Cholera Toxin-conjugated Horseradish Peroxidase Research Articles

Differential effects of exercise and hormone treatment on spinal cord injury-induced changes in micturition and morphology of external urethral sphincter motoneurons.

Spinal cord injury (SCI) results in lesions that destroy tissue and spinal tracts, leading to deficits in locomotor and autonomic function. We have previously shown that after SCI, surviving motoneurons innervating hindlimb muscles exhibit extensive dendritic atrophy, which can be attenuated by treadmill training or treatment with gonadal hormones post-injury. We have also shown that following SCI, both exercise and treatment with gonadal hormones improve urinary function. Animals exercised with forced running wheel training show improved urinary function as measured by bladder cystometry and sphincter electromyography, and treatment with gonadal hormones improves voiding patterns as measured by metabolic cage testing. The objective of the current study was to examine the potential protective effects of exercise or hormone treatment on the structure and function of motoneurons innervating the external urethral sphincter (EUS) after contusive SCI. Gonadally intact young adult male rats received either a sham or a thoracic contusion injury. Immediately after injury, one cohort of animals was implanted with subcutaneous Silastic capsules filled with estradiol (E) and dihydrotestosterone (D) or left blank; continuous hormone treatment occurred for 4 weeks post-injury. A separate cohort of SCI-animals received either 12 weeks of forced wheel running exercise or no exercise treatment starting two weeks after injury. At the end of treatment, urinary void volume was measured using metabolic cages and EUS motoneurons were labeled with cholera toxin-conjugated horseradish peroxidase, allowing for assessment of dendritic morphology in three dimensions. Locomotor performance was improved in exercised animals after SCI. Void volumes increased after SCI in all animals; void volume was unaffected by treatment with exercise, but was dramatically improved by treatment with E + D. Similar to what we have previously reported for hindlimb motoneurons after SCI, dendritic length of EUS motoneurons was significantly decreased after SCI compared to sham animals. Exercise did not reverse injury-induced atrophy, however E + D treatment significantly protected dendritic length. These results suggest that some aspects of urinary dysfunction after SCI can be improved through treatment with gonadal hormones, potentially through their effects on EUS motoneurons. Moreover, a more comprehensive treatment regime that addresses multiple SCI-induced sequelae, i.e., locomotor and voiding deficits, would include both hormones and exercise.

EMG Testing throughout behavioral recovery after rat sciatic nerve crush injury results in exuberant motoneuron dendritic hypertrophy.

Peripheral nerve injury (PNI) is the most common type of nerve trauma yet, while injured motoneurons exhibit a robust capacity for regeneration, behavioral recovery is protracted and typically poor. Neurotherapeutic approaches to PNI and repair have primarily focused on the enhancement of axonal regeneration, in terms of rate, axonal sprouting, and reconnection connectivity. Both electrical stimulation (ES) and treatment with androgens [e.g., testosterone propionate (TP)] have been demonstrated to enhance axonal sprouting, regeneration rate and functional recovery following PNI. To date, very little work has been done to examine the effects of ES and/or TP on dendritic morphology and organization within the spinal cord after PNI. The objective of the current study was to examine the impact of treatment with TP and ES, alone or in combination, on the dendritic arbor of spinal motoneurons after target disconnection via sciatic nerve crush injury in the rat. Rats received a crush injury to the sciatic nerve. Following injury, some animals received either (1) no further treatment beyond implantation with empty Silastic capsules, (2) electrical nerve stimulation immediately after injury, (3) implantation with Silastic capsules filled with TP, or (4) electrical nerve stimulation immediately after injury as well as implantation with TP. All of these groups of axotomized animals also received bi-weekly electromyography (EMG) testing. Additional groups of intact untreated animals as well as a group of injured animals who received no further treatment or EMG testing were also included. Eight weeks after injury, motoneurons innervating the anterior tibialis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. After nerve crush and ES and/or TP treatment, motoneurons innervating the anterior tibialis underwent marked dendritic hypertrophy. Surprisingly, this dendritic hypertrophy occurred in all animals receiving repeated bi-weekly EMG testing, regardless of treatment. When the EMG testing was eliminated, the dendritic arbor extent and distribution after nerve crush in the treated groups did not significantly differ from intact untreated animals. The ability of repeated EMG testing to so dramatically affect central plasticity following a peripheral nerve injury was unexpected. It was also unexpected that gonadal steroid hormones and/or ES, two neurotherapeutic approaches with demonstrated molecular/behavioral changes consistent with peripheral improvements in axonal repair and target reconnection, do not appear to impact central plasticity in a similar manner. The significance of peripheral EMG testing and resulting central plasticity reorganization remains to be determined.

Intraneural Topography of Rat Sciatic Axons: Implications for Polyethylene Glycol Fusion Peripheral Nerve Repair.

Peripheral nerve injuries are the most common type of nerve trauma. We have been working with a novel repair technique using a plasmalemmal fusogen, polyethylene glycol (PEG), to re-fuse the membranes of severed axons. PEG-fusion repair allows for immediate re-innervation of distal targets, prevents axonal degeneration, and improves behavioral recovery. PEG-fusion of severed axons is non-specific, and we have previously reported that following injury and PEG-fusion misconnections between spinal motoneurons and their distal targets were present. Surprisingly, appropriately paired proximal and distal motor axons were observed in all PEG-fused animals. We hypothesized that a topographic organization of axons contributing to the sciatic nerve could explain the incidence of appropriate connections. We traced the course of specific axon populations contributing to the sciatic nerve in young adult male and female rats. Following intraneural injection of Fast Blue into the tibial branch, labeled axons were confined to a discrete location throughout the course of the nerve. Following intramuscular injection of cholera toxin-conjugated horseradish peroxidase into the anterior tibialis, labeled axons were confined to a smaller but still discrete location throughout the nerve. In both cases, the relative locations of labeled axons were consistent bilaterally within animals, as well as across animals and sexes. Thus, the relatively consistent location of specific axon populations could allow for realignment of appropriate populations of axons, and enhanced behavioral recovery seen in PEG-fused animals. Knowing the organization of axons within the sciatic nerve promotes accurate territory realignment during repair, therefore aiding in recovery outcomes.

Exercise is neuroprotective on the morphology of somatic motoneurons following the death of neighboring motoneurons via androgen action at the target muscle.

Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that an androgen-dependent effect of exercise following injury can be neuroprotective against this dendritic atrophy. In this study, we explored where the necessary site of androgen action is for exercise-driven neuroprotective effects on induced dendritic atrophy. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected animals were given implants of the androgen receptor antagonist hydroxyflutamide, either directly at the adjacent vastus lateralis musculature ipsilateral to the saporin-injected vastus medialis or interscapularly as a systemic control. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the same vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Dendritic arbor lengths of saporin-injected animals allowed to exercise were significantly longer than those not allowed to exercise. Androgen receptor blockade locally at the vastus lateralis muscle prevented the protective effect of exercise. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites, which acts via androgen receptor action at the target muscle.

Neuroprotective Effects of Exercise on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons

Background. Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that treatment with androgens can be neuroprotective against this dendritic atrophy. Exercise has also been shown to be protective following a variety of neural injury models and, in some cases, is dependent on androgen action. Objective. In this study, we explored whether exercise shows the same neuroprotective effect on induced dendritic atrophy as that seen with androgen treatment. Methods. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in 3 dimensions. Results. Dendritic arbor lengths of animals allowed to exercise were significantly longer than those not allowed to exercise. Conclusions. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites comparable to that seen with exogenous androgen treatment.

Neuroprotective Effects on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons: A Role for Microglia?

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone protects motoneurons from induced dendritic atrophy. We explored a potential mechanism for this induced atrophy and protection by testosterone, examining the microglial response to partial depletion of motoneurons. Motoneurons innervating the vastus medialis muscles of adult male rats were killed by intramuscular injection of cholera toxin-conjugated saporin; some saporin-injected rats were treated with testosterone. Microglia were later visualized via immunohistochemical staining, classified as monitoring or activated, and counted stereologically. Partial motoneuron depletion increased the number of activated microglia in the quadriceps motor pool, and this increase was attenuated with testosterone treatment. The attenuation in microglial response could reflect an effect of testosterone on suppressing microglia activation, potentially sparing motoneuron dendrites. Alternatively, testosterone could be neuroprotective, sparing motoneuron dendrites, secondarily resulting in reduced microglial activation. To discriminate between these hypotheses, following partial motoneuron depletion, rats were treated with minocycline to inhibit microglial activation. Motoneurons innervating the ipsilateral vastus lateralis muscle were later labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed. Reduction of microglial activation by minocycline did not prevent induced dendritic atrophy following partial motoneuron depletion. Further, reduction of microglial activation by minocycline treatment resulted in dendritic atrophy in intact animals. Together, these findings indicate that the neuroprotective effect of testosterone on dendrites following motoneuron death is not due to inhibiting microglial activation, and that microglial activity contributes to the normal maintenance of dendritic arbors.

Neuroprotective effects of testosterone metabolites and dependency on receptor action on the morphology of somatic motoneurons following the death of neighboring motoneurons.

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone is neuroprotective, attenuating induced dendritic atrophy. In this study we examined whether the protective effects of testosterone could be mediated via its androgenic or estrogenic metabolites. Furthermore, to assess whether these neuroprotective effects were mediated through steroid hormone receptors, we used receptor antagonists to attempt to prevent the neuroprotective effects of hormones after partial motoneuron depletion. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected rats were treated with either dihydrotestosterone or estradiol, alone or in combination with their respective receptor antagonists, or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal animals, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Dendritic atrophy was attenuated with both dihydrotestosterone and estradiol treatment to a degree similar to that seen with testosterone, and attenuation of atrophy was prevented by receptor blockade. Together, these findings suggest that neuroprotective effects on motoneurons can be mediated by either androgenic or estrogenic hormones and require action via steroid hormone receptors, further supporting a role for hormones as neurotherapeutic agents in the injured nervous system. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 691-707, 2017.

Inhibition of Cytosolic Phospholipase A2 Has Neuroprotective Effects on Motoneuron and Muscle Atrophy after Spinal Cord Injury.

Surviving motoneurons undergo dendritic atrophy after spinal cord injury (SCI), suggesting an important therapeutic target for neuroprotective strategies to improve recovery of function after SCI. Our previous studies showed that cytosolic phospholipase A2 (PLA2) may play an important role in the pathogenesis of SCI. In the present study, we investigated whether blocking cytosolic PLA2 (cPLA2) pharmacologically with arachidonyl trifluoromethyl ketone (ATK) or genetically using cPLA2 knockout (KO) mice attenuates motoneuron atrophy after SCI. C57BL/6 mice received either sham or contusive SCI at the T10 level. At 30 min after SCI, mice were treated with ATK or vehicle. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. ATK administration reduced percent lesion volume and increased percent volume of spared white matter, compared to the vehicle-treated control animals. SCI with or without ATK treatment had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with ATK. Similarly, vastus lateralis muscle weights of untreated SCI animals were smaller than those of sham surgery controls, and these reductions were prevented by ATK treatment. No effects on fiber cross-sectional areas, motor endplate area, or density were observed across treatment groups. Remarkably, genetically deleting cPLA2 in cPLA2 KO mice attenuated dendritic atrophy after SCI. These findings suggest that, after SCI, cord tissue damage and regressive changes in motoneuron and muscle morphology can be reduced by inhibition of cPLA2, further supporting a role for cPLA2 as a neurotherapeutic target for SCI treatment.

Neuroprotective effects of testosterone on motoneuron and muscle morphology following spinal cord injury.

Treatment with testosterone is neuroprotective/neurotherapeutic after a variety of motoneuron injuries. Here we assessed whether testosterone might have similar beneficial effects after spinal cord injury (SCI). Young adult female rats received either sham or T9 spinal cord contusion injuries and were implanted with blank or testosterone-filled Silastic capsules. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. Contusion injury resulted in large lesions, with no significant differences in lesion volume, percent total volume of lesion, or spared white or gray matter between SCI groups. SCI with or without testosterone treatment also had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with testosterone. Similarly, the vastus lateralis muscle weights and fiber cross-sectional areas of untreated SCI animals were smaller than those of sham-surgery controls, and these reductions were both prevented by testosterone treatment. No effects on motor endplate area or density were observed across treatment groups. These findings suggest that regressive changes in motoneuron and muscle morphology seen after SCI can be prevented by testosterone treatment, further supporting a role for testosterone as a neurotherapeutic agent in the injured nervous system.

Overexpression of Androgen Receptors in Target Musculature Confers Androgen Sensitivity to Motoneuron Dendrites

The dendritic arbors of spinal motoneurons are dynamically regulated by a variety of factors, and several lines of evidence indicate that trophic interactions with the target musculature are of central importance. In highly androgen-sensitive motoneuron populations, androgens are thought to regulate motoneuron dendrites through their action at the receptor-enriched target musculature. Using rats transgenically modified to overexpress androgen receptor (AR) in skeletal muscle, we directly tested the hypothesis that the enhanced expression of AR in the target musculature can underlie the androgenic regulation of motoneuron dendritic morphology. The morphology of motoneurons innervating the quadriceps muscle was examined in wild-type (WT) rats as well as in rats that had been transgenically modified to overexpress ARs in their skeletal musculature. Motoneurons innervating the vastus lateralis muscle of the quadriceps in gonadally intact male rats, and castrated males with or without androgen replacement, were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. In WT rats, quadriceps motoneuron dendrites were insensitive to hormonal manipulation. In contrast, quadriceps motoneuron dendrites in gonadally intact transgenic males were larger than those of WT males. Furthermore, overexpression of ARs in the quadriceps muscle resulted in androgen sensitivity in dendrites, with substantial reductions in dendritic length occurring after castration; this reduction was prevented with testosterone replacement. Thus, it appears that the androgen sensitivity of motoneuron dendrites is conferred indirectly via the enrichment of ARs in the musculature.

Neuroprotective effects of testosterone on the morphology and function of somatic motoneurons following the death of neighboring motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have previously shown that partial depletion of motoneurons from sexually dimorphic, highly androgen-sensitive spinal motor populations induces dendritic atrophy in remaining motoneurons, and this atrophy is attenuated by treatment with testosterone. To test whether testosterone has similar effects in more typical motoneurons, we examined potential neuroprotective effects in motoneurons innervating muscles of the quadriceps. Motoneurons innervating the vastus medialis muscle were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected rats were given implants containing testosterone or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal males, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons, and this atrophy was attenuated by testosterone treatment. To examine the functional consequences of the induced dendritic atrophy, and its attenuation with testosterone treatment, the activation of remaining quadriceps motoneurons was assessed using peripheral nerve recording. Partial motoneuron depletion resulted in decreased amplitudes of motor nerve activity, and these changes were attenuated by treatment with testosterone, providing a functional correlate to the neuroprotective effects of testosterone treatment on quadriceps motoneuron morphology. Together these findings suggest that testosterone has neuroprotective effects on morphology and function in both highly androgen-sensitive as well as more typical motoneuron populations, further supporting a role for testosterone as a neurotherapeutic agent in the injured nervous system.

Central neural activation of hyperdynamic circulation in portal hypertensive rats depends on vagal afferent nerves

Background:Hyperdynamic circulation in portal hypertensive rats depends on central neural c-fos gene expression, but how the portal hypertensive signal activates central c-fos expression remains obscure.Aim:To elucidate the afferent pathway from...

Androgen-sensitivity of somata and dendrites of spinal nucleus of the bulbocavernosus (SNB) motoneurons in male C57BL6J mice

In rats, androgens in adulthood regulate the morphology of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), including the size of their somata and the length of their dendrites. There are conflicting reports about whether androgens exert similar influences on SNB motoneurons in mice. We castrated or sham-operated C57BL6J mice at 90 days of age and, thirty days later, injected cholera toxin conjugated horseradish peroxidase into the bulbocavernosus muscle (to label SNB motoneurons) on one side, and into intrinsic foot muscles contralaterally (to label motoneurons of the retrodorsolateral nucleus (RDLN)). Castrated mice had significantly smaller SNB somas compared to sham-operated mice while there were no differences in soma size of RDLN motoneurons. Dendritic length in C57BL6J mice, estimated in 3-dimensions, also decreased significantly after adult castration. In rats, androgens act directly through androgen receptors (AR) in SNB motoneurons to control soma size and nearly all SNB motoneurons contain AR. Since SNB somata in C57BL6J mice shrank after adult castration, we used immunocytochemistry to characterize AR expression in SNB cells as well as motoneurons in the RDLN and dorsolateral nucleus (DLN). A pattern of labeling matched that seen previously in rats: the highest percentage of AR-immunoreactive motoneurons are in the SNB (98%), the lowest in the RDLN (25%) and an intermediate number in the DLN (78%). This pattern of AR labeling is consistent with the possibility that androgens also act directly on SNB motoneurons in mice to regulate soma size in mice.

Fine structural survey of the intermediate subnucleus of the nucleus tractus solitarii and its glossopharyngeal afferent terminals

The intermediate subnucleus of the nucleus tractus solitarii (imNTS) receives somatosensory inputs from the soft palate and pharynx, and projects onto the nucleus ambiguus, thus serving as a relay nucleus for swallowing. The ultrastructure and synaptology of the rat imNTS, and its glossopharyngeal afferent terminals, have been examined with cholera toxin-conjugated horseradish peroxidase (CT-HRP) as an anterograde tracer. The imNTS contained oval or ellipsoid-shaped, small to medium-sized neurons (18.2 x 11.4 microm) with little cytoplasm, few cell organelles and an irregularly shaped nucleus. The cytoplasm often contained one or two nucleolus-like stigmoid bodies. The average number of axosomatic terminals was 1.8 per profile. About 83% of them contained round vesicles and formed asymmetric synaptic contacts (Gray's type I), while about 17% contained pleomorphic vesicles and formed symmetric synaptic contacts (Gray's type II). The neuropil contained small or large axodendritic terminals, and about 92% of them were Gray's type I. When CT-HRP was injected into the nodose ganglion, many labeled terminals were found in the imNTS. All anterogradely labeled terminals contacted dendrites but not somata. The labeled terminals were usually large (2.69+/-0.09 mum) and exclusively of Gray's type I. They often contacted more than two dendrites, were covered with glial processes, and formed synaptic glomeruli. A small unlabeled terminal occasionally made an asymmetric synaptic contact with a large labeled terminal. The large glossopharyngeal afferent terminals and the neurons containing stigmoid bodies characterized the imNTS neurons that received pharyngeal afferents.

Purkinje cell activity in the cerebellar anterior lobe after rabbit eyeblink conditioning

The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus. Rabbits were trained in an interstimulus interval discrimination procedure in which one tone signaled a 250-msec conditioned stimulus-unconditioned stimulus (CS-US) interval and a second tone signaled a 750-msec CS-US interval. All rabbits showed conditioned responses to each CS with mean onset and peak latencies that coincided with the CS-US interval. Many anterior lobe Purkinje cells showed significant learning-related activity after eyeblink conditioning to one or both of the CSs. More Purkinje cells responded with inhibition than with excitation to CS presentation. In addition, when the firing patterns of all conditioning-related Purkinje cells were pooled, it appeared that the population showed a pattern of excitation followed by inhibition during the CS-US interval. Using cholera toxin-conjugated horseradish peroxidase, Purkinje cells in recording areas were found to project to the interpositus nucleus. These data support previous studies that have suggested a role for the anterior cerebellar cortex in eyeblink conditioning as well as models of cerebellar-mediated CR timing that postulate that Purkinje cell activity inhibits conditioned response (CR) generation during the early portion of a trial by inhibiting the deep cerebellar nuclei and permits CR generation during the later portion of a trial through disinhibition of the cerebellar nuclei.

Cells of origin of vagal motor neurons projecting to different parts of the stomach in the rat: confocal laser scanning and electron microscopic study.

Parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) innervate the stomach by way of the gastric and hepatic branches of the vagus nerve. To investigate whether single neurons of the DMV provide collateral innervations to various parts of the stomach, we injected the retrograde tracer Fluoro-Gold (FG) into the cardia and the retrograde tracer cholera toxin subunit b (CTb) into the antrum or the pylorus of the same animal. Both retrogradely FG-labeled and CTb-labeled neurons were found throughout the DMV. Almost all CTb-labeled neurons (97%) were double-labeled with FG after injection of FG into the cardia and CTb into the antrum, while only a few CTb-labeled neurons (11%) were double-labeled with FG after injection of FG into the cardia and CTb into the pylorus. Thus, the cardia and the antrum received collateral projections, but the pylorus received projections mainly from different neurons in the DMV. These results indicate that different neurons in the DMV activate either the cardia or the pyloric sphincter muscles. We also labeled, retrogradely, the neurons projecting to the cardia and the pylorus in the DMV with cholera toxin-conjugated horseradish peroxidase (CT-HRP) to examine their ultrastructural characteristics. Although the neurons projecting to the cardia (21.6x15.0 microm, 248.0 microm2 per section) were significantly smaller than the neurons projecting to the pylorus (27.5x15.9 microm, 323.2 microm2 per section), their ultrastructural appearances were similar. Both types of neurons were small-to-medium sized, round or oval in shape, and generally had a small amount of cytoplasm containing a few Nissl bodies and a round nucleus. The average number of axosomatic terminals per section was low in the neurons projecting to the cardia (2.3) and the neurons projecting to the pylorus (3.0). Almost all axon terminals contacting these motor neurons contained round synaptic vesicles and made asymmetric synaptic contacts (Gray's type I).

Ultrastructure of the central subnucleus of the nucleus tractus solitarii and the esophageal afferent terminals in the rat.

The central subnucleus of the nucleus tractus solitarii (ceNTS) receives afferent projections from the esophageal wall and projects to the nucleus ambiguus, thus serving as a relay nucleus for peristalsis of the esophagus. Here we examine the synaptic organization of the ceNTS, and its esophageal afferents by using transganglionic anterograde transport of cholera toxin-conjugated horseradish peroxidase (CT-HRP). When CT-HRP was injected into the subdiaphragmatic esophagus, many anterogradely labeled terminals were found only in the ceNTS. The ceNTS was composed of round or oval-shaped, small neurons (14.7x8.7 micro m) containing sparse organelles and an irregularly shaped nucleus. The average number of axosomatic terminals was only 1.3 per section cut through the nucleolus. Most of them (92%) contained round vesicles and formed asymmetric synaptic contacts (Gray's type I), and a few (8%) contained pleomorphic vesicles and formed symmetric synaptic contacts (Gray's type II). All anterogradely labeled terminals contacted dendrites but not the neuronal somata. The labeled terminals were large (2.55+/-0.07 micro m) and exclusively Gray's type I. More than half of them (60%) contacted small dendrites (less than 1 micro m in diameter), and contained dense-cored vesicles. More than 40% of the labeled terminals contacted two to four dendrites, thus forming a synaptic glomerulus. Sometimes a labeled terminal that contacted an unlabeled terminal by an adherent junction was found within the glomerulus. The large terminals and these complex synaptic relations appeared to characterize the esophageal afferent projections in the ceNTS.

Ultrastructure and synaptic organization of the spinal accessory nucleus of the rat.

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.

Regulation of motor neuron dendrite growth by NMDA receptor activation.

Spinal motor neurons undergo great changes in morphology, electrophysiology and molecular composition during development. Some of this maturation occurs postnatally when limbs are employed for locomotion, suggesting that neuronal activity may influence motor neuron development. To identify features of motor neurons that might be regulated by activity we first examined the structural development of the rat motor neuron cell body and dendritic tree labeled with cholera toxin-conjugated horseradish peroxidase. The motor neuron cell body and dendrites in the radial and rostrocaudal axes grew progressively over the first month of life. In contrast, the growth of the dendritic arbor/cell and number of dendritic branches was biphasic with overabundant growth followed by regression until the adult pattern was achieved. We next examined the influence of neurotransmission on the development of these motor neuron features. We found that antagonism of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor inhibited cell body growth and dendritic branching in early postnatal life but had no effect on the maximal extent of dendrite growth in the radial and rostrocaudal axes. The effects of NMDA receptor antagonism on motor neurons and their dendrites was temporally restricted; all of our anatomic measures of dendrite structure were resistant to NMDA receptor antagonism in adults. These results suggest that the establishment of mature motor neuron dendritic architecture results in part from dendrite growth in response to afferent input during a sensitive period in early postnatal life.

Distribution and morphology of motoneurons innervating different muscle fiber types in an amphibian muscle complex

The distribution and morphology of motoneurons innervating specific types of muscle fibers in the levator scapulae superior (LSS) muscle complex of the bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) were studied by retrograde labelling with cholera toxin-conjugated horseradish peroxidase (CT-HRP). The LSS muscle complex in both of these amphibians has a segregated pattern of muscle-fiber types (tonic; fast oxidative-glycolytic twitch [FOG]; fast glycolytic twitch [FG]) along an anteroposterior axis. The entire motor pool was labelled by injection of CT-HRP into the whole LSS muscle complex. The motoneurons innervating specific fiber types were labelled by injection of CT-HRP into certain muscle regions. The organization of the motoneuron pool of the LSS complex of both species was arranged in two columns--one ventrolateral and one medial. In bullfrogs, the ventrolateral column contains motoneurons innervating FG and tonic fiber types and the medial column contains motoneurons innervating FOG fiber types. In tiger salamanders, the ventrolateral column contains motoneurons innervating FG fiber types and the medial column contains motoneurons innervating FOG and tonic fiber types. The different motoneuron types also have different soma sizes and patterns of dendritic arborization. In both species, FG motoneurons are the largest, whereas FOG motoneurons are intermediate in size and tonic motoneurons are the smallest. In bullfrogs, the main dendrites of FG motoneurons extend into the dorsolateral and the ventrolateral gray matter of the spinal cord, whereas the dendrites of FOG motoneurons extend into the ventral and medial cord. In the tiger salamander, dendrites of FG motoneurons extend into the ventrolateral spinal cord and dendrites of the FOG motoneurons extend more generally into the ventral cord. Dendrites of tonic motoneurons in both amphibians were small and short, and difficult to observe. These results establish that motoneurons innervating different types of muscle fibers in the LSS muscle complex are segregated spatially and display consistent morphological differences.