Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.     

TrkA-expressing trigeminal sensory neurons display both neurochemical and structural plasticity despite a loss of p75NTR function: responses to normal and elevated levels of nerve growth factor

In neural crest-derived sensory ganglia, approximately half of the neuronal population expresses the transmembrane trkA receptor that is required for neuronal binding of target-derived nerve growth factor (NGF). These same neurons also express the p75 neurotrophin receptor (NTR) that increases the affinity of trkA for NGF. Depleting p75NTR expression reduces both the survival of trkA-positive sensory neurons and their afferent innervation of peripheral targets. In this investigation, we assessed the neurochemical and structural plasticity of trigeminal sensory neurons in p75NTR-deficient mice in response to either normal or elevated levels of NGF during postnatal development and into adulthood. Although p75NTR-deficient mice have 30% fewer trigeminal neurons, levels of trkA mRNA expression are modestly elevated in these mutant mice as compared to control mice. The density of central afferent axons and local levels of NGF are, however, comparable between mutant and control animals. Thus, despite the survival of fewer trigeminal neurons, neither ganglionic levels of trkA mRNA expression nor the density of central afferent projections are depleted in p75NTR-deficient mice. In response to elevated levels of NGF protein, transgenic mice with and without p75NTR expression display both increased levels of trkA mRNA expression and a greater density of trigeminal central afferent axons as compared to control mice. These data further reveal that an absence of p75NTR function in trigeminal sensory neurons does not diminish their capacity for NGF-dependent plasticity, namely trkA mRNA expression and collateral growth of central afferent axons.     

Long-Term Estrogen Replacement Coordinately Decreases trkA and β-PPT mRNA Levels in Dorsal Root Ganglion Neurons

A population of adult dorsal root ganglion (DRG) neurons bind NGF with high affinity and express the trkA gene. In these cells, NGF regulates gene expression and function. Recently, a number of laboratories reported the presence of estrogen receptors in DRG neurons and profound effects of estrogen on DRG gene expression. Our laboratory, for example, has reported a significant and coordinate decrease in DRG trkA and beta-preprotachykinin (beta-PPT) mRNA levels following 90 days of daily estrogen injections to ovariectomized (OVX) rats. These data suggest, as has been suggested for medial septal cholinergic neurons, that estrogen may collaborate with NGF in the regulation of DRG neuronal gene expression and function. The current study examined further this potential collaboration in the DRG by determining the effect of short-term estrogen replacement in OVX rats on DRG trkA mRNA levels following sciatic nerve transection and the resulting removal of a vital source of NGF for those cells. In OVX rats, about 40% of lumbar DRG neurons contained trkA mRNA. Short-term estrogen replacement had no effect on the percentage of neurons containing trkA mRNA, but increased the mean trkA mRNA level in uninjured DRGs of OVX rats by 23%. Axotomy in OVX rats reduced the mean trkA mRNA level by 55% but did not significantly decrease the percentage of neurons containing the mRNA. Estrogen replacement, 7 days after axotomy, partially and significantly restored the mean trkA mRNA level. It was 49% greater than that of the untreated axotomized DRGs. It did not, however, significantly increase the percentage of DRG neurons containing trkA in axotomized DRGs. These observations show that short-term estrogen has an opposite effect on DRG neuronal trkA mRNA levels as compared to that of long-term estrogen demonstrated in our previous study. Moreover, the current data show that estrogen regulates trkA mRNA levels in the absence of target-derived NGF. These data suggest that estrogen may collaborate with NGF in the maintenance of normal adult DRG gene expression and function. Furthermore, these data suggest that loss of estrogen, such as that associated with menopause, may contribute to a decline in DRG neuronal function and an exacerbation of ongoing neuropathic processes.     

Nitric Oxide-NGF Mediated PPTA/SP,ADNP, and VIP Expression in the Peripheral Nervous System

Nerve growth factor (NGF)-deprivation or axotomy of dorsal root ganglion (DRG) neurons causes stress, which they cope by triggering various mechanisms. Among several molecular changes, in the present study, we demonstrate preprotachykinin-A–substance P (PPTA–SP) and activity-dependent neuroprotective protein–vasoactive intestinal peptide (ADNP–VIP) expression pattern using DRG neurons–Schwann cells coculture and axotomy model. In the presence of NGF, DRG cultures showed high levels of PPTA and ADNP mRNA expression, which were significantly suppressed in the absence of NGF and/or nitric oxide synthase (NOS) inhibition by N ^G-nitro-l-arginine methyl ester (l-NAME), suggesting that both NGF and nitric oxide (NO) can regulate PPTA and ADNP expression. However, treating coculture with NO donor, diethylenetriamine nitric oxide (DETA–NO) did not increase PPTA and ADNP expression in the presence or absence of NGF, although there was a marginal increase in ADNP expression in the absence of NGF. NGF-deprivation increases endogenous NO; thus, DETA–NO had no further effect on PPTA and ADNP expression. Alternatively, NGF produced from NO-stimulated Schwann cells influence gene expression. In addition, interestingly, DETA–NO treatment of Schwann cells alone suppresses both PPTA and ADNP, suggesting differential response of DRG neurons–Schwann cells coculture to DETA–NO. SP and ADNP immunostaining of axotomized DRGs revealed significant reduction in SP and ADNP compared to intact DRG, which was partially recovered in neuronal NOS blocker, 7-nitroindazole (7-NI)-treated DRGs, particularly intense ADNP staining in satellite glia. As ADNP is VIP-responsive gene, we further explored VIP expression in DRGs. Axotomy increased VIP in DRG neurons, but 7-NI treatment caused intense VIP staining in satellite glia. These observations suggest a complex interaction of NO–NGF with PPTA/SP and ADNP–VIP in neuron–glial communication when neurons are stressed.     

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.     

Postnatal Changes in the Expression of the trkA High-affinity NGF Receptor in Primary Sensory Neurons

In development ∼70–80% of dorsal root ganglion (DRG) cells are dependent on nerve growth factor (NGF) for their survival, while in the adult only some 40% of DRG cells express the high-affinity NGF receptor, trkA. This discrepancy suggests that trkA expression, and therefore neurotrophin sensitivity, may alter as the animal matures. We have tested this possibility by counting the number of L4/5 DRG neurons showing immunoreactivity for trkA in rats from the day of birth to postnatal day 14. We also examined changes in p75 and IB4 labelling. On the day of birth, 71% of DRG cells were found to express trkA. However, this percentage gradually fell with age and reached adult levels at postnatal day 14. The expression of p75 did not parallel that of trkA, remaining relatively constant at between 45 and 50% of cells from birth to postnatal day 14. Over the same period there was a marked increase in the proportion of cells which bind the lectin IB4 from 9 (day of birth) to 40% (day 14). Since in the adult the 1B4 population consists of small cells which mostly do not express trkA, this finding suggests that the postnatal down-regulation of trkA occurs in this population. Consistent with this suggestion are the results of double labelling for trkA and IB4, which confirmed that at times intermediate between birth and postnatal day 14 there was a high degree of coexpression between these markers (which is absent in the adult). This result also suggests that the down-regulation of trkA is unlikely to be directly responsible for the emerging IB4 binding.     

Age-dependent effect of nitric oxide on subventricular zone and olfactory bulb neural precursor proliferation

Nitric oxide (NO) synthase (NOS) is developmentally regulated in the embryonic brain, where NO participates in cell proliferation, survival, and differentiation. In adults, NO inhibits neurogenesis under physiological conditions. This work investigates whether the NO action is preserved all along development up to adulthood or whether its effects in adults are a new feature acquired during brain maturation. The relationship between nitrergic neurons and precursors, as well as the functional consequences of pharmacological NOS inhibition, were comparatively analyzed in the subventricular zone (SVZ) and olfactory bulb (OB) of postnatal (P7) and adult (>P60) mouse brains. The SVZ was markedly reduced between P7 and adults, and, at both ages, neurons expressing neuronal NOS (nNOS) were found in its striatal limits. In postnatal mice, these nitrergic neurons contained PSA-NCAM, and their projections were scarce, whereas, in adults, mature nitrergic neurons, devoid of PSA-NCAM, presented abundant neuropil. In the OB, local proliferation almost disappeared in the transition to adulthood, and periglomerular nitrergic neurons, some of which were PSA-NCAM positive, were found in postnatal and adult mice. Administration of the NOS inhibitor L-NAME did not affect cell proliferation in the SVZ or in the OB of postnatal mice, whereas it significantly enhanced the number of mitotic cells in both regions in adults. Thus, the NO action on SVZ neurogenesis is a phenomenon that appears after the postnatal age, which is probably due to the germinal layer size reduction, allowing exposure of the NO-sensitive neural precursors to the NO produced in the SVZ-striatum limits.     

Nerve growth factor sensitivity is broadly distributed among myenteric neurons of the rat colon

Nerve growth factor (NGF) acts on the two-receptor system of trkA and p75 to mediate neuroprotection and influence phenotype and function in the peripheral nervous system, but the effects of NGF on the enteric nervous system (ENS) are virtually unknown. To establish a basis for enteric responsiveness to NGF, we studied the presence and distribution of NGF-sensitive receptors in the myenteric neurons of the normal rat colon and examined their activation via trkA phosphorylation. Fluorescent immunocytochemistry on wholemounts showed that the two NGF receptors were abundantly present in the ENS, with 71% of all neurons positive for trkA and 78% for p75. More thanr 60% of the myenteric neurons expressed both receptors, and exogenous application of NGF resulted in trkA phosphorylation, evidence for high NGF sensitivity within the ENS. trkA was co-expressed with choline acetyltransferase (61% of trkA-positive neurons), neuronal nitric oxide synthase (22%), or calbindin (10%), suggesting widespread potential for NGF action. We conclude that functional receptors for NGF are widely distributed among the diverse enteric phenotypes and argue for a novel NGF-mediated regulatory system within the ENS.     

Neuroprotective and neurodestructive functions of nitric oxide after spinal cord hemisection

Nitric oxide (NO) may subserve different functions in different central neurons subjected to axotomy. The difference may depend on whether the neurons basally express neuronal nitric oxide synthase (nNOS), a biosynthetic enzyme of NO. This is supported by our previous finding that suggests the differential role of NO in neurons of nucleus dorsalis (ND) and red nucleus (RN) which have different basal expression of nNOS. This study aimed to establish firmly the functions of NO, as revealed by nNOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, by the administration of endogenous NO donor, l-arginine (l-arg), and NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME). To relate the role of NO to glutamate receptors (GluR), the distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) in the two nuclei were revealed by immunohistochemical techniques. nNOS immunoreactivity was void in ND neurons, but expressed weakly in the RN normally. It was induced in ipsilateral ND neurons and upregulated on both sides of RN after spinal cord hemisection. Neuronal loss in the ipsilateral ND was augmented by l-arg, but reduced by l-NAME. In the contralateral RN, l-arg attenuated neuronal loss. NMDAR1 was present in most neurons in ND. After axotomy, some NMDAR1 immunoreactive neurons of the ipsilateral ND were induced to express NOS, whereas RN neurons showed strong staining for NMDAR1 and all the AMPA subunits. Most of the NOS-positive neurons in the RN were coexistent with GluR2 in normal rats and those subjected to axotomy. The present data demonstrated that NO exerted neurodestructive function in the non-NOS-containing ND neurons characterized by NMDAR as the predominant glutamate receptor. NO might be beneficial to the NOS-containing RN neurons. This could be attributed to the presence of GluR2. Possible diverse synthesizing pathways of NO in two different central nuclei were suggested from the observation that NOS was colocalized with NADPH-d in ND neurons, but not in RN neurons.     

Short-term estrogen replacement increases beta-preprotachykinin mRNA levels in uninjured dorsal root ganglion neurons, but not in axotomized neurons.

Dorsal root ganglion (DRG) neurons that mediate nociception express the high affinity NGF receptor (trkA) gene and the preprotachykinin (PPT) gene. NGF has been shown to regulate both of these DRG neuronal genes. Our laboratory has shown that these genes are also regulated by estrogen. Long-term daily estrogen replacement, in adult ovariectomized (OVX) rats, causes a coordinate decline in trkA and beta-PPT mRNA levels in lumbar DRG neurons, while short-term estrogen replacement increases trkA mRNA levels in uninjured as well as in axotomized lumbar DRG neurons. The purpose of the current study was to test the hypothesis that short-term estrogen replacement increases DRG beta-PPT mRNA levels in lumbar DRG neurons of OVX rats and that the increase is dependent on target-derived NGF. Sciatic nerve transection (SNT) was used to eliminate target-derived NGF in L4 and L5 DRGs in adult OVX rats. Seven days later, one-half of the SNT and one-half of the animals that had received sham sciatic nerve transactions (SHAM) received two daily injections of estradiol benzoate (EB). The remaining rats received two daily injections of vehicle alone. Quantitative in situ hybridization analyses of sections from L4 and L5 DRGs showed that two daily injections of EB significantly increased beta-PPT mRNA levels in DRGs of SHAM animals, but had no effect on beta-PPT mRNA levels in DRGs from SNT animals. These data coupled with our earlier observations of the effect of short-term estrogen replacement on DRG trkA mRNA levels, indicate that the regulation of DRG beta-PPT mRNA levels by estrogen requires target-derived NGF.     

Developing patterns of nitric oxide synthesizing neurons in the rat striatum: histochemical analysis

The prenatal and postnatal development of NADPH-diaphorase (NADPH-d)/neuronal nitric oxide synthase (nNOS) positive neurons was studied in the striatum of rats. NADPH-d was demonstrated enzyme histochemically and nNOS immunohistochemically using a polyclonal antibody. NADPH-d neurons appeared in the ventrolateral part of the striatum on embryonic day 18 (E18). Thereafter, the number of NADPH-d neurons increased and began to distribute homogeneously in the striatum. The density of NADPH-d neurons became highest at postnatal day 5 (P5) and then decreased as the volume of the striatum continued to increase. The number of NADPH-d neurons reached its peak around 3-4 weeks after birth. The sizes of NADPH-d neurons were measured. The NADPH-d neurons grew larger until P14 (mean area 260 microm(2)) and became smaller thereafter (mean area 170 microm(2)). Patches of high NADPH-d activity and tyrosine hydroxylase (TH) immunoreactivity were also examined in the developing striatum. The distributions of NADPH-d patches overlapped with those of TH-immunoreactive patches by P10. The spatiotemporal appearance of nNOS and overlapping of nNOS patchy distribution with TH point to an important role of NO and to an interaction between nNOS and DA fibers during development of the striatum.     

Local isoform‐specific NOS inhibition: A promising approach to promote motor function recovery after nerve injury

Physical injury to a nerve is the most frequent cause of acquired peripheral neuropathy, which is responsible for loss of motor, sensory and/or autonomic functions. Injured axons in the peripheral nervous system maintain the capacity to regenerate in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Therefore, the identification of molecules that regulate degenerative and regenerative processes is indispensable in developing therapeutic tools to accelerate and improve functional recovery. Here, I consider the role of nitric oxide (NO) synthesized by the three major isoforms of NO synthases (NOS) in motor neuropathy. Neuronal NOS (nNOS) seems to be the primary source of NO that is detrimental to the survival of injured motoneurons. Endothelial NOS (eNOS) appears to be the major source of NO that interferes with axonal regrowth, at least soon after injury. Finally, NO derived from inducible NOS (iNOS) or nNOS is critical to the process of lipid breakdown for Wallerian degeneration and thereby benefits axonal regrowth. Specific inhibitors of these isoforms can be used to protect injured neurons from degeneration and promote axonal regeneration. A cautious proposal for the treatment of acquired motor neuropathy using therapeutic tools that locally interfere with eNOS/nNOS activities seems to merit consideration. © 2010 Wiley‐Liss, Inc.     

NGF对鸡胚背根神经节神经突起生长作用的机制研究

目的探讨神经生长因子(nerve growth factor,NGF)促进鸡胚背根神经节(dorsal root ganglion,DRG)神经突起生长的作用机制。方法实验采用9 d的鸡胚分离背根神经节,原代培养法,观察鸡胚DRG的体外生长情况。通过半定量PCR检测诱导型一氧化氮合酶(iNOS)mRNA表达,采用NO检测试剂盒检测NO释放水平。结果 NGF能明显促进鸡胚背根神经节神经突起生长,同时可见NGF抑制iNOS mRNA表达,NO检测结果显示,添加NGF培养的背根神经节上清NO分泌水平明显降低,与阴性对照组比较差异显著(P0.05)。结论 NGF可促进鸡胚背根神经节神经突起生长,其作用与其下调iNOS mRNA表达及抑制神经损伤因子NO释放有关。     

Histochemical and immunocytochemical localization of nitric oxide synthase in the supramedullary neurons of the pufferfish Tetraodon fluviatilis

The presence of the nitric oxide (NO) converting enzyme, constitutive neuronal NO synthase (nNOS), was investigated in the supramedullary neurons (SN) cluster of the pufferfish Tetraodon fluviatilis. The identification of NADPH diaphorase- (NADPHd-) positivity and the demonstration of nNOS with the BAS technique and with immunofluorescence together, strongly indicate the presence of a constitutive NO converting enzyme in SN cellular bodies and axons, and provides evidence that the SN cluster represents a distinct nitrergic neuronal system in the vertebrate CNS. The possible roles of NO in the cluster are discussed, including an involvement in communication among neurons and between neurons and the glial cells in the cluster.     

Overexpression of Nerve Growth Factor in Skin Increases Sensory Neuron Size and Modulates Trk Receptor Expression

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4–positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.     

L-arginine uptake, the citrulline-NO cycle and arginase II in the rat brain: an in situ hybridization study.

Nitric oxide (NO) is synthesized from a unique precursor, arginine, by nitric oxide synthase (NOS). In brain cells, arginine is supplied by protein breakdown or extracted from the blood through cationic amino acid transporters (CATs). Arginine can also be recycled from the citrulline produced by NOS activity, through argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) activities, and metabolized by arginase. NOS, AS and AL constitute the so-called citrulline-NO cycle. In order to better understand arginine transport, recycling and degradation, we studied the regional distribution of cells expressing CAT1, CAT3, AS, AL, neuronal NOS (nNOS) and arginase II (AII) in the adult rat brain by non-radioisotopic in situ hybridization (ISH). CAT1, AL and AII presented an ubiquitous neuronal and glial expression, whereas CAT3 and AS were confined to neurons. nNOS was restricted to scattered neurons and a few brain nuclei and layers. We demonstrate by this study that cells expressing nNOS all appear to express the entire citrulline-NO cycle, whereas numerous cells expressing AL do not express AS. The differential expression of these genes within the same anatomical structure could indicate that intercellular exchanges of citrulline-NO cycle metabolites are relevant. Thus vicinal interactions should be taken into account to study their regulatory mechanisms.     

Guiding adult Mammalian sensory axons during regeneration

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.     

Increased expression of neuronal nitric oxide synthase in bladder afferent cells in the lumbosacral dorsal root ganglia after chronic bladder outflow obstruction

Nitric oxide (NO), a neurotransmitter in autonomic reflex pathways, plays a role in functional neuroregulation of the lower urinary tract. Upregulation of the levels of neuronal nitric oxide synthase (nNOS), the enzyme system responsible for NO synthesis, has been documented in the peripheral, spinal and supraspinal segments of the micturition reflex in diseases such as cystitis, bladder/sphincter dyssynergia following spinal cord injury and bladder overactivity after cerebral infarction. These observations suggest that NO might play a role in the development of bladder overactivity. In this study, nNOS-immunoreactivity (IR) was evaluated in bladder afferent and spinal neurons following bladder outflow obstruction (BOO) in male and female rats. Chronic BOO was induced by placing lumen reducing ligatures around the proximal urethra. Six weeks following the obstructive or sham surgery, bladder function was evaluated by awake cystometry. Bladder afferent neurons in L1, L2, L6 and S1 dorsal root ganglia (DRG) were identified by retrograde neuronal labeling with injection of Fast Blue into the bladder smooth muscle. A differential distribution of nNOS-IR was subsequently evaluated in bladder afferent neurons in the DRG and in the associated spinal cord segments. The percentage of bladder afferent neurons expressing nNOS-IR was increased in L6 (1.8-fold in males and 1.9-fold in females) and S1 (2.8-fold in males and 5.3-fold in females) DRG. In contrast, no changes in nNOS-IR in neurons or fiber distribution were observed in any spinal cord segments examined.     

Differing patterns of neurotrophin-receptor expressing neurons allow distinction of the transient Frorieps' ganglia from normal DRG before morphological differences appear

The Frorieps' ganglia are dorsal root ganglia (DRG) that form and then degenerate during normal embryonic development of amniotes. Their degeneration or survival has been shown to be modulated by modifying expression of Hox-family and other genes involved in pattern formation, and by the mesodermal microenvironment of the cranial somites in which they develop. In ovo application of the neurotrophin NGF partially rescues DRG2 from degeneration. To further examine the potential role of neurotrophins in the life cycle of Frorieps' DRG we have now quantified the numbers of neurons expressing neurotrophin receptors trkA and trkC in avian Frorieps' ganglia (DRG2) and normal cervical DRG (DRG5). We have found that the Frorieps' DRG are different from normal DRG in terms of the numbers of neurons expressing these receptors. trkC-expressing neurons are generally lacking in DRG2, this is the earliest (St 18, E2.5) described difference between DRG2 and normal DRG, preceding morphological differences between these ganglia that appear at St 20. The difference between DRG2 and DRG5 in terms of numbers of trkA-expressing neurons is evident only at later embryonic stages, where DRG2 contains a higher proportion of trkA neurons than normal cervical DRG. The few trkC+ neurons present late in DRG2 development are not concentrated in the VL portion of the ganglion, the zone where trkC+ neurons are generally found in normal DRG. We also find that DRG2 neurons are smaller than those of normal DRG, this is true for both trkA+ and trkC+ populations. These data together therefore suggest that the neurons that survive in the Frorieps' ganglia at later stages belong almost exclusively to the trkA-expressing DM class DRG neurons. We further find that the differences in the populations of trkA/trkC between DRG2 and DRG5 result from signals from the mesodermal microenvironment, since DRG arising in cranial somites transplanted caudally contain few trkC+ neurons and a higher proportion of trkA+ cells than contralateral controls.