Trophic effects of androgen: Development and hormonal regulation of neuron number in a sexually dimorphic vocal motor nucleus

In Xenopus laevis, the laryngeal motor nucleus (n. of cranial nerves IX‐X) is part of a sexually differentiated, androgen sensitive neuromuscular system devoted to vocalization. Adult males have more n. IX‐X neurons than females; however, during development of n. IX‐X, the rate of neurogenesis does not appear to differ between the sexes. In this study, we explored the role of naturally occurring cell death in the development of this nucleus and asked whether cell death might be involved in establishing the sex difference in neuron number. Counts of n. IX‐X neurons reveal that at tadpole stage 56, males and females have similar numbers of n. IX‐X neurons, but by stage 64 male neuron numbers are greater. This sex difference arises owing to a greater net loss of neurons in females—males lose ∼25% of their n. IX‐X neurons between stages 56 and 64, while females lose ∼47%. Sexual differentiation of n. IX‐X neuron number coincides with a period of developmental cell death, as evidenced by terminal transferase‐mediated dUTP nick‐end labeling and the presence of pyknotic nuclei in n. IX‐X. A role for gonadal hormones in controlling cell number was examined by treating tadpoles with exogenous androgen and determining the number of n. IX‐X neurons at stage 64. Dihydrotestosterone (DHT) treatment from the beginning of the cell death period (stage 54) until stage 64 had no effect on the number of n. IX‐X neurons in males but did significantly increase n. IX‐X neuron number in females. This increase was sufficient to abolish the sex difference normally observed at stage 64. Although DHT induced increases in female neuron number, it did not induce increases in cell proliferation or addition of newly born neurons to n. IX‐X. DHT may therefore have increased neuron number by protecting cells from death. We conclude that androgens can influence the survival of n. IX‐X neurons during a period of naturally occurring cell death, and that this action of androgen is critical to the development of sex differences in n. IX‐X neuron number. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 375–385, 1999     

Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia

The molecular mechanisms responsible for specifying the dorsal-ventral pattern of neuronal identities in dorsal root ganglia (DRG) are unclear. Here we demonstrate that Sonic hedgehog (Shh) contributes to patterning early DRG cells. In vitro, Shh increases both proliferation and programmed cell death (PCD). Increasing Shh in vivo enhances PCD in dorsal DRG, while inducing greater proliferation ventrally. In such animals, markers characteristic of ventral sensory neurons are expanded to more dorsal positions. Conversely, reducing Shh function results in decreased proliferation of progenitors in the ventral region and decreased expression of the ventral marker trkC. Later arising trkA⁺ afferents make significant pathfinding errors in animals with reduced Shh function, suggesting that accurate navigation of later arising growth cones requires either Shh itself or early arising, Shh-dependent afferents. These results indicate that Shh can regulate both cell number and the distribution of cell types in DRG, thereby playing an important role in the specification, patterning and pathfinding of sensory neurons.     

Permeability of the normal rat brain, spinal cord and dorsal root ganglia microcirculations to immunoglobulins G

The distribution of blood-borne immunoglobulins G (IgG) was studied in the cerebral cortex, pineal gland, spinal cord and dorsal root ganglia of normal Lewis rats using the detection of autologous anti-horseradish peroxidase (HRP) antibodies. This detection was performed by means of light and electron microscopy. This study demonstrated that, in the cerebral cortex and the spinal cord microcirculations, endothelial cells are a restrictive barrier against IgG while IgG are able to diffuse into the perivascular parenchyma of the pineal gland and spinal ganglia.     

Calretinin-immunoreactive nerves in the uterus, pelvic autonomic ganglia, lumbosacral dorsal root ganglia and lumbosacral spinal cord

Nerves containing the calcium-binding protein calretinin have been reported in several organs but not in female reproductive organs and associated ganglia. This study was undertaken to determine if nerves associated with the uterus contain calretinin and the source(s) of calretinin-synthesizing nerves in the rat (are they sensory, efferent, or both?). Calretinin-immunoreactive nerves were present in the uterine horns and cervix where they were associated with arteries, uterine smooth muscle, glands, and the epithelium. Calretinin-immunoreactive terminals were apposed to neurons in the paracervical ganglia; in addition, some postganglionic neurons in this ganglion were calretinin positive. Calretinin perikarya were present in the lumbosacral dorsal root ganglia, no-dose ganglia, and lumbosacral spinal cord. Retrograde axonal tracing, utilizing Fluorogold injected into the uterus or paracervical parasympathetic ganglia, revealed calretinin-positive/Fluorogold-labeled neurons in the dorsal root and nodose ganglia. Also, capsaicin treatment substantially reduced the calretinin-positive fibers in the uterus and pelvic ganglia, thus indicating the sensory nature of these fibers. The presence of calretinin immunoreactivity identifies a subset of nerves that are involved in innervation of the pelvic viscera and have origins from lumbosacral dorsal root ganglia and vagal nodose ganglia. Though the exact function of calretinin in these nerves is not currently known, calretinin is likely to play a role in calcium regulation and their function.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Prenatal development of the rat dorsal root ganglia

Summary This study describes three-dimensional aspects of the development and pseudo-unipolarization of neuroblasts and the maturation of satellite cells in prenatal rat dorsal root ganglia, using scanning electron microscopy, after removal of extracellular connective tissue components by trypsin digestion and HC1 hydrolysis.At 14 days of gestation, the vast majority of neurons are spindle-shaped or bipolar and only 3% are unipolar, while at 16 and 18 days this percentage has increased to 30% and 91%, respectively. The initial portions of the central and peripheral neuronal processes gradually approach each other and form a common initial portion. Finally, the cytoplasm of this common initial portion becomes thinner and elongates to form the stem process of the mature cell.Satellite cells are present from the beginning of the period studied, but intricate networks of branching satellite cell processes only develop after about day 17.     

Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death

Only male zebra finches sing, and several brain regions implicated in song behavior exhibit marked sex differences in neuron number. In one region, the high vocal center (HVC), this dimorphism develops because the incorporation of new neurons is greater in males than in females during the first several weeks after hatching. Although estrogen (E₂) exposure stimulates neuron addition in females, it is not known where (E₂) acts, or to what extent sexual differentiation influences the production, specification, or survival of HVC neurons. In the present study we first reassessed sex and (E₂)-induced differences in cell degeneration within the HVC using the TUNEL technique to identify cells undergoing DNA fragmentation indicative of apoptosis. HVC neuron number, as well as the density and number of TUNEL-labeled and pyknotic cells within the HVC were measured in normal 20- and 30-day-old males and females, and in 30-day-old females implanted with E₂ on posthatch day 18. Although HVC neuron number was greater in males than in females, and was masculinized in E₂ females, no group differences were evident in the absolute number of dying cells. These results indicate that sex differences in cell survival within the HVC do not entirely account for sexually dimorphic neuron addition to this region. Rather, sexual differentiation acts on some HVC neurons before they complete their migration and/or early differentiation. Although the migratory route of HVC neurons is not known, a large number of E₂ receptor-containing cells (ER cells) reside just ventromedial to the HVC and adjacent to the proliferative ventricular zone. Next, we investigated whether these ER cells contribute to early-arising sex differences in HVC neuron addition. By combining [³H] thymidine autoradiography with immunocytochemistry for ERs, we first established that ER-expressing cells are not generated during posthatch sexually dimorphic HVC neuron addition, and thus are not young HVC neurons that transiently express ERs during their migration. Furthermore, in 25-day-old birds we found no sex difference in the density of pyknotic cells among this group of ER cells, suggesting that these cells do not promote the differential survival of HVC neuronal precursors migrating through this region. Rather, ER cells or other cell populations may establish sex differences in HVC neuron number by creating dimorphisms in cellular specification. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 61–71, 1997     

Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia.

Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions.     

Effects of testosterone on the development of a sexually dimorphic neuromuscular system in ciliary neurotrophic factor receptor knockout mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) innervate the perineal muscles, bulbocavernosus (BC), and levator ani (LA). Testosterone regulates the survival of SNB motoneurons and BC/LA muscles during perinatal life. Previous findings suggest that effects of testosterone on this system may be mediated by trophic factors—in particular, by a factor acting through the ciliary neurotrophic factor α‐receptor (CNTFRα). To test the role of CNTFRα in the response of the developing SNB system to testosterone, CNTFRα +/+ and −/− mice were treated with testosterone propionate (TP) or oil during late embryonic development. BC/LA muscle size and SNB motoneuron number were evaluated on the day of birth. Large sex differences in BC and LA muscle size were present in newborn mice of both genotypes, but muscle volumes were reduced in CNTFRα −/− animals relative to same‐sex, wild‐type controls. Prenatal testosterone treatment completely eliminated the sex difference in BC/LA muscle size in wild‐type animals, and eliminated the effect of the CNTFRα gene deletion on muscle size in males. However, the effect of TP treatment on BC and LA muscle sizes was blunted in CNTFRα −/− females. SNB motoneuron number was sexually dimorphic in oil‐treated, wild‐type mice. In contrast, there was no sex difference in SNB motoneuron number in oil‐treated, CNTFRα knockout mice. Prenatal treatment with testosterone did not increase SNB motoneuron number in CNTFRα −/− mice, but also did not significantly increase SNB motoneuron number in newborn wild‐type animals. These findings confirm the absence of a sex difference in SNB motoneuron number in CNTFRα −/− mice. Moreover, the CNTFRα gene deletion influences perineal muscle development and the response of the perineal muscles to testosterone. Prenatal TP treatment of CNTFRα −/− males overcomes the effects of the gene deletion on the BC and LA muscles without a concomitant effect on SNB motoneuron number. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 317–325, 1999     

Synaptic connections in a dissociated mixed culture of rat dorsal root ganglion and spinal cord neurons

Postsynaptic currents and action potentials recorded from neurons in a mixed culture of rat dorsal root ganglion and spinal cord cells are described. The existence of mutual synaptic connections between the above two types of neurons is demonstrated. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 358–360, July–August, 2006.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKζ and DGKι in the DRG. By immunohistochemical analysis, DGKζ immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKι immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKζ in the nucleus and DGKι in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKζ between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.The work was supported by grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan (M.T., K.G.) and from the Ono Medical Research Foundation, Kato Memorial Bioscience Foundation, and Janssen Pharmaceutical (K.G.) and by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Enhanced neuronal regeneration by retinoic acid of murine dorsal root ganglia and of fetal murine and human spinal cord in vitro

Summary This study demonstrates that retinoic acid (RA), an active metabolite of vitamin A, can act to enhance regeneration of neurites, at physiologic concentrations, in vitro. Explanted fragments of mouse dorsal root ganglia (DRG) and mouse and human spinal cord (SC) were maintained, in vitro, for periods up to 11 d. Murine DRG neurons were exposed to RA concentrations ranging from 100 μM to 1 nM, whereas neurons within murine and human SC explants were exposed to 10 μM to 10 nM RA. Results show that RA significantly (P<0.001) increases mean neurite length but not neurite number. Specifically, murine DRG neurons showed increases in mean neurite length of 30.7% with individual explants showing increases of up to 133.5%. Murine and human SC showed mean enhancements of 43.4 and 58.1%, respectively, but did so at lower concentrations of RA. The results indicate that RA may play a potentially critical role in neuronal regeneration.     

Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD(65) and GAD(67), EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD(65) encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD(65). Goldfish GAD(67) encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD(67). Goldfish GAD(65) and GAD(67) are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD(65) mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD(67) mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD(65) and GAD(67) mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

Axotomy of developing rat spinal motoneurons: Cell survival,soma size,muscle recovery,and the influence of testosterone

During the period of synapse elimination, motoneurons are impaired in their ability to generate or regenerate axonal branches: following partial denervation of their target muscle, young motoneurons do not sprout to nearby denervated fibers and after axonal injury, they fail to reinnervate the muscle. In the rat levator ani (LA) muscle, which is innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB), synapse elemination ends relatively late in development and can be regulated by testosterone. We took advantage of this system to determine if the end of synapse elimination and the development of regenerative capabilities by motoneurons share a common mechanism, or, alternatively, if these two events can be dissociated in time. Axotomy on or before postnatal day 14 (P14) caused the death of SNB motoneurons. By P21, toward the end of synapse elimination in the LA muscle, SNB motoneurons had developed the ability to survive axonal injury. Altering testosterone levels by castration on P7 followed by 4 weeks of either testosterone propionate or control injections did not change the ability of SNB motoneurons to survive axonal injury during development, although these same treatments alter the time course of synapse elimination in the LA muscle. Thus, we dissociated the inability of SNB motoneurons to recover from axonal injury from their developmental elimination of synaptic terminals. We also measured the effect of early axotomy on motoneuronal soma size and on target muscle weight. Axotomy on P14 caused a long-lasting decrease in the soma size of surviving SNB motoneurons, whereas motoneurons axotomized on P28 recovered their normal soma size. Axotomy on or before P7 caused severe atrophy of the target muscles, matching the extensive loss of motoneurons. However, target muscle recovery after axotomy on P14 was as good as recovery after axotomy at later ages, despite greater motoneuronal death after axotomy on P14. This result may reflect an increase in motor unit size, a decrease in polyneuronal innervation by SNB motoneurons that survive axotomy on P14, or a combination of the two. © 1995 John Wiley & Sons, Inc.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.