Expression of NGF receptor and NGF receptor mRNA in the developing and adult rat retina

Nerve growth factor (NGF) has been recently found to rescue axotomized retinal ganglion cells (RGCs) of the adult rat from degeneration. Because the trophic effect of NGF involves a receptor-coupling event, the characterization and cellular localization of the NGF receptor (NGFR) in the retina are essential to understanding the possible specific action of NGF in this district of the central nervous system. We report here that the NGFR mRNA is expressed in fetal, neonatal, and adult rat retina. Using monoclonal antibody 192-IgG to immunoprecipitate and immunohistochemically identify NGFR, we also found that the NGFR from the retina has a molecular weight identical to that of the NGFR from PC12 cells. The NGFR is localized on RGCs and Müller cells. Finally, following ligation of the optic nerve, NGFR-immunopositive material was found to accumulate both distal and proximal to the site of ligation, suggesting that RGC axons anterogradely and retrogradely transport the NGFR. These data raise the possibility that NGF may play a specific role in rat RGCs.     

Expression of NGF receptor in the developing and adult primate central nervous system

Monoclonal antibodies against human NGF receptor have been used for immunocytochemical localization of NGF receptors in the CNS of macaques and baboons at various stages of development. In the adult, neurons in most brain regions are devoid of detectable NGF receptors. However, abundant NGF receptor immunoreactivity is present on a population of neurons in basal forebrain, which, on the basis of appearance and pattern of distribution, probably correspond, at least in part, to magnocellular cholinergic neurons of this region. NGF receptors were also associated with the vasculature in most brain regions. NGF receptor immunoreactivity is present on Mueller glia of neural retina. In macaque fetuses, approximately 1 month prenatally, retinal Mueller glia possess lower levels of receptor, while higher levels of receptor are present in the retinal nerve fiber layer. In fetal cerebellum, abundant receptor immunoreactivity is present on Purkinje cells, granule cells of the premigratory zone of the external granule layer, and neurons of the deep nuclei. Immunoreactivity decreases with subsequent development and is absent in the adult. In cerebellum, levels of NGF receptor assayed by affinity crosslinking to radioiodinated NGF, and levels of NGF receptor mRNA assayed by Northern blot analysis decrease dramatically during the last month of fetal life.     

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Hippocampus and cortex

N-methyl-D-aspartate receptor (NR) activation in the hippocampus and neocortex plays a central role in memory and cognitive function. We analyzed the cellular expression of the five NR subunit (NR1 and NR2A-D) mRNAs in these regions with in situ hybridization and human ribonucleotide probes. Film autoradiograms demonstrated a distinct pattern of hybridization signal in the hippocampal complex and the neocortex with probes for NR1, NR2A, and NR2B mRNA. NR2C and NR2D probes yielded scattered signals without a distinct organization. At the emulsion level, the NR1 probe produced high-density hybridization signals across the hippocampal complex. NR2A mRNA was higher in dentate granule cells and pyramidal cells in CA1 and subiculum compared to hilus neurons. NR2B mRNA expression was moderate throughout, with higher expression in dentate granule cells, CA1 and CA3 pyramidal cells than in hilus neurons. In the hippocampal complex, the NR2C probe signal was not different from background in any region, whereas the NR2D probe signal resulted in low to moderate grain densities. We analyzed NR subunit mRNA expression in the prefrontal, parietal, primary visual, and motor cortices. All areas displayed strong NR1 hybridization signals. NR2A and NR2B mRNAs were expressed in cortical areas and layers. NR2C mRNA was expressed at low levels in distinct layers that differed by region and the NR2D signal was equally moderate throughout all regions. Pyramidal cells in both hippocampus and neocortex express NR1, NR2A, NR2B, and, to a lesser extent, NR2D mRNA. Interneurons or granular layer neurons and some glial cells express NR2C mRNA. J. Comp. Neurol. 390:75–90, 1998. © 1998 Wiley-Liss, Inc.     

Sexually dimorphic expression of the NGF receptor gene in the developing rat brain.

To define relations between trophic molecules and known sexually dimorphic traits in brain, we examined possible sex differences in nerve growth factor (NGF) and NGF receptor (NGF-R) gene expression in the rat cholinergic basal forebrain (BF)-hippocampal system. Hippocampal NGF mRNA levels did not differ between sexes; in contrast, BF NGF-R mRNA levels were greater in neonatal females than males, paralleling the known dimorphic development of cholinergic enzyme activity. Cerebellar NGF-R mRNA levels were also dimorphic in the neonate, suggesting that sex-specific influences may regulate trophic receptor gene expression in diverse brain systems.     

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Striatum and globus pallidus

N-methyl-D-aspartate receptors (NRs) play an important role in basal ganglia function. By using in situ hybridization with ribonucleotide probes, we investigated the regional and cellular distribution of NR subunit mRNA expression in the human basal ganglia: caudate nucleus, putamen, lateral globus pallidus (LGP), and medial globus pallidus (MGP). Analysis of both film autoradiograms and emulsion-dipped slides revealed distinct distribution patterns for each subunit. On film autoradiograms, the signal for NR1, NR2B, and NR2C in the striatum (STR) was higher than in globus pallidus (GP). The NR2D probe gave a stronger signal in GP than in STR. For NR2A we found a signal in all regions. Analysis of emulsion-dipped sections demonstrated that in striatal neurons, the NR2B signal was higher than in GP neurons. In GP neurons, NR2D was more abundant than in striatal neurons. Despite the relatively low signal on film for NR2C in GP, we found a slightly higher signal in GP per neuron than in STR since in the pallidal areas neurons were sparse but intensely labeled. NR1 and NR2A were more evenly distributed over neurons of STR and GP. Between the different parts of STR and GP, we observed only minor differences in the expression of NRs. In MGP a subpopulation of neurons exhibiting low NR2D signals could be separated from the majority of neurons showing an intense NR2D signal. Since the physiological properties of NRs are dependent on subunit composition, these data suggest a high degree of regional specialization of NR properties in the human basal ganglia. J. Comp. Neurol. 390:63–74, 1998. © 1998 Wiley-Liss, Inc.     

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.     

NGF expression in the developing rat brain: effects of maternal separation

A number of studies have shown that mothering style in rodents can produce neuroendocrine, neurochemical and behavioural changes in the adult, although the basic mechanisms initiating this cascade of events still need to be investigated. Long term changes in neuronal function might be due to alterations in the expression of neurotrophins which have been shown to promote neuronal survival, differentiation and function during development, such as Nerve Growth Factor (NGF). NGF is essential for proper development of sympathetic and neural crest-derived sensory neurons of the peripheral nervous system as well as of central cholinergic neurons. In previous studies, using a maternal separation paradigm, we have shown that NGF expression is increased in the dentate gyrus and the hilus of the hippocampus as a result of brief (45 min) maternal separations. In the present study neonatal rats were separated for longer periods of time (up to 3 h) and at different ages during development (9 and 16 days postnatally). Results indicate that the effects of maternal separation on NGF expression are stronger with longer separations and are not restricted to the hippocampal region but can be seen also in other brain areas. Overall these results indicate that external factors, such as the presence/absence of the mother, can modify neurotrophic factor's availability in the brain, thus indicating NGF as a potential player in environmentally-mediated brain plasticity during development.     

NGF receptor immunoreactivity in aged rat brain

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.     

Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum

The peptide sequence of autocrine motility factor (AMF), a tumor secreted cytokine that induces cell motility, corresponds to that of the previously identified cytokine/enzyme, neuroleukin/glucose-6-phosphate isomerase. Neuroleukin is a neurotrophic factor that promotes neuronal survival and sprouting at the neuromuscular junction. The AMF receptor (AMF-R) has been identified and shown to be highly expressed in malignant tumors with minimal expression in adjacent normal tissue. Neuroleukin mRNA is highly expressed in the cerebellum and we therefore undertook a developmental study of AMF-R expression in rat cerebellum. As determined by immunoblot, AMF-R is expressed at equivalent high levels in brain and cerebellum of postnatal day 5 (P5) and 12 (P12) rats and at significantly reduced levels in the adult. Coimmunofluorescence studies with MAP-2 and gamma-actin revealed that at P12, AMF-R was mainly localized to Purkinje and granule cells. Moreover, the premigratory cells of the external granular layer were also immunoreactive for AMF-R suggesting a role for AMF-R in granule cell migration during cerebellar development in the first two weeks after birth. In the adult, AMF-R distribution was similar to P12, although weaker, and was localized to Purkinje and granule cells. AMF-R labeling of GFAP positive glial processes could not be detected in cerebellar sections although in cerebellar primary cultures, both neurons and glial cells were labeled for AMF-R. In neurons, AMF-R labeling was present in the cell body, neurites and growth cones. These data indicate that regulation of the neurotrophic function of neuroleukin might be regulated spatially and temporally by expression of its receptor, AMF-R, in developing and adult cerebellum.     

Phytoestrogen action in the adult and developing brain

Abstract Soy isoflavonoids are plant phytoestrogens available as dietary supplements and are increasingly advocated as a natural alternative to oestrogen replacement therapy. As weak oestrogen agonists/antagonists with a range of other enzymatic activities, the isoflavonoids provide a useful model to investigate the actions of endocrine disruptors. Here, the activational and organisational effects of these compounds on the brain are reviewed. In spite of their preferential affinity for oestrogen receptor (ER)beta in vitro, isoflavonoids act in vivo through both ERalpha and ERbeta. Their neurobehavioural actions are largely anti-oestrogenic, either antagonising or producing an action in opposition to that of oestradiol. Small, physiologically relevant exposure levels can alter oestrogen-dependent gene expression in the brain and affect complex behaviour in a wide range of species. The implications for these findings in humans, and particularly in infants, largely remain uninvestigated but are a subject of increasing public interest.     

Distribution of non-NMDA glutamate receptor mRNAs in the developing rat cochlea

In situ hybridization was used to document the distribution of mRNA encoding six subunit isoforms of non-N-methyl D-aspartic acid (NMDA) glutamate receptors (GluR1, GluR2, GluR3, GluR4, GluR5 and GluR6) in the inner ears of embryonic, postnatal and adult rats. GluR2 and GluR3 expression in the spiral ganglion appeared well before birth, and reached adult levels several days before the onset of function in the cochlea. In the spiral limbus, expression of GluR2 and GluR3 mRNA reached very high levels at around the time of birth, then declined after a few days. Low levels of GluR1, GluR4 and GluR6 expression were detected in various tissues of the cochlea during development. In the adult cochlea, GluR expression was limited to GluR2 and GluR3 mRNAs in the spiral ganglion neurons and GluR2 mRNA in fibrocytes of the spiral limbus, a non-neural tissue. The ontogenetic expression of additional GluR subunit genes and their appearance in different cochlear tissues could reflect different roles for these genes during development, or less precise regulation of gene expression within the GluR family. In particular, the very high levels of GluR gene expression in the spiral limbus during the perinatal period support a non-neural function, perhaps as cell surface receptors during tissue differentiation. © 1995 Wiley-Liss, Inc.     

High and low affinity NGF receptor mRNAs in human foetal spinal cord and ganglia.

The cellular localization of mRNAs encoding the low affinity NGF receptor (here referred to as LANR) and the putative high affinity receptor for NGF, trk, have been studied in the human foetal spinal and sympathetic ganglia, and spinal cord, using in situ hybridization. The receptor mRNAs were highly expressed in the spinal and sympathetic ganglia, with most but not all neurons expressing both LANR and trk mRNA. Spinal nerve rootlets distal to the spinal ganglia expressed LANR but not trk mRNA, confirming the presence of the low affinity receptor in developing Schwann cells. In the spinal cord, LANR mRNA was found throughout the medial and lateral motor columns while, trk mRNA was detected in scattered cells in the dorsal aspect of developing grey matter.     

Transport and elimination of recombinant human NGF during long-term delivery to the brain

The gene for human nerve growth factor (NGF) has been cloned into a mammalian cell line and large quantities of recombinant human NGF (rhNGF) can now be produced for clinical use, but little is known about the fate of rhNGF following delivery to the brain. In this study, we implanted polymer matrices containing¹²⁵l-labeled rhNGF into the brains of adult rats and measured spatial distributions of the released protein for 8 weeks after implantation. NGF content in the tissue was determined by counting gamma radiation in thick (1 mm) sections and by autoradiography of thin (20 μm) sections. For the first several days, the rate of NGF release from the polymer matrix was high ( 100 ng/day); maximal NGF concentrations, measured at the polymer-tissue interface, were correspondingly high (> 20 μg/ml) through day 4. At later times, the release rate decreased (2–10 ng/day) and lower maximal concentrations were observed (1–10 μg/ml). NGF levels were always highest in the tissue sections closest to the polymer; during the 8 weeks of the experiment, NGF levels measured in thick sections decreased 100-fold, from 30 ng/section at day 2 to 0.3 ng/section at day 54. The first 10-fold decrease occurred during the first 10 days of the study; a further 6 weeks was required to achieve the second 10-fold decrease. Throughout the experiment, the majority of NGF remained within a restricted zone around the polymer at all times; the mass of NGF decreased to 10% of the maximal level within 2–3 mm of the polymer matrix. At early times (< 1 week), radiolabel corresponding to > 20 pg of NGF was also detected in regions of the brain further removed from the polymer. Comparison of local rhNGF concentration profiles with a simple mathematical model indicated that rhNGF diffuses through the brain interstitial space and is eliminated with a half-life of 45 min, although elimination appears to be substantially slower in white matter regions. This limited ability of NGF to penetrate and be retained within the brain tissue indicates that NGF will need to be delivered almost directly to the target tissue for efficacy.     

视神经不完全损伤动物模型视神经TrkA表达及局部缓释NGF对其表达的影响

目的 观察TrkA在不完全损伤视神经上的表达及医用生物蛋白胶局部缓释神经生长因子对其表达的影响.方法 雄性SD大鼠按随机数字表法分为3组,损伤组双侧视神经均用动脉瘤夹临时阻断夹钳夹30 s,神经生长因子组双侧视神经均用动脉瘤夹临时阻断夹钳夹30 s后局部应用含有神经生长因子的医用生物蛋白胶,正常组不做任何处理.以RT-PCR方法检测各组伤后3、5、7、10d视神经TrkAmRNA的表达情况.结果 正常组相对值为0.44±0.06.损伤组和神经生长因子组3 d后表达有明显增高,其中损伤组第3、5、7、10天分别增高至正常的7.1、35.0、19.6和9.5倍,NGF组第3、5、7、10天分别增高至正常的8.0、32.4、20.4和8.5倍;两组之间不同时间测定值差异均无统计学意义(P>0.05).结论 视神经不完全损伤后TrkA表达短期内增高,局部应用医用生物蛋白胶缓释神经生长因子不影响其表达.     

Expression of NG2 and platelet-derived growth facto receptor alpha in the developing neonatal rat brain

Platelet-derived growth factor receptor alpha(PDGFRα) is a marker of oligodendrocyte precursor cells in the central nervous system. NG2 is also considered a marker of oligodendrocyte precursor cells. However, whether there are differences in the distribution and morphology of oligodendrocyte precursor cells labeled by NG2 or PDGFRα in the developing neonatal rat brain remains unclear. In this study, by immunohistochemical staining, NG2 positive(NG2~+) cells were ubiquitous in the molecular layer, external pyramidal layer, internal pyramidal layer, and polymorphic layer of the cerebral cortex, and corpus callosum, external capsule, piriform cortex, and medial septal nucleus. NG2~+ cells were stellate or fusiform in shape with long processes that were progressively decreased and shortened over the course of brain development. The distribution and morphology of PDGFRα positive(PDGFRα~+) cells were coincident with NG2~+ cells. The colocalization of NG2 and PDGFRα in the cell bodies and processes of some cells was confirmed by double immunofluorescence labeling. Moreover, cells double-labeled for NG2 and PDGFRα were predominantly in the early postnatal stage of development. The numbers of NG2~+/PDGFRα~+ cells and PDGFRα~+ cells decreased, but the number of NG2~+ cells increased from postnatal days 3 to 14 in the developing brain. In addition, amoeboid microglial cells of the corpus callosum, newborn brain macrophages in the normal developing brain, did not express NG2 or PDGFRα, but NG2 expression was detected in amoeboid microglia after hypoxia. The present results suggest that NG2 and PDGFRα are specific markers of oligodendrocyte precursor cells at different stages during early development. Additionally, the NG2 protein is involved in inflammatory and pathological processes of amoeboid microglial cells.     

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.