitecture of the extranuclear and commissural dendrites of hypoglossal nucleus neurons as revealed by conjugates of horseradish peroxidase with cholera toxin

The ipsilateral extranuclear dendrites of hypoglossal nucleus (HN) neurons of the rat, as revealed by conjugates of horseradish peroxidase (HRP) with cholera toxin (CTHRP), were distributed to four distinct regions: (i) to the reticular formation proper (“external dendrites” of Cajal); (ii) to the solitary nucleus and tract; (iii) to the medial longitudinal fasciculus; and (iv) to the nucleus raphe obscurus. The latter two distributions are described for the first time. They were best revealed by CTHRP; and less adequately by a wheat germ agglutinin HRP conjugate (WGHRP). They could not be demonstrated by free HRP. Furthermore, CTHRP best revealed the distribution of commissural dendrites of HN neurons, the tuftlike structures at the termination of some external dendrites of Cajal, and the few dorsal protoplasmic commissural dendrites coursing dorsal to the central canal and to the contralateral solitary nucleus and the area postrema. To further clarify the distribution of the commissural dendrites of the HN, cholera toxin HRP was injected into the tongue of rats after one hypoglossal nerve had been severed. We found that the retrogradely labeled commissural dendrites crossed the midline horizontally to enter the contralateral HN and the solitary nucleus and tract as well as the reticular formation and the medial longitudinal fasciculus. The method greatly expanded our knowledge of the course and terminations of the commissural dendrites of the “protoplasmic commissure” of Van Gehuchten. We conclude that the cholera toxin HRP conjugate was a sensitive probe for the demonstration of the dendritic arborization of retrogradely labeled neurons.

Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP

Horseradish peroxidase (HRP) conjugates of 8 different lectins (wheat germ agglutinin, ricinus communis I and II, peanut agglutinin, lens culinaris, soybean agglutinin, limulus polyhemus, ulex europaeus I) and cholera toxin (CT) or free HRP (FHRP) were individually injected into the submandibular gland (SMG) or anterior chamber (AC) of the eye and the retrogradely labeled neurons in the superior cervical ganglion (SCG) were quantitated. The effect of using 3 different cross-linking reagents (glutaraldehyde, p-benzoquinone and periodic acid) on the results obtained with HRP conjugates of wheat germ agglutinin (WG) was also examined. The results in 100 rats demonstrated the superior sensitivity of ligand-HRP conjugates over FHRP as retrogradely transported markers. After SMG injections, HRP conjugates of CT, WG and soybean agglutinin were 20–50 times more sensitive than FHRP. After AC injections, HRP conjugates of CT and WG consistently yielded labeled SCG neurons while FHRP failed to do so even when the amount of FHRP injected was increased 10-fold. The sensitivity of HRP conjugates of WG was similar after SMG injections using each of the 3 cross-linking reagents, but AC injections of conjugates produced with p-benzoquinone yielded twice as many labeled SCG neurons as the other WG conjugates. Mixtures of conjugates with and without FHRP were no more sensitive than the most sensitive individual ligand-HRP conjugates, except after SMG injections of a conjugate mixture with FHRP. Additional experiments demonstrated the specificity of the ligand-‘receptor’ interaction of WG and CT and that the superior sensitivity of these ligand-HRP conjugates does not depend on the tissue destruction produced by the injection procedure.

Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL.

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.