Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons.

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.

Nerve growth factor and brain-derived neurotrophic factor mRNAs are regulated in distinct cell populations of rat heart after ischaemia and reperfusion.

Neurotrophins play a crucial role in the development of the peripheral nervous system and their mRNAs are often regulated after several types of tissue injury. This study has investigated the regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNAs 30 min after myocardial ischaemia followed by reperfusion, by northern blotting, and in situ hybridization in a rat model. Between 2 and 120 h of reperfusion, Ngf mRNA levels showed two- to four-fold up-regulation compared with sham-operated hearts. Scattered Ngf-expressing cells, probably pericytes, were detected in the viable border zone of the myocardium in close association with capillaries, venules, and arterioles. In addition, diffuse Ngf expression was seen in the infarct area after 120 h of reperfusion. Bdnf mRNA showed transient up-regulation after 2 and 5 h of reperfusion and remained at control levels thereafter. Bdnf was expressed in the myocytes of the viable border zone. Nt-3 expression showed no significant changes compared with sham-operated hearts. These results suggest a role for NGF and/or BDNF in the pathogenesis of reperfusion injury or in the alterations of cardiac sensory and sympathetic neuronal function after myocardial ischaemia and reperfusion.

GDNF family receptors in the embryonic and postnatal rat heart and reduced cholinergic innervation in mice hearts lacking ret or GFRalpha2.

Members of the GDNF family, which are important during peripheral nervous system development and kidney organogenesis, signal via Ret and GFRalpha receptors. Here we have studied their possible role in heart development. Gfra1 was expressed in the endocardial cushion mesenchyme at E12 and later, in the developing and mature valves, and in the walls of the aorta and the pulmonary trunk. Gfra2 was expressed in the outer layers of the aorta and pulmonary trunk and in the valves at E18-P60. Endocardial cells showed moderate Gfra2 mRNA and protein expression between E12 and E15. Gfra3 mRNA was detected, mainly postnatally, in scattered cells of the atria and the great vessels. In embryonic and postnatal rat cardiac ganglia, Ret and Gfra2 transcripts were seen in the neurons, whereas Gfra1 and Gfra3 mRNA were preferentially found in non-neuronal cells within the ganglia. GFRalpha2 immunoreactivity was seen in both cardiac ganglion neurons and their nerve fibers. There were no obvious non-neuronal defects in hearts of Ret-, GFRalpha1-, or GFRalpha2-deficient mice, suggesting that these receptors are not essential for gross cardiac development. However, E18 Ret-deficient mice exhibited a reduced volume of cardiac ganglia and cholinergic innervation of the ventricular conduction system. Moreover, adult Gfra2(-/-) mice showed reduced cholinergic innervation by 40% in their ventricles and by 60% in the ventricular conduction system. These findings indicate that GFRalpha2/Ret signaling is required for normal cholinergic innervation of heart.

Neurturin is a neurotrophic factor for penile parasympathetic neurons in adult rat.

Neurturin (NRTN), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. Recent gene ablation studies have shown that NRTN is a neurotrophic factor for many cranial parasympathetic and enteric neurons, whereas its significance for the sacral parasympathetic neurons has not been studied. NRTN signals via a receptor complex composed of the high-affinity binding receptor component GFRalpha2 and the transmembrane tyrosine kinase Ret. The aim of this study was to determine whether NRTN could be an endogenous trophic factor for penis-projecting parasympathetic neurons. NRTN mRNA was expressed in smooth muscle of penile blood vessels and corpus cavernosum in adult rat as well as in several intrapelvic organs, whereas GFRalpha2 and Ret mRNAs were expressed in virtually all cell bodies of the penile neurons, originating in the major pelvic ganglia. (125)I-NRTN injected into the shaft of the penis was retrogradely transported into the major pelvic and dorsal root ganglia. Mice lacking the GFRalpha2 receptor component had significantly less nitric oxide synthase-containing nerve fibers in the dorsal penile and cavernous nerves. In conclusion, these data suggest that NRTN acts as a target-derived survival and/or neuritogenic factor for penile erection-inducing postganglionic neurons.

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves.

Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.

Developmental expression of palmitoyl protein thioesterase in normal mice.

Deficiency in palmitoyl protein thioesterase (PPT) results in the rapid death of neocortical neurons in human. Very little is known about the developmental and cell-specific expression of this lysosomal enzyme. Here we show that PPT is expressed as a major 2.65 kb and a minor 1.85 kb transcript in the mouse brain. Transcript levels gradually increase between postnatal days 10 and 30. In situ hybridization analysis revealed that PPT transcripts are found widely but not homogeneously in the brain. The most intense signal was detected in the cerebral cortex (layers II, IV-V), hippocampal CA1-CA3 pyramidal cells, dentate gyrus granule cells and the hypothalamus. Immunostaining of PPT was localized in the cell soma, axons and dendrites, especially in the pyramidal and granular cells of the hippocampus, correlating well, both spatially and temporally, with the immunoreactivity of a presynaptic vesicle membrane protein, synaptophysin. In whole embryos, at embryonic day 8, the PPT mRNA expression was most apparent throughout the neuroepithelium, and from day 9 onwards it was seen in all tissues. The expression pattern of PPT suggests its general significance for the brain cells and reflects the response to maturation and growth of the neural networks. Strong PPT immunoreactivity in the axons and dentrites would imply that PPT may not be exclusively a lysosomal enzyme. A notable correlation with synaptophysin would suggest that PPT may have a role in the function of the synaptic machinery.

Toward understanding the neuronal pathogenesis of aspartylglucosaminuria: expression of aspartylglucosaminidase in brain during development.

The deficiency of a lysosomal enzyme, aspartylglucosaminidase, results in a lysosomal storage disorder, aspartylglucosaminuria, manifesting as progressive mental retardation. To understand tissue pathogenesis and disease progression we analyzed the developmental expression of the enzyme, especially in brain, which is the major source of the pathological symptoms. Highest mRNA levels in brain were detected during embryogenesis, the levels decreased neonatally and started to increase again from Day 7 on. In Western analyses, a defective processing of aspartylglucosaminidase was observed in brain as compared to other tissues, resulting in very low levels of the mature, active form of the enzyme. Interestingly immunohistochemical analyses of mouse brain revealed that aspartylglucosaminidase immunoreactivity closely mimicked the myelin basic protein immunostaining pattern. The only evident neuronal staining was observed in the developing Purkinje cells of the cerebellum from Days 3 to 10, reflecting well the mRNA expression. In human infant brain, the immunostaining was also present in myelinated fibers as well as in the Purkinje cells and, additionally, in the soma and extensions of other neurons. In the adult human brain neurons and oligodendrocytes displayed immunoreactivity whereas myelinated fibers were not stained. Our results of aspartylglucosaminidase immunostaining in myelinated fibers of infant brain might imply the involvement of aspartylglucosaminidase in the early myelination process. This is consistent with previous magnetic resonance imaging findings in the brains of aspartylglucosaminuria patients, revealing delayed myelination in childhood.

Distribution of GABA receptor rho subunit transcripts in the rat brain.

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.

The messenger RNAs for both glial cell line-derived neurotrophic factor receptors, c-ret and GDNFRalpha, are induced in the rat brain in response to kainate-induced excitation.

Glial cell line-derived neurotrophic factor (GDNF) has two receptors, receptor-tyrosine kinase c-ret and glycosylphosphatidylinositol-linked cell surface receptor GDNFRalpha. Kainate-induced seizures, a widely studied model of neuronal plasticity and human epilepsy, have been shown to increase gene expression of several trophic factors, including GDNF, in the rat hippocampus. Here we show that systemic kainate-induced excitation leads to a transient increase of both c-ret and GDNFRalpha messenger RNAs in the rat brain. Northern analysis demonstrated that, in the hippocampus, the maximal 2.5-fold increase of c-ret and four-fold increase of GDNFRalpha messenger RNAs was observed after 12 h of kainate injection, in contrast to GDNF messenger RNA, which reaches its maximum in 4-6 h. The blocking of de novo protein synthesis by cycloheximide inhibited the induction of GDNF receptors by kainate, whereas blocking of the N-methyl-D-aspartate-type glutamate receptors by the antagonist dizocilpine maleate did not significantly alter the response. Thus, GDNF receptor messenger RNA increase by kainate depends on protein synthesis, but is not mediated by the N-methyl-D-aspartate receptor. GDNFRalpha and c-ret show distinct, but partially overlapping, patterns of expression in the brain after kainate treatment. GDNFRalpha messenger RNA was prominently induced in the dentate gyrus of the rat hippocampus, less in the habenular and reticular thalamic nuclei and cerebral cortex as revealed by in situ hybridization. C-ret transcripts were induced in the hilus of the hippocampus, several thalamic and amygdala nuclei and in superficial layers of the piriform cortex. These data suggest that GDNF and its receptors may play a local role in neuronal plasticity and in neuronal protection following epileptic insults.

Expression of mRNAs for neurotrophins and their receptors in developing rat heart.

Because the neurotrophic system has not been systematically studied in developing heart, we studied the expression of mRNAs for neurotrophins and their high- and low-affinity receptors by radioactive in situ hybridization in the rat heart from embryonic day 9 (E9) to parturition. The neurotrophin-3 (NT-3) transcripts were seen in the group of Leu-7 immunoreactive cells in the ventricular region from E11 to parturition, suggesting that NT-3 is expressed in the part of the developing conduction system, mRNAs for truncated trk receptors, trkC.TK- and trkB.T1, were expressed in the outflow tract at E12 and in the walls of developing aorta and pulmonary trunk from E13 to parturition, whereas the mRNA for catalytic trkC.TK+ was revealed in the walls of aorta and pulmonary trunk from E13 to parturition and in the cardiac ganglion neurons from E14 to adult stage. Transcripts for low-affinity neurotrophin receptor (p75) were transiently seen in the distal outflow tract from E11 to E13, declining by E14. At E18, p75 transcripts were also seen in the cardiac ganglia. Transcripts for nerve growth factor, neurotrophin-4/5, trkA, or trkB.TK+ were not detected. Expression of NT-3 mRNA in the developing conduction system and of trkC.TK + mRNA in the cardiac neurons suggests a role for NT-3 in the innervation of the conduction system. Expression of trkC.TK+ in the wall of aorta and pulmonary trunk suggests that NT-3 also may affect the development of the smooth muscle cells.

Localization of glial cell line-derived neurotrophic factor (GDNF) mRNA in embryonic rat by in situ hybridization.

The localization of glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by in situ hybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx. Also mesenchymal cells of the gut and mesonephric tubules contain GDNF mRNA. At E13, expression is observed in the mesenchymal cell layers of the oesophagus, intestine and stomach, the mesenchymal cells around the condensing cartilages and metanephric kidney mesenchyme. Also, the epithelia of Rathke's pouch and pharynx are intensely labelled. High expression of GDNF mRNA continues at E15 in kidney, gastrointestinal tract and cartilage. At that stage, GDNF mRNA is seen also in whisker pad and skeletal muscles. The distribution of GDNF mRNA in embryonic rat suggests important roles for GDNF in the early differentiation of the kidney tubules, the innervation of the gastrointestinal tract and the differentiation process of the cartilage and muscle. Our results indicate novel functions for GDNF outside the nervous system.