A perioculomotor nitridergic population in the macaque and cat.

PURPOSE: We determined the distribution of cells containing synthetic enzymes for the unconventional neurotransmitter, nitric oxide, with respect to the known populations within the oculomotor complex. METHODS: The oculomotor complex was investigated in monkeys and cats by use of histochemistry to demonstrate nicotinamide adenine dinucleotide phosphate diaphorase positive (NADPHd(+)) cells and antibodies to localize neuronal nitric oxide synthase positive (NOS(+)) cells. In some cases, wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into extraocular muscles to allow comparison of retrogradely labeled and NADPHd(+) cell distributions. RESULTS: The distribution of the NADPHd(+) and NOS(+) neurons did not coincide with that of preganglionic and extraocular motoneurons in the oculomotor complex. However, labeled perioculomotor neurons were observed. Specifically, in monkeys, they lay in an arc that extended from between the oculomotor nuclei into the supraoculomotor area (SOA). Comparison of WGA-HRP-labeled medial and superior rectus motoneurons with NADPHd staining confirmed that the distributions overlapped, but showed that the C- and S-group cells were not NADPHd(+). This suggested that NADPHd(+) cells are part of the centrally projecting Edinger-Westphal population (EWcp). Examination of the NADPHd(+) cell distribution in the cat showed that these cells were indeed found primarily within its well-defined EWcp. CONCLUSIONS: Based on their similar distributions, it appears that the peptidergic EWcp neurons, which project widely in the brain, also may be nitridergic. While the preganglionic and C- and S-group motoneuron populations do not use this nonsynaptic neurotransmitter, nitric oxide produced by surrounding NADPHd(+) cells may modulate the activity of these motoneurons.

Developmental changes in brainstem neurons regulating lower airway caliber.

Premature infants are at risk for lower airway obstruction; however, maturation of reflex pathways regulating lower airway patency is inadequately studied. We hypothesized that postnatal maturation causes developmental change in brainstem efferent airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) that project to the airways and in pulmonary afferent fibers that terminate in the nucleus tractus solitarius (NTS). Ferrets aged 7, 14, 21, and 42 d received intrapulmonary injection of cholera toxin (CT)-beta subunit, a transganglionic retrograde tracer. Five days later, their brainstem was processed for dual immunolabeling of CT-beta and the cholinergic marker, choline acetyl transferase. CT-beta-labeled AVPNs and CT-beta-labeled afferent fiber optical density (OD) were analyzed. There was a significantly higher CT-beta-labeled cell number within the rNA at the youngest compared with older ages. All efferent CT-beta-labeled cells expressed choline acetyl transferase. OD of CT-beta-labeled afferent fibers was also higher at 7 d compared with 14 d. We conclude that the number of efferent AVPNs and afferent fiber OD both diminish over the second postnatal week. We speculate that exposure to injurious agents in early postnatal life may inhibit natural remodeling and thereby enhance later vulnerability to airway hyperreactivity.

Cdk5 selectively affects the migration of different populations of neurons in the developing spinal cord.

It has been shown that cyclin-dependent kinase 5 (Cdk5) is crucial for neuronal migration and survival in the brain. However, the role of Cdk5 in neuronal migration in the spinal cord has never been investigated. The present study is the first to show that Cdk5 affects the migration of different populations of neurons in the developing spinal cord. In the absence of Cdk5, at least four neuronal populations failed to migrate to their final destinations: sympathetic and parasympathetic preganglionic neurons, as well as dorsally originating and ventrally originating (U-shaped group) diaphorase-positive dorsal horn interneurons. In contrast, the migration of somatic motor neurons and various types of ventral and dorsal interneurons was unaffected by the absence of Cdk5. Moreover, our results suggest that Cdk5-dependent migration in the developing spinal cord is axon- or glial fiber-mediated. Finally, our results show that sympathetic preganglionic neurons and somatic motor neurons in Cdk5-deficient mice continue to extend processes and project toward their normal target areas, suggesting that Cdk5 has no obvious effects on axonal outgrowth and guidance mechanisms of these two neuronal populations in spinal cord development.

Death of preganglionic sympathetic neurons after surgical or immunologic lesion of peripheral processes.

Three months after systemic injection of antibody to acetylcholinesterase (AChE), there is a 60% decrease in the population of preganglionic sympathetic neurons expressing choline acetyltransferase (ChAT) in the intermediolateral (IML) nucleus of the rat spinal cord. In principle, the disappearance of identifiable cholinergic neurons might reflect either outright cell death or severe atrophy with downregulation of cholinergic markers. To distinguish between these possibilities, preganglionic neurons were labeled with the retrograde tracer dye, Fast Blue, 1 week before antibody injection or surgical transection of the cervical sympathetic trunk. Three months after either treatment, the thoracic IML contained 40-60% fewer Fast Blue-labeled neurons than in controls. Therefore, preganglionic sympathetic neurons do degenerate after antibody injection or axotomy. To clarify the role of axonal damage in this process, the effects of three different mechanical lesions were examined. A lumbar ganglionectomy designed to interrupt most sympathetic axons emanating from L2 IML caused 92% loss of ChAT-positive cells observed 10 weeks later at that site. In comparison, transection of the cervical sympathetic trunk, which spared some distally directed axonal branches from the thoracic IML, caused only a 46% loss of ChAT-positive neurons at T1. Still smaller effects were seen after the same nerve was crushed, a lesion that is less destructive. Thus, the ability of central sympathetic neurons to survive a peripheral lesion may be related to the degree of axonal damage and to the opportunity for axonal regrowth.

The regional distribution of nitric oxide synthase activity in the spinal cord of the dog.

The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme's activity, measured by the conversion of [3H]arginine to [3H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1-L3, L4-L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1-C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme's activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme's activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4-L7 segments in the dorsal horn and in all the above mentioned white matter columns.

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Acetylcholinesterase immunolesioning: regional vulnerability of preganglionic sympathetic neurons in rat spinal cord.

Rats given antibodies against acetylcholinesterase (AChE) develop sympathetic dysfunction stemming from losses of preganglionic neurons in spinal cord. Central effects of AChE antibodies are surprising since IgG does not readily cross the blood-brain barrier, and lesions of peripheral terminals should not cause cell death. This study was designed to explore the distribution of central neural damage and to investigate features that might account for vulnerability. Rat spinal cord and brainstem were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity. Four months after administration of AChE antibodies, ChAT-positive neurons in the intermediolateral nucleus (IML) were 61-66% fewer throughout the thoracolumbar cord (T1, T2, T8, T12, L1). NOS-positive neurons in these loci were affected to the same extent by antibody-treatment, although they were only two-thirds as numerous. By contrast, neurons in the central autonomic nucleus of the thoracolumbar cord were scarcely affected. These results point to immunochemical differences in the central autonomic outflow, which may partially explain the puzzling selectivity of neural damage in AChE immunolesioning. Different results were obtained after guanethidine sympathectomy, which ablated nearly all neurons in the superior cervical ganglion without any effect on preganglionic neurons in the IML. Therefore, if the central effects of antibodies are indirectly mediated by loss of trophic support from the periphery, this support cannot arise from adrenergic neurons but must come from other ganglionic cells.

Neuroprotective fibroblast growth factor type-2 down-regulates the c-Jun transcription factor in axotomized sympathetic preganglionic neurons of adult rat.

The immediate-early gene encoded transcription factor c-Jun is highly inducible following axotomy and therefore serves as a valuable marker in neuronal de- and regeneration. As the signals that may trigger c-Jun expression are still obscure, molecules derived from lesioned neurons and/or their targets such as growth factors or cytokines have been proposed as candidates for interneuronal transcriptional regulation in vivo. We therefore tested whether local administration of the neuroprotective cytokine fibroblast growth factor type-2 in vivo has an effect on the axotomy-induced nuclear expression patterns of the activator protein-1 transcription factors c-Fos and JunB, or c-Jun in the spinal cord-intermedolateral nucleus-adrenal axis lesion paradigm in the rat. Partial axotomy of preganglionic nerve fibres by selective unilateral removal of the adrenal medulla resulted in strong staining patterns of c-Jun in the nuclei of preganglionic cell bodies located in the spinal intermediolateral cell column identified by in vivo retrograde prelabelling with the fluorescent tracer Fast Blue prior to lesion. Axotomy-induced nuclear c-Jun expression was highly increased when compared with the moderate baseline expression in normal or sham-operated animals. In animals treated with fibroblast growth factor-2 gelfoams implanted to the lesioned adrenal gland the nuclear c-Jun staining pattern is reduced or even absent from these neurons. By contrast, c-Fos and JunB induction did not occur in the intermediolateral nucleus in the lesion paradigm investigated. These results support the idea of functional links between neurotrophic cytokines such as fibroblast growth factor-2 and transcriptional effectors such as c-Jun. The target derived fibroblast growth factor-2 thus may signal the intactness of the neuron-target axis resulting in suppression of central extrinsic neurons and promotion of neuroprotective gene activation. Neuronal survival in absence of c-Jun indicates that c-Jun exerts negative actions in vulnerated neurons.

Periaqueductal gray matter projection to vagal preganglionic neurons and the nucleus tractus solitarius.

The periaqueductal gray matter (PAG) has been implicated in a variety of different functions, including autonomic regulation. Chemical stimulation of the lateral PAG produces hypertension and tachycardia while activation of the ventrolateral PAG produces the opposite effect. While these effects are the result of alterations in sympathetic activity, little is known about whether the PAG can modulate vagal functions as well. The anterograde axonal tracing method using the plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) was used to determine whether both of the lateral and ventrolateral PAG columns project to vagal preganglionic neurons and/or to the nucleus tractus solitarius (NTS). Highly restricted PHA-L injections were made in all four PAG columns throughout their rostrocaudal extent in rats. Labeled fibers were visualized by immunohistochemistry and studied in relationship with choline acetyltransferase (ChAT) immunostained parasympathetic preganglionic neurons of the dorsal motor vagal nucleus (DMV) and nucleus ambiguous (NA). The lateral PAG projects to the lateral DMV and to the caudal part of the external NA. The ventrolateral PAG innervates the same regions and also projects to the rostral part of the external NA -- a site that contains cardiac parasympathetic preganglionic neurons. Both the lateral and ventrolateral PAG project to the NTS in a similar fashion innervating the medial, ventrolateral and commissural subnuclei. In summary, the lateral and ventrolateral PAG have similar patterns of innervation of the NTS and DMV, but their projection to the NA is different: the rostral external NA receives innervation only from the ventrolateral PAG and the lateral PAG innervates the caudal part.

Cytochrome oxidase staining in the major pelvic ganglion of the male rat.

Cytochrome oxidase staining was used as a marker of metabolic activity in neural elements in the rat major pelvic ganglion. Many neurons in the ventral pole of the ganglion have little cytochrome oxidase activity, while neurons in other locations show gradations in staining intensity. Punctate staining around principal neurons may represent preganglionic terminals, since it was greatly reduced after denervation of the ganglion. Image analysis was used to compare neuronal size to staining intensity. There was a negative correlation between cell size and staining intensity; the largest neurons were only lightly stained for cytochrome oxidase, while the medium and the small neurons showed a full range of metabolic activity. To study metabolic activity of an identified neuronal population, the seminal vesicles were injected with a retrograde tracer. The largest seminal vesicles neurons (1500 to 3200 microns2) had low enzyme activity, whereas the majority of neurons to this organ were smaller with gradations in staining. These results are indicative of the metabolic activity of the autonomic innervation to various pelvic tissues. Cytochrome oxidase histochemistry should prove valuable in assessing the demands placed on autonomic ganglia in differing functional and dysfunctional states.

Catecholamine enzymes and neuropeptides are expressed in fibres and somata in the intermediate gray matter in chronic spinal rats.

Spinal cord injury disrupts control of sympathetic preganglionic neurons because bulbospinal input has been lost and the remaining regulation is accomplished by spinal circuits consisting of dorsal root afferent and spinal neurons. Moreover, an initial retraction and regrowth of dendrites of preganglionic neurons in response to deafferentation creates the potential for remodelling of spinal circuits that control them. Although catecholamines and neuropeptide Y are found in descending inputs to the preganglionic neurons, their presence in spinal circuits has not been established. Spinal circuits controlling preganglionic neurons contain substance P but participation of these peptidergic neurons in remodelling responses has not been examined. Therefore, we compared immunoreactivity for the catecholamine-synthesizing enzyme dopamine beta-hydroxylase, for neuropeptide Y and for substance P in the intermediate gray matter of the spinal cord in control rats and in rats seven or fourteen days after transection at the fourth thoracic cord segment. Sympathetic preganglionic neurons were retrogradely labelled by intraperitoneal injection of the tracer FluoroGold. These experiments yielded three original findings. 1) At one and two weeks after cord transection, fibres and terminals immunoreactive for dopamine beta-hydroxylase and neuropeptide Y were consistently found in the intermediolateral cell column in segments caudal to the transection. The area of fibres and terminals containing these immunoreactivities was markedly reduced compared to control rats or to segments rostral to the transection in the spinal rats. 2) Immunoreactivity for substance P was increased after cord transection and the distribution of fibres immunoreactive for this peptide in segments caudal to the transection extended more widely through the intermediate gray matter. These reactions demonstrated a plastic reaction to cord transection by spinal neurons expressing substance P. 3) Dopamine beta-hydroxylase expression was up-regulated in somata within the intermediate gray matter of spinal segments caudal to the transection. The numbers of somata immunoreactive for this enzyme increased six-fold by 14 days after cord transection, compared to the few somata counted in control rats. In conclusion, the presence of a catecholamine synthesizing enzyme and neuropeptides in fibres surrounding sympathetic preganglionic neurons caudal to a cord transection suggests a source of catecholamines and these peptides within spinal circuits in the chronic spinal rat. The presence of dopamine beta-hydroxylase in a markedly greater number of neuronal somata after cord transection reflects significant up-regulation of gene expression and may indicate a switch by these neurons to an adrenergic phenotype, revealing a plastic response to injury within the spinal cord.

Reduced acetylcholinesterase (AChE) activity in adrenal medulla and loss of sympathetic preganglionic neurons in TrkA-deficient, but not TrkB-deficient, mice.

TrkA high-affinity receptors are essential for the normal development of sympathetic paravertebral neurons and subpopulations of sensory neurons. Paravertebral sympathetic neurons and chromaffin cells of the adrenal medulla share an ontogenetic origin, responsiveness to NGF, and expression of TrkA. Which aspects of development of the adrenal medulla might be regulated via TrkA are unknown. In the present study we demonstrate that mice deficient for TrkA, but not the neurotrophin receptor TrkB, show an early postnatal progressive reduction of acetylcholinesterase (AChE) enzymatic activity in the adrenal medulla and in preganglionic sympathetic neurons within the thoracic spinal cord, which are also significantly reduced in number. Quantitative determinations of specific AChE activity revealed a massive decrease (-62%) in the adrenal gland and a lesser, but still pronounced, reduction in the thoracic spinal cord (-40%). Other markers of the adrenal medulla and its innervation, including various neuropeptides, chromogranin B, secretogranin II, amine transporters, the catecholamine-synthesizing enzymes tyrosine hydroxylase and PNMT, synaptophysin, and L1, essentially were unchanged. Interestingly, AChE immunoreactivity appeared unaltered, too. Preganglionic sympathetic neurons, in contrast to adrenal medullary cells, do not express TrkA. They must, therefore, be affected indirectly by the TrkA knock-out, possibly via a retrograde signal from chromaffin cells. Our results suggest that signaling via TrkA, but not TrkB, may be involved in the postnatal regulation of AChE activity in the adrenal medulla and its preganglionic nerves.

PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy.

Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.

Preganglionic parasympathetic salivatory neurons in the brainstem contain markers for nitric oxide synthesis in the rabbit.

The present study investigated the possible presence of markers for nitric oxide synthesis in brainstem preganglionic parasympathetic neurons involved in control of the submandibular and sublingual salivary glands of rabbits and rats. Retrograde axonal tracing was performed with biotinylated dextran to identify preganglionic parasympathetic salivatory neurons and combined with NADPH diaphorase histochemistry or nitric oxide synthase immunohistochemistry. The results of the double-labelling experiments demonstrated that most of the retrogradely labelled preganglionic parasympathetic neurons in rabbits contained the markers for nitric oxide synthesis, whereas, in rats most retrogradely labelled neurons lacked the markers for nitric oxide synthesis. These observations suggest that nitric oxide could influence ganglionic transmission in parasympathetic pathways controlling salivary secretion in the rabbit, but not in the rat.

Axonal transport of nitric oxide synthase in autonomic nerves.

By using mechanical nerve ligation or nerve pinch technique, we provide evidence that nitric oxide synthase (NOS) is transported in the preganglionic sympathetic axons, while postganglionic axons lack NOS transport. This finding corroborates the preganglionic sympathetic terminal as the site of NO synthesis, which is known to affect ganglionic transmission. Both vasoactive intestinal polypeptide (VIP) and substance P (SP) containing neurons of the nodose ganglion transport NOS in their axons. These results therefore suggest that NOergic innervation of autonomically innervated tissues is of parasympathetic and/or sensory, rather than sympathetic, origin.

Direct nitric oxide-containing innervation from the rat spinal cord to the penis.

Nitric oxide synthase (NOS)-immunoreactive pathways to the rat penis were studied by combining retrograde tracing by Fluorogold with NOS immunohistochemistry. Injection of the tracer into the penile shaft resulted in Fluorogold labelling in a vast number of NOS-immunoreactive neurons in the major pelvic ganglion. In addition, some neurons in the spinal cord intermediolateral column consistently contained Fluorogold label in the upper lumbar levels. Most of these neurons were also NOS-immunoreactive. These findings suggest that the sympathetic preganglionic, NOS-containing neurons project to the penis, and they may participate in nitric oxide-mediated penile erection.

A comparative study of NADPH-diaphorase in the sympathetic preganglionic neurons of the upper thoracic cord between spontaneously hypertensive rats and Wistar-Kyoto rats.

With retrograde tracing using fluorogold injection into the superior cervical ganglion and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the present comparative study revealed that the retrogradely labelled neurons in n. intermediolateralis pars funicularis (ILf) and n. intermediolateralis pars principalis (ILp) of the autonomic region in the upper thoracic cord exhibited a much stronger reactivity for NADPH-diaphorase in Wistar-Kyoto (WKY) rats than those in spontaneously hypertensive rats (SHR). It was found that in ILf in WKY rats, 77.62% of the fluorogold-labelled neurons were NADPH-d positive, while in SHR, only 56.43% of the labelled neurons were NADPH-d positive. The frequency distribution of NADPH-d positive retrogradely labelled neurons was significantly reduced in ILf of the spinal cord of SHR (U-test: P < 0.01). In ILp in WKY rats, 65.25% of fluorogold-labelled neurons were NADPH-d positive in WKY rats, while in SHR, only 56.28% of the labelled neurons were NADPH-d positive. Although the difference (P > 0.05) in the frequency of NADPH-d positive neurons in ILp between the two strains of rats was not significant, the reductions in SHR seemed considerable. Examination of the preganglionic sympathetic trunk and the superior cervical ganglion between SHR and WKY rats revealed that virtually all the NADPH-d positive fibers were derived from the sympathetic preganglionic neurons. In SHR, the NADPH-d positive fibers were not as intensely stained as those of WKY rats. This preliminary results suggest that nitric oxide, as an inhibitory neurotransmitter, may be implicated in the onset of hypertension.

Transient and continuous expression of NADPH diaphorase in different neuronal populations of developing rat spinal cord.

Nitric oxide is a novel intercellular messenger whose role in neuronal development is not yet known. As a first step toward elucidating its developmental function, we examined the pattern of NADPH diaphorase histochemical staining, an indicator of the presence of nitric oxide synthase, in the rat spinal cord at pre and postnatal ages. Some types of neurons expressed diaphorase activity transiently during development. For example, a subset of somatic motor neurons, located in the ventrolateral corner of a few caudal segments of the cervical spinal cord, were diaphorase-positive beginning on E15, but gradually became diaphorase-negative by birth. In contrast, other spinal neurons expressed diaphorase activity continuously from development into adulthood. Preganglionic autonomic motor neurons became diaphorase-positive early in their development, as they were migrating toward their adult positions. Other spinal neurons, such as those in superficial dorsal horn, first expressed diaphorase relatively late in their development, after reaching their final location. The transient expression in some cell types, as well as the early expression in others, suggest that nitric oxide may have an important role(s) during development, which may differ from its functions in the adult nervous system.

Immuno-electron microscopic study of tyrosine hydroxylase in the cat urinary bladder and proximal urethra.

The distribution and density of tyrosine hydroxylase (TH)-immunoreactive nerves in the cat urinary bladder and proximal urethra were similar to those of glyoxylic acid fluorescent nerves. Both TH-immunoreactive and fluorescent nerve fibres markedly decreased after 6-hydroxydopamine treatment. Hence, immuno-electron histochemistry of TH was employed to investigate adrenergic termination to the muscle layers of the cat urinary bladder and proximal urethra. A total number of 3728 TH-immunoreactive axon terminals in the lower bladder body, lateral bladder base, trigone, bladder neck and proximal urethra were examined. In the bladder base (the lateral bladder base, trigone and bladder neck) and proximal urethra, most of the TH-immunoreactive axon terminals (57.1-89.2%) lay outside smooth muscle cell fascicles, while most (69.7%) in the bladder body were inside muscle cell fascicles. The proportions of TH-immunoreactive axon terminals accompanying non-TH-immunoreactive axon terminals in the five regions were almost the same (about 70%). The present study demonstrated that the mode of adrenergic termination in the bladder base and proximal urethra notably differed from that in the bladder body, and that approximately 70% of adrenergic axon terminals were associated with non-adrenergic or cholinergic axon terminals in each region.

The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat.

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densest in the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were surrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.