Pre- and postnatal development of opiate receptor subtypes in rat spinal cord

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.     

Localization of components of glycinergic synapses during rat spinal cord development

The sequence of events leading to the chemical matching of presynaptic neurotransmitters and postsynaptic transmitter receptors is investigated here in vivo for the spinal glycine receptor (GlyR) by using immunocytochemical methods. In the ventral horn of adult rat spinal cord, GlyRs are only present at glycinergic postsynaptic differentiations where they are stabilized by the associated protein gephyrin. With quantitative confocal microscopy, we found that gephyrin is detected before GlyRs at embryonic day (E)13–E14 and at E15, respectively, inside the cytoplasm and at plasmalemmal loci. Around the time of birth, the number of cell surface gephyrin-immunoreactive (-IR) spots exceeds that of GlyR. They first match 10 days after birth. The densities of postsynaptic gephyrin- and GlyR-IR were quantified between birth and the adult stage with post-embedding immunogold staining. Immunostaining for gephyrin and GlyR was not detected in the extrasynaptic membrane. The density of staining in postsynaptic membrane increased progressively with development. The inhibitory amino-acid content of the presynaptic terminal boutons opposed to gephyrin-IR sites was also analyzed. In the newborn, postnatal day 10, and adult, more than 90% of these boutons were immunostained for glycine. As seen with serial sections, 38% and 51.2% of the terminals also contained γ-aminobutyric acid (GABA) in neonate and adult, respectively. These data indicate that around the time of birth, most glycine-containing boutons, some also containing GABA, are opposed to gephyrin-IR postsynaptic densities, whereas GlyRs are not present. Our results suggest that gephyrin determines subsynaptic loci on the plasma membrane where GlyR will subsequently accumulate. J. Comp. Neurol. 398:359–372, 1998. © 1998 Wiley-Liss, Inc.     

Ionotropic glutamate receptor expression in human spinal cord during first trimester development

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.     

Bisphenol a regulates the estrogen receptor alpha signaling in developing hippocampus of male rats through estrogen receptor

Bisphenol A (BPA), one of the most common environmental endocrine disruptors, has been recognized to have wide adverse effects on the brain development and behavior. These adversities are related to its ability to bind estrogen receptor (ER) with subsequent alteration of its expression in the target areas. However, very little is known about whether BPA exposure also affects ER phosphorylation and its translocation to nucleus during postnatal development, two critical steps for its function. Here, we found that during development from postnatal day 7 (P7) to P21, the alpha subtype of ER (ERα) in the hippocampus of male rats experienced remarkable alterations in terms of its expression, phosphorylation and translocation to nucleus. Exposure to low level of BPA had bidirectional, development‐dependent effects on the expression of ERα mRNA and protein, but decreased ERα phosphorylation and impaired its translocation to nucleus throughout the period investigated. Treatment with low dose of ICI 182,780 (ICI), an ER antagonist to block the binding of ER with BPA, reversed the altered ERα following BPA exposure, highlighting critical involvement of ER. Moreover, ICI treatment rescued the hippocampus‐dependent behavioral deficits in the adult rats experiencing early‐life BPA exposure. Overall, our results indicate that BPA interferes with the ERα signaling in the developing hippocampus in an ER‐dependent manner, which may underlie its adverse behavioral and cognitive outcomes in adult animals. © 2014 Wiley Periodicals, Inc.     

Immunohistochemical localization of enkephalin in rat brain and spinal cord.

The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretreated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nucleus tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.     

Immunohistochemical study of catecholaminergic cell bodies in the rat spinal cord

Immunohistochemistry of three specific synthesizing catecholamine enzymes was used in the rat spinal cord to determine precisely the distribution of catecholaminergic perikarya and the nature of the neurotransmitter they contain. Single and double labeling experiments were performed on cryostat sections from perfused rats. The peroxidase anti-peroxidase (PAP) and the indirect fluorescence techniques were used for labeling spinal catecholaminergic somata and separated into two completely different populations. The first is located in the upper cervical cord and includes three apparently distinct groups: a lateral cluster, of probably a noradrenergic nature, and two central subgroups where noradrenergic and dopaminergic neurons are intermingled. It is likely that these cervical cells represent caudal extensions of the medullary catecholaminergic cell groups. In the remaining cord, only tyrosine hydroxylase immunoreactive cell bodies have been found. Accordingly, this second population is probably dopaminergic. It is present almost exclusively in the first sacral segments, where it is located in the commissural (mostly lateral) grey matter and in the marginal dorsal horn.     

Expression of L1 decreases during postnatal development of rat spinal cord

L1 is a cell adhesion molecule that is highly expressed on developing axons and is associated with neurite outgrowth, guidance, and fasciculation. In this study we systematically examined L1 expression at all spinal levels across eight postnatal ages to detect regional and developmental differences. We observed striking changes in the developmental pattern of L1 expression between birth (P0) and adult ages, with intense L1-immunopositive axons prevalent throughout the funiculi at P0 compared with predominantly L1-immunonegative funicular axons in adults. At all ages and spinal levels examined, some L1-positive dorsal root afferents entered the spinal cord, coursed in Lissauer's tract, and projected into the superficial dorsal horn and the dorsal columns, as well as across the dorsal commissure. Additional L1-positive axons were detected consistently around the perimeter of the spinal cord, in the dorsolateral funiculus, and adjacent to the central canal. While specific L1-labeled axons were detected at all ages, a pattern of segmental variation was observed within animals, with the highest levels of L1 expression detected in lumbar and sacral segments and the lowest in cervical spinal cord. The pattern of L1 immunoreactivity was compared to that of the growth-associated protein GAP-43 and the results indicated colabeling of most axons. These observations demonstrate that L1 is expressed on immature axons well into postnatal development, possibly until they have completed their differentiation. Furthermore, the L1-positive axons that continue to be detected in adults are likely to be either unmyelinated or sprouting axons.     

Occurrence of glutamate receptor subunit 1-containing aggresome-like structures during normal development of rat spinal cord interneurons.

During a developmental study of the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptor subunits in rat spinal cord, we observed the existence of cytoplasmic inclusion bodies with positive immunoreactivity to glutamate receptor subunit 1 (GluR1) but not to other glutamate receptor subunits. GluR1-positive bodies have a diameter of between 1 and 3 microm and can be seen widely distributed throughout spinal cord gray matter, with the exception of the ventral horn region. They transiently appear within a definite developmental time-period between embryonic day 19 and postnatal day 17 and are only associated with neuronal cells. Ultrastructural analysis revealed that these inclusions were located adjacent to the nucleus and consisted of amorphous material without any limiting membrane. Immunocytochemical analysis revealed that the inclusions displayed positive immunoreactivity to ubiquitin, HSP70, and 20S proteasome. All these data indicate that GluR1-containing inclusions display all the ultrastructural and immunocytochemical characteristics of the recently described structure, which have been given the name aggresomes. Further studies are needed to determine the biological significance of these normally occurring aggresome-like inclusions, because aggresomes are usually considered in a pathologic context.     

Immunohistochemical localization of substance P in postmortem rat and human spinal cord

The stability of substance P-like immunoreactivity was examined in postmortem rat and human spinal cord using radioimmunoassay and indirect fluorescence immunohistochemistry. The distribution of fluorescence in rat and human spinal cord was unchanged at intervals up to 48 hours (h) and 87 h, respectively. Fine linear fluorescent fibers were seen only in rat spinal cord processed at time intervals up to two h postmortem; they were never observed in human spinal cord, which was routinely obtained more than 6 h postmortem. The content of substance P-like material in human spinal cord nd its immunohistochemical appearance was not affected by age, the nature of the terminal illness, or autopsy delay. However, no correlation was found between intensity of fluorescent staining and content as measured by radioimmunoassay. It may be possible to use postmortem analysis of substance P in diseases of the central nervous system, but radioimmunoassay and immunohistochemistry should be used concurrently.     

Immunohistochemical demonstration of serotonin neurons in autonomic regions of the rat spinal cord

The immunohistochemical distribution of serotonin neurons in normal and transected spinal cords of rats was examined. Intraspinal serotonin neurons were immunostained as far rostral and caudal as T3 and Co1, respectively. All serotonin neurons were located in lamina VII and X, and most were located in spinal autonomic areas. Both bipolar and multipolar neurons were observed with many of the neurons oriented longitudinally to the long axis of the cord. Spinal neurons immunostained for serotonin were visible with and without L-tryptophan and monoamine oxidase inhibitor pretreatment.     

Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development

Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERβ mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.     

Ontogeny of the peptidergic system in the rat spinal cord: Immunohistochemical analysis

The ontogeny of neuropeptides, such as somatostatin (SRIF), substance P (SP), leucine-enkephalin (LE), and neurotensin (NT) in the spinal cord (including the spinal ganglion) of the rat, was examined by means of the indirect immunofluorescence method. SRIF and SP appear in the early fetal period before the establishment of the spinal synaptic transmission system, and their appearance precedes that of LE and NT, thus suggesting that SRIF and SP might have some important role in the development of the spinal cord. Furthermore, a number of SRIF-positive structures are found during the fetal period in the spinal cord; however, SRIF-positive fibers in the ventral horn, lamina V, VI, and X tend to decrease remarkably in number after birth, while those found in the dorsal horn maintain their immunoreactivity even in the adult rats. These facts suggest that SRIF in the latter area might function as a neurotransmitter, whereas in the former areas, SRIF might have another role in the development of the spinal cord. SP-positive structures also made their appearance during the fetal period. SP-positive fibers continue to increase in number after birth, and they can be seen throughout the entire spinal cord even in the adult rats. It becomes difficult to identify SP-positive neurons as the rats grow. Numerous SP-positive cells are demonstrated, however, by colchicine pretreatment, thus suggesting that this system is functioning actively in the adult rats. LE-and NT-positive structures appear at perinatal stages and they continue to increase in number after birth. These facts suggest that these peptides (SP, LE and NT) might act as neurotransmitters.     

Immunohistochemical changes of the blood-brain barrier in rat spinal cord after heavy-ion irradiation

Changes in the blood-brain barrier (BBB) of the rat spinal cord after irradiation with heavy ion were investigated ultrastructurally and immunohistochemically by using SMI 71, a monoclonal antibody against rat endothelial barrier antigen (EBA); anti-ZO-1, a polyclonal antibody against endothelial tight junctions, anti-rat serum to extravasated serum, and anti-vascular endothelial growth factor (VEGF). The lower thoracic and lumbar cord of male Wistar rats was singly irradiated with a carbon beam at a dose of 30 Gy. Rats were sacrificed before or after the onset of hind limb paralysis. Histologically, white-matter vacuolization was observed from 13 weeks after irradiation, and white-matter necrosis was first noted at 17 weeks. SMI 71 staining was decreased or lost 13 weeks after irradiation, just prior to the formation of white-matter necrosis, and was almost completely lost in the center and periphery of the white-matter necrosis. Although ZO-1 expression and tight junctions in the ultrastructure were preserved at that time, serum leakage occurred almostly completely in parallel with the changes in EBA. Therefore, carbon-ion irradiation at a dose of 30 Gy induces BBB breakdown 13 weeks after irradiation. The SMI 71-negative blood vessels were sparsely distributed throughout the entire white and gray matter, and there was no evidence of preferential localization. Immunostaining of smooth muscle actin showed that most of the SMI 71-negative blood vessels were veins or capillaries. These findings suggest that the hyper-permeability of the veins and/or capillaries that occurs after a certain latent period is one of the important factors in the pathogenesis of delayed radiation injury by carbon ions, the same as by X-rays. Radiation-induced functional disturbances of the endothelium and involvement of cytokines such as VEGF are suspected of being the cause of such vascular hyper-permeability.     

Progestin receptor expression in the developing rat brain depends upon activation of estrogen receptor alpha and not estrogen receptor beta

Perinatal 17beta-estradiol (E2) rapidly and markedly affects the morphological and neurochemical organization of the vertebrate brain. For instance, the sex difference in perinatal progestin receptor (PR) immunoreactivity in the medial preoptic nucleus (MPN) of the rat brain is due to the intracellular conversion of testosterone into E2 in males. Neonatal alpha-fetoprotein prevents circulating estrogens from accessing the brain, therefore, to overcome alpha-fetoprotein sequestration of E2, estrogen replacement studies during development have used natural and synthetic estrogen dosages in the milligram to microgram range. These levels could be considered as supraphysiological. Moreover, it is not clear through which ER subtype E2 acts to induce PR expression in the neonatal rat MPN because E2 binds similarly to estrogen receptor (ER)alpha and ERbeta. Consequently, we investigated whether nanogram levels of E2 affected PR protein and mRNA levels in the neonatal MPN. Furthermore, propylpyrazole-triol (PPT), a highly selective agonist for ERalpha, and diarylpropionitrile (DPN), a highly selective agonist for ERbeta, were used to determine if E2-dependent PR expression in the neonatal rat is mediated through ERalpha and/or ERbeta. Immunocytochemistry and quantitative real-time RT-PCR determined that as little as 100 ng E2 significantly induced PR protein and mRNA in the female and neonatally castrated male MPN on PN 4, indicating that the neonatal rat brain is highly sensitive to circulating estrogens. PPT, but not DPN, induced PR expression in the neonatal MPN and arcuate nucleus (Arc), demonstrating that PR expression in the neonatal rat brain depends solely on E2 activated ERalpha. In the lateral bed nucleus of the stria terminalis (BSTL), neither PPT nor DPN affected PR expression, suggesting the presence of a gonadal hormone-independent PR regulatory mechanism.     

Solubilization of the glycine receptor from rat spinal cord

Glycine receptors, as detected by glycine-displaceable [3H]strychnine binding, were solubilized from a membrane fraction of rat spinal cord by the non-ionic detergent Triton X-100. The solubilized material retained its high affinity for [3H]strychnine and exhibited the typical pharmacological properties of the membrane-bound glycine receptor. On sucrose density gradients, the solubilized receptor had a sedimentation coefficient of 8.3 +/- 0.4 S. Gel exclusion chromatography on Sepharose 6 B in the presence of phosphatidylcholine gave a Stokes radius of 7.3 +/- 0.3 nm.     

Traumatic injury alters opiate receptor binding in rat spinal cord

Recent studies with dynorphin (an endogenous ligand for the kappa-opiate receptor) and receptor-selective opiate antagonists have indicated a role for the kappa-receptor in the pathophysiology of spinal cord injury. However, no studies have specifically examined opiate receptor binding in relation to spinal injury. In the present experiments, opiate receptor binding was measured in spinal cord after traumatic injury in rats using the selective radioligands [3H] [D-Ala2, D-Leu5]enkephalin (delta-receptor agonist); [3H] [D-Ala2,MePhe4,Gly-(ol)5]enkephalin (mu-receptor agonist); and [3H]ethylketocyclazocine (kappa-receptor agonist). The specific binding of ethylketocyclazocine, but not the other agonists, showed a significant, time-dependent, and localized increase at the injury site. Since dynorphin, which has been implicated as an injury factor after spinal trauma, shows similar localized increases after spinal injury, the present data are consistent with the hypothesis that up-regulation of the kappa-receptor after injury may contribute to the subsequent secondary injury process.