P2X2 and P2X3 receptor expression in postnatal and adult rat urinary bladder and lumbosacral spinal cord

P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R(2) = 0.96, P 相似文献   

Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain

Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP-binding cassette (ABC) transporter subfamilies ABCA1-4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up-regulation of ABCA2 occurred in the white matter at 14 days-of-age when various myelin protein genes are known to be up-regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio-temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.     

The ontogeny of cytosol and nuclear progestin receptors in male rat brain and its male-female differences

The fetal and postnatal development of the progestin receptor systems in the intact male rat brain was investigated by means of the in vitro cytosol binding and the nuclear exchange assay using [³H]R5020 ([17α-methyl-³H]17α,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione). The cortical cytosol receptors, first detectable at day 0, rapidly increased at day 7, reaching a maximum at day 10, then gradually declined thereafter. The receptors in the HPOA appeared clearly at day 1, increased during the first 10 days, then remained constant at days 14–21. The postnatal developmental patterns of cytosol brain progestin receptors in males were essentially similar to those in females, but there were some differences between both sexes. The male HPOA at days 10–14 contained more receptors than the female one. Nuclear progestin binding was low in the neonatal male brain at days 1–3. Despite the low level of serum progesterone, the cortical nuclear binding suddenly increased at days 7–10, then remained high at days 14–21. A similar, though less pronounced, pattern was seen in the HPOA. The male pattern of nuclear binding, thus, essentially resembled the female one. However, lower binding in the cortex and, possibly, HPOA was found in males than in females at days 10–21. After progesterone injection postnatal male rats accumulated a lower concentration of progestin receptors in the cortex and, possibly, HPOA than similarly-treated females.It is concluded from these results that progestin receptors in male rat brain appear immediately after birth and develop differentially in the cortex and HPOA. The sudden onset of increased nuclear translocation of endogenous progestin receptor complexes may occur in the brain at around days 7–10. There is a marked sex difference in the nuclear progestin receptor system in the postnatal brain, particularly the cortex. Moreover, the postnatal male brain has lower capacities of nuclear receptor translocation than does the female one. The progestin receptor system in the cortex and, possibly, HPOA of rats in the early postnatal life might be involved with some processes in the mechanism of sexual differentiation of the brain.     

Autoradiographic study of the ontogeny of the imipramine receptors in the cerebral cortex of rats

Localization of 3H-imipramine binding sites in the cerebral cortex of rats was studied on the 19th day of prenatal development and on the 3d and 14th days of postnatal ontogenesis. Imipramine was injected into rats on days 17, 18 and 19 of gestation. The maximal imipramine receptor density was found in cortex layers IV-V. The therapeutic doses of imipramine provoked marked stimulation of the formation of imipramine receptors on the 3d day of postnatal development. However, on the 14th day the 3H-imipramine binding level was stabilized.     

Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.     

Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing.

The expression of two alternative splice variants, Flip and Flop, in mRNAs encoding the four AMPA-selective glutamate receptors (GluR-A, -B, -C, and -D) was studied in the developing brain by in situ hybridization. These receptors are expressed prominently before birth, and patterns of distribution for Flip versions remain largely invariant during postnatal brain development. In contrast, the Flop versions are expressed at low levels prior to postnatal day 8. Around this time, the expression of Flop mRNAs increases throughout the brain, reaching adult levels by postnatal day 14. Thus, receptors carrying the Flop module appear to participate in mature receptor forms.     

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.     

Postnatal expression of neurotransmitters, receptors, and cytochrome oxidase in the rat pre-B?tzinger complex.

The pre-B?tzinger complex (PBC) is postulated as the center of respiratory rhythmogenesis. Previously, we found a reduction or plateau of cytochrome oxidase (CO) activity in the PBC and other respiratory nuclei at postnatal days 3-4, despite a general increase of CO with age, suggesting a period of synaptic readjustment. The present study examined the expression of CO and a number of neurochemicals in the PBC at closer time intervals. At postnatal days 3-4 and, more prominently, at postnatal day 12, expression of CO, glutamate, and N-methyl-D-aspartate receptor subunit 1 was reduced, whereas expression of GABA, GABA(B) receptor, glycine receptor, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit 2 was increased. These findings are consistent with our hypothesis that decreased CO activity is associated with an increase in inhibitory drive (mediated by GABA and glycine, their receptors, and possibly blockage of Ca(2+) entry by glutamate receptor subunit 2) and a decrease in excitatory drive (mediated by glutamate and its receptors). Our findings point to two critical periods during postnatal development of the rat when their respiratory system may be more vulnerable to respiratory insults.     

生后早期听觉剥夺、经验改变大鼠听皮层NMDA受体NR2B蛋白表达

应用蛋白质印迹检测技术,研究早期听觉剥夺、经验对大鼠听皮层NMDA受体NR2B蛋白表达的影响.结果表明,听觉剥夺使生后14天龄组和28天龄组动物听皮层NR2B蛋白表达水平明显下降(P<0.05,P<0.01),听觉剥夺7天后再给予纯音暴露则又使NR2B表达水平明显提高(P<0.05),呈现双向调节趋势.听觉剥夺和纯音暴露对生后42天龄组大鼠听皮层NR2B表达不再产生明显调节作用(P>0.05).结果提示,在大鼠生后发育关键期,听觉剥夺、经验可改变听皮层NMDA受体NR2B蛋白表达水平.研究结果为研究感觉功能发育可塑性的机制提供了重要实验资料.     

Amino Acid Levels in the Guinea Pig Spinal Gray Matter After Axotomy of Primary Sensory and Descending Tracts

This study attempts to determine if the axonal endings of dorsal root sensory fibers and of descending axons to the spinal gray matter in the guinea pig store glutamate and/or aspartate. Bilateral dorsal rhizotomy (spinal segments C5-T1) and partial cordotomy (segment C5, right side) were used to interrupt primary sensory and descending tracts, respectively. At 1 and 2 days after surgery, amino acid levels were determined in regions microdissected from areas of the gray matter of spinal segment C7 that receive heavy projections from the primary sensory and the descending tracts. These regions were identified by visualizing the degeneration of axons and their terminal fields in silver-impregnated light microscopic preparations of the spinal cord. After dorsal rhizotomy, the heaviest degeneration in the spinal gray appeared centrally in laminae II-IV and medially in laminae IV-VI. The levels of aspartate, glutamate, and gamma-aminobutyrate were reduced by 34, 21, and 26% in laminae II-IV and 28, 33, and 23% in medial laminae IV-VI. The levels of glycine, alanine, and threonine-serine-glutamine (unseparated) were increased. After partial cordotomy, the heaviest degeneration in the spinal gray appeared laterally in laminae IV-VI, dorsolaterally in lamina VII, and in lamina IX. The levels of aspartate and glutamate were reduced by 22 and 28% in lateral laminae IV-VI and by 26 and 28% in dorsolateral laminae VII and IX. Glycine levels were reduced by 9% in dorsolateral laminae VII and IX. The levels of gamma-aminobutyrate, alanine, and threonine-serine-glutamine were either unchanged or raised. These findings suggest that the axonal endings of the primary sensory and of one or more of the descending tracts probably contain relatively high levels of glutamate and aspartate, and that they may use these amino acids as transmitters. The partial deafferentation of spinal interneurons and the destruction of some propriospinal fibers probably caused the losses of gamma-aminobutyrate and glycine, and contributed modestly to those of aspartate.     

Fimbria-Fornix Transections Selectively Down-Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.     

Decreased Release of D-Aspartate in the Guinea Pig Spinal Cord After Lesions of the Red Nucleus

This study attempts to determine if fibers that project from the guinea pig red nucleus to the spinal cord use L-glutamate and/or L-aspartate as transmitters. Unilateral injections of kainic acid were placed stereotaxically in the red nucleus to destroy the cells of origin of the rubrospinal tract. Six days after the injection, Nissl-stained sections through the lesion site showed that the majority of neurons in the red nucleus ipsilateral to the kainic acid injection were destroyed. In addition, the lesioned area included parts of the surrounding midbrain reticular formation. Silver-impregnated, transverse sections of the cervical spinal cord revealed the presence of degenerating fibers contralaterally in laminae IV-VII of the gray matter. Ipsilaterally, very sparse degeneration was evident in laminae VII and VIII of the gray matter. Two to six days after surgery, the electrically evoked, Ca2(+)-dependent release of both D-[3H]aspartate, a marker for glutamatergic/aspartatergic neurons, and gamma-amino[14C]-butyric acid ([14C]GABA) was measured in dissected quadrants of the spinal cervical enlargement. Lesions centered on the red nucleus depressed the release of D-[3H]aspartate by 25-45% in dorsal and ventral quadrants of the cervical enlargement contralaterally. The release of [14C]GABA was depressed by 27% in contralateral ventral quadrants. To assess the contribution of rubro- versus reticulospinal fibers to the deficits in amino acid release, unilateral injections of kainic acid were placed stereotaxically in the midbrain reticular formation lateral to the red nucleus. Nissl-stained sections through the midbrain revealed the presence of extensive neuronal loss in the midbrain and rostral pontine reticular formation, whereas neurons in the red nucleus remained undamaged. In the spinal cord, degenerating axons were present ipsilaterally in laminae VII and VIII of the gray matter. Some fiber degeneration was also evident contralaterally in laminae V and VI of the gray matter. This lesion did not affect the release of either D-[3H]aspartate or [14C]GABA in the spinal cord. The substantial decrements in D-[3H]aspartate release following red nucleus lesions suggests that the synaptic endings of rubrospinal fibers mediate the release of D-[3H]aspartate in the spinal cord. Therefore, these fibers may be glutamatergic and/or aspartatergic. Because other evidence suggests that rubrospinal neurons are probably not GABAergic, the depression of [14C]GABA release probably reflects changes in the activity of spinal interneurons following the loss of rubrospinal input.     

Immunohistochemical investigations of excitotoxicity in experimental spinal cord trauma

The concept of "excitotoxicity" assumes that high concentration of glutamate (main excitatory neuromediator) acting through specific receptors leads to damage of cells due to an influx of calcium ions. Proteins called "excitatory amino acid transporters" (EAATs), present in astroglia, play important role in the removal of glutamate. We investigated the expression of GluR2 (glutamate receptor), EAAT1, and EAAT2 by immunohistochemistry in formalin-fixed, paraffin-embedded rat spinal cords, previously subjected to experimental mechanical trauma. In the injured spinal cords, an elevated immunoreactivity of GluR2 was noted even 10 min after trauma and was still observed 2 days after injury. Strong immunoreactivity was observed not only in many cells in gray matter but also in some cells in white matter (probably glial cells). In the injured spinal cords, we observed stronger (as compared with controls) expression of EAATs in the white matter, especially 6 hours after injury. The results support the role of excitotoxicity in mechanical trauma of spinal cord suggesting a possibility of long lasting elevated expression of glutamate receptor. It may help to understand and to explain beneficial action of "anti-glutamate" drugs, reported by other investigators.     

H3-dopamine and H3-quinuclidinylbenzilate binding by autonomic ganglia tissue in rats of various age

Binding of tritiated ligands of muscarinic and dopamine receptors was analysed in rats 1, 7, 14, 28, 60 days and 24-30 months old. The following ganglia were studied: the nodose ganglion, the lumbar ganglia of sympathetic chain, the main pelvic ganglion in male rats and the paracervical ganglion in female rats. The same level was found for binding of each of ligands for all investigated ganglia. Parameters of postnatal dynamics of development M-cholino- and dopamine reception systems prove to be quite similar, but not identical. Both of the systems reach matured level during first 2 postnatal weeks. In comparison with quinuclidinylbenzilate binding failure of dopamine binding in aged rats is to be more pronounced.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

Differential Modulation of TREM2 Protein during Postnatal Brain Development in Mice

During postnatal development, microglia, the resident innate immune cells of the central nervous system are constantly monitoring the brain parenchyma, cleaning the cell debris, the synaptic contacts overproduced and also maintaining the brain homeostasis. In this context, the postnatal microglia need some control over the innate immune response. One such molecule recently described to be involved in modulation of immune response is TREM2 (triggering receptor expressed on myeloid cells 2). Although some studies have observed TREM2 mRNA in postnatal brain, the regional pattern of the TREM2 protein has not been described. We therefore characterized the distribution of TREM2 protein in mice brain from Postnatal day (P) 1 to 14 by immunostaining. In our study, TREM2 protein was expressed only in microglia/macrophages and is developmentally downregulated in a region-dependent manner. Its expression persisted in white matter, mainly in caudal corpus callosum, and the neurogenic subventricular zone for a longer time than in grey matter. Additionally, the phenotypes of the TREM2+ microglia also differ; expressing CD16/32, MHCII and CD86 (antigen presentation markers) and CD68 (phagocytic marker) in different regions as well as with different intensity till P7. The mannose receptor (CD206) colocalized with TREM2 only at P1–P3 in the subventricular zone and cingulum, while others persisted at low intensities till P7. Furthermore, the spatiotemporal expression pattern and characterization of TREM2 indicate towards its other plausible roles in phagocytosis, progenitor’s fate determination or microglia phenotype modulation during postnatal development. Hence, the increase of TREM2 observed in pathologies may recapitulate their function during postnatal development, as a better understanding of this period may open new pathway for future therapies.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Effects of prenatal cocaine, morphine, or both on postnatal opioid (mu) receptor development

We studied the effects of prenatal cocaine and morphine given separately and in combination on the (1) postnatal brain mu-opioid receptor development and (2) interaction of dopamine with mu receptors. Pregnant rats received single daily intraperitoneal (I.P.) injections of saline, cocaine (20 mg/kg), morphine (2 mg/kg), or the combination of both drugs from day 13 to day 20 of gestation. Postnatal days (P) 1, 7, 14, and 28, whole brains were analyzed for opioid receptor binding and mu mRNA. Prenatal cocaine administered by itself had no significant effect on the ontogeny of brain mu receptors on all the days studied when compared to controls. The morphine-treated group showed a significant increase in mu receptor binding on P1 and P7. Exposure to both cocaine and morphine showed a significant increase in mu receptor density on P1 and P7. In addition, there was also a significant increase in MOR mRNA in both the morphine alone and combination groups. Pretreatment with dopamine D2 receptor antagonist (sulpiride, 20 mg/kg) prior to drug administration showed decreased mu receptor binding on P1 and P7. These results suggest that prenatal exposure to morphine or a combination of cocaine and morphine significantly increases mu receptor density. By P14, mu-opioid receptor binding was no longer different than the control. This may suggest that the effect on receptor may be short-lived and that other key intracellular events may be activated to mediate the long-term effects. Also, the data show that dopaminergic mechanisms are (or opioid-dopamine interaction is) involved in the effects of morphine alone or morphine in combination with cocaine on mu receptor regulation.     

The influence of transient spinal ischemia on substance P positive nerve structures

The aim of this study was to investigate, if transient spinal ischemia and a period of 4-day reperfusion will change the distribution pattern of substance P in the spinal cord of rabbits. Strongly enhanced staining of substance P positive nerve structures appeared in the superficial dorsal horn (laminae I, II), the Lissauer's tract, the pericentral region (lamina X), and in the areas of autonomic nuclei (sympathetic-intermediolateral--IML nucleus and parasympathetic-sacral parasympathetic nucleus--SPN) in the control group. Transient spinal ischemia was produced by occlusion of the abdominal aorta just below the left renal artery. Neuropathology of the lesion 4 days after transient ischemia was characterized by selective necrosis of gray matter in the central part of dorsal horn and medial portions of anterior gray matter. Areas with the most dense accumulation of substance P positive structures stayed almost intact. Therefore, no significant change in the distribution pattern of substance P was found in the spinal cord of animals with ischemia-reperfusion-induced injury.