Changes in the number and volume of NPY and VIP neurons from periprostatic accessory vegetative ganglia in pre- and peripubertal rats. A stereological study.

The amount of neurons of periprostatic accessory ganglia in pre- and peripubertal rats was studied to ascertain whether the development of these autonomic ganglia is androgen-dependent. Stereological estimates of the volumes and number of neurons immunoreactive to protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) were carried out. Immunostaining of androgen receptors (AR) in the ganglia was also performed. The ganglionic neurons from the two groups studied were immunoreactive to PGP 9.5, NPY, and VIP. Almost all the neurons were immunostained for AR. The ganglionic volume showed a significant increase in peripubertal prostate in comparison with the prepubertal gland. No significant changes were observed with respect to the absolute number of neurons immunoreactive to all the antigens. The neuronal volume was significantly increased in peripubertal rats in comparison with prepubertal animals. These findings led us to the following conclusions: There is no evidence of neurogenesis during pubertal development in the periprostatic accessory ganglia of the rat. The increase of ganglionic volume in puberty is due to the growth in neuronal volume. There were no differences between the sizes of NPY and VIP neurons in pubertal periprostatic accessory ganglia. The development of periprostatic vegetative neurons is androgen-dependent.

Neurturin regulates postnatal differentiation of parasympathetic pelvic ganglion neurons, initial axonal projections, and maintenance of terminal fields in male urogenital organs.

We have investigated the development of autonomic nerves in the urogenital tract of male mice and the effect of neurturin gene deletion on this process. At birth, autonomic innervation of the reproductive organs was sparse, but urinary bladder smooth muscle was well innervated. Further innervation of reproductive tissues occurred until P21, but noradrenergic axons established their complete terminal field later than nitrergic cholinergic axons: in adults the former are more prevalent, yet this became apparent only at P7 (vas deferens, seminal vesicles), P14 (prostate) or after P14 (penis). Neurturin was essential for initial projection of axons (mucosa of vas deferens), maintenance of terminal fields (prostate and seminal vesicles), or both functions (cavernosum of penis). In contrast, some targets (e.g., bladder muscle and suburothelium, vas deferens smooth muscle) were unaffected by neurturin gene deletion. Pelvic ganglion neurons more than doubled between birth and adulthood, probably as aresult of continued maturation of p75-positive undifferentiated neuronal precursors rather than cell division. The adult number of neurons was achieved by P7 (sympathetic) or P21 (parasympathetic). In adult neurturin knockout mice, there were approximately 25% fewer parasympathetic neurons compared with wild types, because of failure of differentiation after P14. This study revealed the complexity of postnatal maturation of urogenital innervation, with each organ showing a distinct chronology of innervation and different requirement for neurturin. Our results also indicate that in adults there will be distinct differences in neurturin dependence between organs, such that proregenerative therapies may have to be tailored specifically for the nerve pathway of interest.

Postnatal maturational changes in rat pelvic autonomic ganglion cells: a mixture of steroid-dependent and -independent effects.

Androgens have potent effects on the maturation and maintenance of a number of neural pathways involved in reproductive behaviors in males. Most studies in this area have focused on central pathways, but androgen receptors are expressed by many peripheral neurons innervating reproductive organs, and previous studies have demonstrated structural and chemical changes in these neurons at puberty and after castration. We have performed the first electrophysiological comparison of pelvic autonomic ganglion neurons in male rats before and after puberty and following pre- or postpubertal castration. Studies were performed in vitro on intact ganglia with hypogastric and pelvic nerves attached to allow synaptic activation of sympathetic or parasympathetic neurons, respectively. Pelvic ganglion neurons underwent many changes in their passive and active membrane properties over the pubertal period, and some of these changes were dependent on exposure to circulating androgens. The most pronounced steroid-dependent effects were on membrane capacitance (soma size) in sympathetic neurons and duration of the action potential afterhyperpolarization in tonic neurons. Our study also showed that rat pelvic ganglion cells and their synaptic inputs were more diverse than previously reported. In conclusion, this study demonstrated that rat pelvic ganglion neurons undergo considerable postnatal changes in their electrophysiological properties. The steroid dependence of some of these changes indicates that circulating androgens may influence reproductive behaviors at many locations within the nervous system not just in the brain and spinal cord.

Testosterone and nerve growth factor have distinct but interacting effects on structure and neurotransmitter expression of adult pelvic ganglion cells in vitro.

Circulating testosterone has potent effects on the structure and function of many pelvic ganglion cells in adult rats in vivo. However not all androgen-sensitive pelvic neurones possess androgen receptors and testosterone effects may therefore be indirect, by an action on the target organs. Here we have examined if testosterone influences neuronal structure in vitro in pelvic ganglion cells cultured from adult male rats. We have also used multiple label immunofluorescence to monitor the expression of transmitter-synthesising enzymes and peptides under various culture conditions. Testosterone was a more potent stimulant of noradrenergic soma growth in culture than nerve growth factor. Whereas nerve growth factor increased the number, branching and length of neurites, testosterone stimulated growth of a small number of very short processes, each of which bore numerous short protrusions. Testosterone also impeded the longer neurite growth induced by nerve growth factor. Many pelvic ganglion cells altered their expression of transmitters/neuropeptides under different culture conditions. In particular, under control conditions or during nerve growth factor treatment, vasoactive intestinal peptide was up-regulated in noradrenergic and cholinergic neurones; testosterone impeded this up-regulation in noradrenergic neurones. Choline acetyltransferase immunoreactivity could only be visualised when nerve growth factor was present in the cultures, and cholinergic neurones showed less neurite outgrowth than noradrenergic neurones under all culture conditions. Nerve growth factor did not stimulate levels of this enzyme as strongly if testosterone was present. This study has shown that testosterone has potent effects on the structure of many pelvic ganglion cells in vitro. It is possible that these effects are mediated indirectly, e.g. by stimulating glial-derived substances, however our results suggest that the effects are not mediated by nerve growth factor. The results also show that testosterone influences some of the actions of nerve growth factor, suggesting that there may be complex interactions between steroid signalling and neurotrophic factors in maintaining neuronal structure and function in vivo.

Ontogeny of neurotransmitter systems in the paracervical ganglion and uterine cervix of the rat.

BACKGROUND: The paracervical ganglia (PG) are components of the pelvic plexus that provides sensory and motor innervation to the reproductive system of the female rat. Several neurotransmitters including norepinephrine (NE), acetylcholine (ACh), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) are present in neurons of the adult PG and in axons innervating the adult uterus and uterine cervix. The current study was undertaken to describe the onset of immunoreactivity of these neurotransmitters and neuropeptides during development. METHODS: Female rats, ages E18 to P36, were prepared for immunohistochemistry for TH (tyrosine hydroxylase, a marker of noradrenergic neurons), NPY, or VIP as well as the histochemical demonstration of acetylcholinesterase (AChE). RESULTS: All four markers were detected in neurons of the PG at E18. Changes in the appearance of these markers from E18 to P36 reflected previously described growth changes in the PG. Axons containing AChE, TH, NPY, or VIP were first detected within the cervix at E20. Immunopositive axons first appeared as thick, unbranched structures at the outermost portion of the cervical myometrium. Over time, these axon bundles ramified to form discrete varicose axons. The ingrowth was similar for axons containing each of the four markers. CONCLUSIONS: The relative density of each neuronal type in the PG was reflected in the density of axons containing the same marker in the cervix. Changes in neurotransmitter/neuropeptide staining of PG neurons or axons in the cervix were not observed as the animals approached puberty.

Organizational role of testosterone on the biochemical and morphological development of the hypogastric ganglion.

Previous reports have demonstrated that after postnatal day 10 testosterone influences hypogastric ganglion (HG) development by 'activating' morphological and biochemical indices. We now report an 'organizational' influence on the developing HG during the first 10 postnatal days. To investigate the organizational effects of testosterone, male rats were castrated within 12 h of birth. Testosterone replacement therapy initiated following castration maintained the normal number of neurons in the HG. Conversely, delayed replacement therapy starting at day 10 or vehicle treatment only, resulted in a significant decrease in neuron number. Castration also produced a significant decrease in somal and nuclear cross-sectional areas. Testosterone replacement, whether initiated immediately or if delayed until day 10, restored somal and nuclear cross-sectional areas to normal. Tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) activities were sensitive to both testosterone dosage and the time of administration. Testosterone decanoate administered subsequent to castration was not able to completely reverse the enzyme activity deficits, while delayed replacement therapy was even less effective in restoring enzyme activities. In contrast, higher doses of testosterone completely reversed enzyme activity deficits, and in fact produced a significant increase in TH activity. Again, delayed testosterone replacement did not fully restore deficits in enzyme activity. In summary, the hormonal environment during the first 10 days of life is critical for the organization of HG cell number; in contrast, nuclear and cell size appear to be dependent on testosterone for activation. TH and ChAT activities also appear to be organized during this dose- and time-dependent developmental period.

Hypogastric ganglion perinatal development: evidence for androgen specificity via androgen receptors.

The hypogastric ganglion (HG) has previously been shown to be sensitive to both the organizational and activational influences of testosterone. The current investigations examined whether testosterone exerts similar effects prenatally, whether these events are specifically controlled by androgen, and whether androgens might directly masculinize the HG. Prenatal treatment with an anti-androgen, flutamide, resulted in significant decreases in the adult levels of tyrosine hydroxylase (TH) activity, an index of postsynaptic noradrenergic ontogeny, and choline acetyltransferase (ChAT) activity, a marker for presynaptic terminal formation. In addition, testosterone propionate and dihydrotestosterone benzoate reversed the effects of neonatal castration on the development of TH and ChAT activities. In contrast estradiol benzoate was unable to restore enzyme activities. To determine whether the above observations might be produced by direct effects on the HG, androgen cytosol receptor characteristics were studied. Competition and saturation analyses demonstrate that the affinity and specificity of the androgen cytosol receptor in the HG are similar to that displayed in the pituitary, which has previously been shown to contain androgen receptors. These results suggest that the adult levels of TH and ChAT activities are organized during prenatal and early postnatal development. In addition, the organization of the HG appears to be androgen specific. The presence of cytosol androgen receptors suggests that the organizational effects of androgens are possibly induced by a direct mechanism.

Biochemical and morphological effects of castration on the postorganizational development of the hypogastric ganglion.

The biochemical and morphological development of the sympathetic hypogastric ganglion (HG) was examined subsequent to postnatal castration at 10-11 days of age. Previous studies suggested that tyrosine hydroxylase (T-OH) activity, an index of noradrenergic maturation, and choline acetyltransferase (ChAT) activity, a marker for preganglionic terminal formation, were dependent on gonadal hormones during normal ontogeny. In the present studies, morphometric analyses of the HG revealed that the cross-sectional area of the cell soma and nucleus were significantly reduced following postnatal castration at day 10. Conversely, castration produced no change in the number of HG neurons. In addition, postnatal castration prevents the development of postsynaptic T-OH activity to a greater extent than ganglionic protein resulting in a significant loss of T-OH specific activity. In contrast, presynaptic ChAT activity was reduced in parallel with ganglionic protein, thus ChAT specific activity was unchanged. Testosterone replacement therapy, even in groups where treatment was delayed for up to 2 weeks after castration, completely reversed deficits in both T-OH and ChAT activities. These studies suggest that altered development of ganglion protein subsequent to postnatal castration is related to decreases in the size of neurons and not to the loss of neurons. The lack of cell loss also suggest that decreased levels of postsynaptic T-OH activity results from a loss of enzyme activity per cell and the decreased levels of ChAT activity probably represent fewer presynaptic terminals per neuron. In addition, delayed testosterone replacement subsequent to castration was effective in restoring enzyme activities suggesting an 'activational' not 'organizational' role for testosterone after postnatal day 10.