Cholera Toxin-horseradish Peroxidase Research Articles

Testosterone prevents synaptic loss in the perineal motoneuron pool in the spinal cord in male rats exposed to chronic stress

Chronic stress is known to induce disorders of reproductive neuroendocrine functions. Motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in male rats play an important role in copulatory behavior. In the present study, it was examined whether chronic stress would alter synaptic organization of the SNB motoneurons and whether androgen would modify the changes under chronic stress. Five male rats were under restraint stress for 5 days per week for 3 weeks, and five males implanted subcutaneously with Silastic capsules containing testosterone were also exposed to stress. Five males served as unstressed controls. After 3 weeks of restraint stress, cholera toxin-horseradish peroxidase (CT-HRP) was injected into the bulbocavernosus muscles and animals were killed 2 days later. The spinal cords containing the SNB were dissected, processed with a modified tetramethylbenzidine (TMB) method for visualization of retrogradely transported CT-HRP, and examined ultrastructurally. Neuronal structures apposing the membranes of 150 SNB motoneurons (total for three groups) were analyzed by measuring the percentage of somatic membranes covered by synaptic contacts. The mean percentage of somatic membranes covered by synapses in males exposed to chronic stress was significantly less than that in controls or stressed males treated with testosterone. Size and number of synaptic contacts per unit length of somatic membranes in males exposed to stress were also significantly less than those in controls or stressed males treated with testosterone. There was no significant difference in any of the parameters between controls and stressed males treated with testosterone. Changes in plasma levels of testosterone showed the same profile as changes in the synaptic contacts. These results suggest that the SNB motoneurons of male rats exposed to chronic stress retain a considerable synaptic plasticity in response to androgen, and that androgen treatment can rescue the SNB system in male rats when under chronic restraint stress.

Brain-derived neurotrophic factor and androgen interact in the maintenance of dendritic morphology in a sexually dimorphic rat spinal nucleus.

Testosterone regulates androgen receptor expression, soma size, and dendritic length in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats. Brain-derived neurotrophic factor (BDNF) is also expressed in SNB motoneurons; BDNF maintains SNB soma size in castrates, and interacts with testosterone to regulate androgen receptor expression in SNB motoneurons. This study tested the hypotheses that BDNF promotes SNB dendritic lengths and that BDNF and testosterone interact to maintain dendritic morphology in SNB motoneurons. Adult male rats were castrated; and, 5 wk later, SNB motoneurons were axotomized bilaterally, and BDNF or PBS was applied via cups sutured to the cut axons. After axotomy plus BDNF or PBS application, castrates received implants of testosterone or blank capsules and were killed 24 d later. Additional males of similar age that received sham castration, sham axotomy, and a blank implant served as sham controls. Two days before death, SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase, and SNB dendritic morphology was reconstructed in three dimensions. Dendritic lengths in blank-implanted castrates treated with PBS were significantly shorter than those of sham controls; treatment with either testosterone or BDNF alone failed to support dendritic length or distribution. However, treatment with both testosterone and BDNF restored dendritic morphology to the level of sham controls. Our findings demonstrate that BDNF interacts with testosterone in the maintenance of SNB dendritic arbors and support the hypothesis that dendritic morphology is regulated by trophic substances that originate in the neuromuscular periphery.

Development of a sexually dimorphic neuromuscular system in male rats after spinal transection: Morphologic changes and implications for estrogen sites of action

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). In male rats, SNB motoneurons exhibit a biphasic pattern of dendritic growth, having an initial period of exuberant growth followed by a period of retraction to mature lengths by 7 weeks of age. This growth is steroid dependent: dendrites fail to grow after castration, but growth is supported in castrates treated with estradiol. In this experiment, we examined whether supraspinal afferent input by means of descending spinal tracts to the SNB was involved in the normal postnatal development of SNB motoneurons, and whether the effect of estradiol on SNB dendritic growth could be explained by an indirect action of estradiol on supraspinal afferents. Motoneuron morphology was assessed in normal males, early- or late-postnatally transected males, castrated males left untreated or treated with estradiol, and transected castrates treated with estradiol. SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase during both the growth and retraction phases of dendritic development and reconstructed in three dimensions. The removal of supraspinal afferents resulted in extremely local effects within the developing SNB arbor, as well as transient alterations in somal growth. Furthermore, spinal transection did not block the trophic effect of estradiol on supporting SNB dendritic growth, indicating that estrogens do not act by means of supraspinal input to support SNB motoneuron development.

Evidence That Androgen Acts Through NMDA Receptors to Affect Motoneurons in the Rat Spinal Nucleus of the Bulbocavernosus

In adult male rats, spinal nucleus of the bulbocavernosus (SNB) motoneurons shrink after castration and are restored in size after androgen treatment. Sixty-day-old Sprague Dawley males were castrated and implanted with SILASTIC capsules containing testosterone (T) or nothing, and osmotic minipumps continuously infusing MK-801, a noncompetitive NMDA receptor antagonist, or saline. Twenty-five days later, bulbocavernosus muscles were injected with the retrograde tracer cholera toxin-horseradish peroxidase conjugate (CT-HRP) to label SNB cells. As seen previously, among saline-treated rats, SNB somata of T-treated castrates were significantly larger than those of castrates receiving blank capsules (p < 0.0001). MK-801 treatment blocked this effect of T on the SNB. MK-801 had no effect on non-androgen-responsive spinal motoneurons in the neighboring retrodorsolateral nucleus (RDLN), nor did the drug affect SNB soma size in the absence of androgen treatment. Motoneuronal soma size in Nissl stain revealed the same pattern of results seen with CT-HRP fills. In situ hybridization indicated that SNB motoneurons express mRNA for the NMDA receptor subunits R1, R2a, and R2b. Castration reduced the expression of R1 mRNA in SNB motoneurons, an effect that was blocked by androgen replacement in castrates. R2A and R2B mRNA expression in SNB cells was not affected by androgen manipulations. Likewise, androgen manipulations had no effect on the expression of any NMDA receptor subtypes in RDLN motoneurons. These results suggest that androgen affects the size of SNB motoneurons by influencing their expression of the NMDA receptor, and therefore the response of the motoneurons to endogenous glutamate.

N-methyl-D-aspartate receptor blockade inhibits estrogenic support of dendritic growth in a sexually dimorphic rat spinal nucleus.

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons requires the action of both androgens and estrogens. Estrogenic effects are limited to the initial growth of SNB dendrites through 4 weeks of age. During this postnatal period, dendritic growth in other spinal motoneurons is regulated by N-methyl-D-aspartate (NMDA) receptor activation. In this study, we tested whether NMDA receptor activation was involved in SNB dendritic growth and whether the estrogenic support of SNB dendritic growth was dependent on the activation of NMDA receptors. Motoneuron morphology was assessed in normal males, intact males treated daily with the NMDA receptor antagonist MK-801, castrated males treated with estradiol benzoate (EB), and castrated males treated with both EB and MK-801. SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase at 4 weeks of age (when dendritic length is normally maximal) and reconstructed in three dimensions. Somal area and dendritic length of SNB motoneurons in MK-801-treated, intact males were below those of normal males. Dendritic growth was partially supported in EB-treated castrates, but this growth was blocked by MK-801 treatment. These results suggest that, as in other motoneurons, dendritic development in the SNB involves NMDA receptors and, furthermore, that the estrogen-sensitive component of SNB dendritic development requires their activation.

Laryngeal and respiratory protective reflexes

Swallowing is a complex motor behavior that relies on an interneuronal network of premotor neurons (PMNs) to organize the sequential activity of motor neurons that are active during the buccopharyngeal and esophageal phases. Swallowing PMNs are highly interconnected to multiple areas of the brain stem and the central nervous system and provide a potential anatomic substrate integration of swallowing activity with airway protective reflexes. Because these neurons have synaptic contact with both afferent inputs and motor neurons and exhibit a true central activity, they appear to constitute the swallowing central pattern generator. We studied the viscerotopic organization of the nucleus of the solitary tract (NTS), the nucleus ambiguus (NA), the dorsal motor nucleus (DMN), and the hypoglossal nucleus (XII) using cholera toxin horseradish peroxidase (CT-HRP), a sensitive antegrade and retrograde tracer that effectively labels afferent terminal fields within the NTS as well as swallowing motor neurons and their dendritic fields within the NA, DMN, and XII. We used CT-HRP to provide a comprehensive description of the dendritic architecture of NA motor neurons innervating swallowing muscles. We also conducted studies using pseudorabies virus (PRV), a swine α-herpesvirus, to map central neural circuits after injection in the peripheral or central nervous systems. One attenuated vaccine strain, Bartha PRV, has preferential affinity for sites of afferent synaptic contact on the cell body and dendrites and a reactive gliosis that effectively isolates the infected neurons and provides a barrier to the nonspecific spread to adjacent neurons. The findings provide a basis for the central integration of swallowing and respiratory protective reflexes.

Androgen stimulates neuronal plasticity in the perineal motoneurons of aged male rats.

Ten aged male rats (24 months of age) were castrated and implanted subcutaneously with Silastic capsules containing testosterone (T)(5 males) or nothing (5 males). Five sham-castrated males (25 months of age) served as controls. Four weeks after castration, cholera toxin-horseradish peroxidase (CT-HRP) was injected into the bulbocavernosus muscles and animals were killed 2 days later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine method for visualization of retrogradely transported CT-HRP, and examined ultrastructurally. Neuronal structures apposing the membranes of 150 CT-HRP-labeled SNB motoneurons were analyzed by measuring the percentage of somatic 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 membranes covered by synapses in castrated, aged males treated with T was significantly greater than that in control or castrated animals. Size and number of synaptic contacts per unit length of somatic membranes in castrated, aged males treated with T were also significantly greater than those in control or castrated animals. Plasma levels of T in castrated, aged males treated with T were significantly greater than that in controls. These results suggest that the SNB motoneurons of aged male rats retain a considerable synaptic plasticity in response to androgen, and that androgen may be, at least in part, involved in the process of aging of the SNB system in male rats.

Brainstem viscerotopic organization of afferents and efferents involved in the control of swallowing

Cholera toxin horseradish peroxidase (CT-HRP), a sensitive antegrade and retrograde tracer, is effective at labeling swallowing motoneurons and their dendritic fields within the nucleus ambiguus (NA), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve, and hypoglossal nucleus. Using this tracer to label motoneurons within the NTS demonstrates that palatal, pharyngeal, and laryngeal afferents overlap considerably within the interstitial and intermediate subnuclei. These afferents have a pattern of distribution within the NTS similar to the labeling observed after application of the same tracer to the superior laryngeal nerve. Esophageal afferents, however, terminate entirely within the central (NTScen) subnucleus and do not overlap their distribution with palatal, pharyngeal, or laryngeal afferents. Within the nodose ganglion (NG), sensory neurons projecting to the soft palate and pharynx are located superiorly, and those projecting to the esophagus and stomach are located inferiorly, an organization that indicates rostrocaudal positioning along the alimentary tract. Sensory neurons within the NG and NTS contain, among others, the major excitatory and inhibitory amino acid neurotransmitters glutamate (Glu) and γ-aminobutyric-acid (GABA). Both Glu and GABA help to coordinate esophageal peristalsis. Using pseudorabies virus as a transsynaptic tracer demonstrates the role of GABA and Glu as mediators of synaptic transmission within the swallowing central pattern generator, a fact further supported by the presence of specific receptors for each neurotransmitter within the NTScen. Anatomic studies using CT-HRP have been effective in revealing the total extent of extranuclear dendritic projections and the organization of dendrites within the confines of a nucleus; further studies have produced the following data. Motoneurons innervating the soft palate, pharynx, larynx, and cervical esophagus have extensive dendrites that extend into the adjacent reticular formation with a distinct pattern for each muscle group. Motoneurons of the musculature active during the buccopharyngeal phase of swallowing (soft palate, pharynx, cricothyroid, and cervical esophagus) have extensive dendritic arborizations that terminate within the adjacent reticular formation of the NA. Swallowing premotor neurons located in the reticular formation surrounding the NA are active during the buccopharyngeal phase of swallowing. These data provide an anatomic basis for interaction of swallowing motoneurons with premotor neurons located in this area. Motoneurons innervating all levels of the esophagus are confined to the compact formation (NAc), whereas those motoneurons projecting to the pharynx and cricothyroid muscle are located in the semicompact formation (NAsc). The intrinsic laryngeal muscles were represented within the loose formation (NAl) and the heart within the external formation. In contrast, the dendrites of motoneurons projecting to the thoracic and subdiaphragmatic esophagus are confined to the NAc. Both the NAsc and NAc have extensive longitudinal bundling of dendrites within the confines of the nucleus, resulting in the formation of a rostrocaudal dendritic plexus where dendrites crisscross between bundles. Intranuclear bundling of dendrites is evident in the soft palate, pharynx, and esophagus and is lacking only for the cricothyroid muscle. Moreover, ventrolateral- and dorsomedial-oriented dendritic bundles are present within the NAsc. In contrast to the longitudinal dendritic bundles, the ventrolateral- and dorsomedial-oriented dendritic bundles exit the NAsc and penetrate the adjacent reticular formation. The extensive bundling of motoneuronal dendrites within the NA supports the hypothesis that these structures serve as networks for the generation of complex motor activities, such as swallowing.

Immunoelectron microscopic study of glutamate inputs from the retrosplenial granular cortex to identified thalamocortical projection neurons in the anterior thalamus of the rat

We have carried out an ultrastructural study to determine the characteristics and distribution of glutamate-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to glutamate and a postembedding immunogold detection method in animals in which the neurons of AD/AV projecting to the cortex had been retrogradely labelled and the terminals of corticothalamic afferents anterogradely labelled by injection of cholera toxin-horseradish peroxidase (HRP) into the retrosplenial granular cortex. The heaviest immunogold labelling was over axon terminals 0.42 to 2.2 μm in diameter containing round synaptic vesicles and establishing Gray type 1 (asymmetric) synaptic contact (type 1 terminals) on HRP-labelled or non-labelled dendrites. Mean gold particle densities over such terminals were 3–4 times higher than the densities over the dendrites to which they were presynaptic and 5–6 times higher than over terminals establishing Gray type 2 (symmetric) synaptic contacts (type 2 terminals). Gold particle densities over neuronal cell bodies and dendrites and over a subpopulation of myelinated axons were intermediate between the densities over type 1 and type 2 terminals. In adjacent serial sections immunoreacted for gamma aminobutyric acid, type 2 terminals were heavily immunolabelled whereas type 1 terminals and other profiles with moderate gold particle densities after glutamate immunoreaction displayed very low labelling. A subpopulation of small type 1 axon terminals (up to 1 μm diameter) contained HRP reaction product identifying them as cortical in origin; they contacted small dendritic profiles (most <1 μm diameter) many of which also contained HRP reaction product. We conclude that terminals of the corticothalamic projection from retrosplenial granular cortex to AD/AV are glutamatergic and innervate predominantly distal dendrites of thalamocortical projection neurons. All rights reserved

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-β-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-β-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.

Aromatase inhibition reduces dendritic growth in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains the steroid-sensitive spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innervate perineal muscles involved in copulatory reflexes. In normal males, SNB motoneuron dendrites grow exuberantly through postnatal (P) day 28. This growth is steroid dependent: Dendrites fail to grow in males castrated at P7, but grow normally in castrates treated with testosterone or its metabolites, dihydrotestosterone combined with estrogen. Treatment with either metabolite alone supports dendritic growth, but not to the level of testosterone-treated or intact males. In this study, we tested the hypothesis that aromatization of androgens to estrogens was involved in the masculine development of SNB dendrites. Motoneuron morphology was assessed in normal males and males treated daily (P7-28) with fadrozole, a potent aromatase inhibitor (0.25 mg/kg, subcutaneously) or saline vehicle (n = 4-6/group). SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase at P28 (when dendritic length is normally maximal) and reconstructed in three dimensions. Comparable labeling was seen across groups; it was equivalent in both the rostrocaudal and radial extents. However, dendritic lengths in fadrozole-treated males were significantly below those of intact or saline-treated males. Neither SNB somata size nor target muscle weight differed across groups. These results suggest that aromatization of androgens to estrogens is necessary for development of masculine SNB dendritic morphology.

Synaptic changes in the perineal motoneurons of aged male rats.

Cholera toxin-horseradish peroxidase (CT-HRP) was injected into the bulbocavernosus muscles of young (2 months of age) and old (19-20 months of age) male rats, and animals were killed 2 days later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine method for visualization of retrogradely transported CT-HRP, and examined ultrastructurally. Neuronal structures apposing the membranes of 120 CT-HRP-labeled SNB motoneurons were analyzed by measuring the percentage of somatic membranes covered by synaptic contacts, synaptoid contacts, and neuron-neuron contacts. Most of the neuronal structures in the young and old SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic membranes covered by synapses in old rats was significantly smaller than that in young ones. Size and number of synaptic contacts per unit length of somatic membranes in old animals were also significantly reduced. Plasma levels of testosterone in old males were significantly smaller than those in young ones. These age-related changes in synaptic inputs to SNB motoneurons and plasma levels of androgen seem to correlate with aging of the SNB system.

Motoneuron development after deafferentation. I. Dorsal rhizotomy does not alter growth in the spinal nucleus of the bulbocavernosus (SNB)

The spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) are sexually dimorphic motor nuclei in the rat lumbar spinal cord. During postnatal development, SNB and DLN motoneurons grow substantially in measures of soma size, dendritic length, and radial dendritic extent. SNB motoneurons exhibit a biphasic pattern of dendritic growth, where there is an initial period of exuberant growth followed by a period of retraction to mature lengths by 7 weeks. In this experiment, we examined whether primary afferent input to the SNB nucleus was necessary for the normal postnatal growth of SNB motoneurons. We partially deafferented the SNB via unilateral dorsal rhizotomy of lumbosacral dorsal roots in male rats at 1 week of age. Using cholera toxin horseradish peroxidase (BHRP) to visualize SNB motoneurons, we examined SNB motoneuron morphology at 4 and 7 weeks of age. SNB motoneurons in rhizotomized males developed normally; measures of dendritic length in rhizotomized males were typically exuberant at 4 weeks of age, and declined significantly to mature lengths by 7 weeks of age. In addition, dorsal rhizotomy did not alter the development of SNB motoneuron soma size or radial dendritic extent. These results are discussed in reference to sensorimotor connections in the SNB, the extent of the deafferentation, and dendrodendritic interactions.

Motoneuron development after deafferentation: II. Dorsal rhizotomy does doe block estrogen-supported growth in the dorsolateral nucleus (DLN)

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.

Nitric oxide-synthesising neurons in the central subnucleus of the nucleus tractus solitarius provide a major innervation of the rostral nucleus ambiguus in the rabbit.

We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin-horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents.

Synaptic terminal coverage of primate triceps surae motoneurons

This study examined the synaptic terminal coverage of primate triceps surae (TS) motoneurons at the electron microscopic level. In three male pigtail macaques, motoneurons were labeled by retrograde transport of cholera toxin-horseradish peroxidase that was injected into TS muscles bilaterally and visualized with tetramethylbenzidine stabilized with diaminobenzidine. Somatic, proximal dendritic, and distal dendritic synaptic terminals were classified by standard criteria and measured. Overall and type-specific synaptic terminal coverages and frequencies were determined. Labeled cells were located in caudal L5 to rostral S1 ventral horn and ranged from 40 to 74 microns in diameter (average, 54 microns). The range and unimodal distribution of diameters, the label used, and the presence of C terminals on almost all cells indicated that the 15 cell bodies and associated proximal dendrites analyzed here probably belonged to alpha-motoneurons. Synaptic terminals covered 39% of the cell body membrane, 60% of the proximal dendritic membrane, and 40% of the distal dendritic membrane. At each of these three sites, F terminals (flattened or pleomorphic vesicles, usually symmetric active zones, average contact length 1.6 microns) were most common, averaging 52%, 56%, and 58% of total coverage and 56%, 57%, and 58% of total number of cell bodies, proximal dendrites, and distal dendrites respectively. S terminals (round vesicles, usually asymmetric active zones, average contact length 1.3 microns) averaged 24%, 29%, and 33% of coverage and 33%, 35%, and 36% of number at these three sites, respectively. Thus, S terminals were slightly more prominent relative to F terminals on distal dendrites than on cell bodies. C terminals (spherical vesicles, subsynaptic cisterns associated with rough endoplasmic reticulum, average contact length 3.5 microns) constituted 24% and 11% of total terminal coverage on cell bodies and proximal dendrites, respectively, and averaged 11% and 6% of terminal number at these two locations. M terminals (spherical vesicles, postsynaptic Taxi bodies, some with presynaptic terminals, average contact length 2.7 microns) were absent on cell bodies and averaged 3% and 7% of total coverage and 2% and 5% of terminals on proximal and distal dendrites, respectively. Except for M terminals, which tended to be smaller distally, terminal contact length was not correlated with location. Total and type-specific coverages and frequencies were not correlated with cell body diameter. Primate TS motoneurons are similar to cat TS motoneurons in synaptic terminal morphology, frequency, and distribution. However, primate terminals appear to be smaller, so that the fraction of membrane covered by them is lower.

Differential effects of dihydrotestosterone and estrogen on the development of motoneuron morphology in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains a sexually dimorphic motor nucleus, the spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innervate perineal muscles involved in copulatory reflexes. Dendritic development of SNB motoneurons is biphasic and androgen dependent. During the first 4 postnatal weeks, SNB dendrites grow exuberantly, and subsequently retract to mature lengths by 7 weeks of age. After early postnatal castration, SNB dendrites fail to grow, and testosterone replacement restores this growth. In other systems, testosterone and its metabolites, dihydrotestosterone and estrogen, are important for somatic and neural sexual differentiation. The purpose of the present study was to examine the effects of castration and dihydrotestosterone or estrogen replacement on the growth of SNB motoneuron somata and dendritic arbors. Male rat pups were castrated on postnatal (P) day 7 and treated daily with either dihydrotestosterone propionate (DHTP; 2 mg) or estradiol benzoate (EB; 100 micrograms) until P28 or P49. By using cholera toxin horseradish peroxidase (BHRP) histochemistry, the soma size, dendritic length, dendritic extent, and arbor area of BHRP-labeled SNB motoneurons were measured and analyzed. Both DHTP and EB treatment supported the initial exuberant growth of SNB dendrites through P28, but EB treatment was ineffective in maintaining mature, adult lengths at P49. The possible sites of hormone action and functional implications of these hormonal treatments are discussed.

Dendritic architecture of hypoglossal motoneurons projecting to extrinsic tongue musculature in the rat.

The tracer, cholera toxin-horseradish peroxidase, was used to determine the dendritic architecture and organization of hypoglossal motoneurons in the rat. In 22 animals, the tracer was injected unilaterally into either the geniohyoid, genioglossus, hyoglossus, or styloglossus muscle. Within the hypoglossal nucleus, motoneurons innervating the extrinsic tongue muscles were functionally organized. Geniohyoid and genioglossus motoneurons were located within the ventrolateral and ventromedial subnuclei, respectively, while hyoglossus and styloglossus motoneurons were located within the dorsal subnucleus. Motoneurons located in all subnuclear divisions were found to have extensive dendrites that extended laterally into the adjacent reticular formation and medially to the ependyma. Less extensive extranuclear dendritic projections were found in the dorsal vagal complex and median raphe. Prominent rostrocaudal and mediolateral dendritic bundling was evident within the ventral subnuclei and dorsal subnucleus, respectively. Dendritic projections were also found extending inter- and intrasubnuclearly with a distinct pattern for each muscle. These data suggest that the varied and extensive dendritic arborizations of hypoglossal motoneurons provide the potential for a wide range of afferent contacts for, and interactions among, motoneurons that could contribute to the modulation of their activity. Specifically, the prominent dendritic bundling may provide an anatomic substrate whereby motoneurons innervating a specific muscle receive and integrate similar afferent input and are thus modulated as a functional unit. In contrast, the extensive intermingling of both inter- and intrasubnuclear dendrites within the hypoglossal nucleus may provide a mechanism for the coordination of different muscles, acting synergistically or antagonistically to produce a tongue movement.

Dendritic Development in Normal Lumbar Motoneurons and Following Neonatal Nerve Crush in the Rat

Motoneurons from the rat were retrogradely labelled with cholera toxin-horseradish peroxidase at intervals during normal postnatal development and following nerve crush at birth. Normal cells displayed a relatively steady increase in total visible dendritic density which was largely confined to the dorsomedial direction. After nerve crush at birth, dorsomedially orientated dendrites failed to achieve normal density, resulting in a significantly smaller dendritic tree by adulthood. There was also a transient, abnormal extension of dendrites in the medioventral direction which had regressed to normal levels by maturity. The predominance of changes in the dorsally directed region of the dendritic tree suggests that dendritic development of motoneurons is influenced by synaptic inputs in the dorsal horn.

Both afferent and efferent connections influence postnatal growth of motoneuron dendrites in the rat.

Spinal motoneurons from mature rats, which had received one of 5 different surgical procedures neonatally, were retrogradely labelled with a cholera toxin-horseradish peroxidase conjugate and their dendritic morphology was analysed. The motoneurons studied were those innervating extensor digitorum longus and the procedures disrupted their motor and sensory connections to varying degrees. Disruption of motor contact with the target muscle retarded dendritic growth in the transverse plane, particularly in the dorso-medial direction. Disruption of sensory as well as motor contact resulted additionally in an increase in dendritic density in the longitudinal plane, largely along the rostral-caudal axis. The findings suggest that dendritic development of motoneurons is influenced by both afferent and efferent target contacts and that these effects can be differentiated.