Expression and localization of dystrophins and β-dystroglycan in the hypothalamic supraoptic nuclei of rat from birth to adulthood

Dystrophins (Dps) are the sub-membranous proteins that work via the dystrophin-associated proteins complex, which comprises β-dystroglycan (β-DG), a cell surface receptor for extracellular matrix. Recently, we have revealed β-DG decrease and central function impairment of supraoptic nucleus (SON) in Dp71 deficient adult mice, opening the question on the profiles of Dps and β-DG during SON development. At birth and the age of 10, 20 and 60 days, we examined the expression by RT-PCR and Western-blotting, and the distribution by immunohistochemistry of Dps and β-DG. Also, we analyzed, by immunohistochemistry and Western-blotting, the neuropeptide, arginine vasopressin (AVP), in the SON at the different ages.At birth, Dp71 and to a lesser extends, Dp140 and Dp427, and also β-DG are revealed in the SON. They are localized in the magnocellular neurons (MCNs), astrocytes and vessels.From birth to adulthood, the AVP raise in the SON coincides with the progressive increase of Dp71 level while the level of Dp140 and Dp427 increased only at D20, D10 post-natal development, respectively, and β-DG expression did not change. Moreover, the location of Dps or/and β-DG in the cell compartments was modified during development: at D10, Dps appeared in the astrocytes end-feet surrounding MCNs, and at D20, Dps and β-DG codistributed in the astrocytes end-feet, surrounding MCNs and vessels. Such a distribution marks the first steps of post-natal SON development and may be considered essential in the establishment of structural plasticity mechanisms in SON, where astrocyte end-feet, vessels, magnocellular neurons, are physiologically associated. The disappearance of β-DG in the MCNs nucleus marks the adulthood SON and suggests that the complex of Dps associating β-DG is required for the nucleoskeleton function in the post-natal development.     

Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina

The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy (DMD) patients and in some genotypes of mice lacking dystrophin has been attributed to altered expression of short products of the dystrophin gene. We have investigated the potential role of Dp71, the most abundant C-terminal dystrophin gene product, in retinal electrophysiology. Comparison of the scotopic electroretinograms (ERG) between Dp71-null mice and wild-type (wt) littermates revealed a normal ERG in Dp71-null mice with no significant changes of the b-wave amplitude and kinetics. Analysis of DMD gene products, utrophin and dystrophin-associated proteins (DAPs), showed that Dp71 and utrophin were localized around the blood vessels, in the ganglion cell layer (GCL), and the inner limiting membrane (ILM). Dp71 deficiency was accompanied by an increased level of utrophin and decreased level of beta-dystroglycan localized in the ILM, without any apparent effect on the other DAPs. Dp71 deficiency was also associated with an impaired clustering of two Müller glial cell proteins-the inwardly rectifying potassium channel Kir4.1 and the water pore aquaporin 4 (AQP4). Immunostaining of both proteins decreased around blood vessels and in the ILM of Dp71-null mice, suggesting that Dp71 plays a role in the clustering and/or stabilization of the two proteins. AQP4 and Kir4.1 may also be involved in the regulation of the ischemic process. We found that a transient ischemia resulted in a greater damage in the GCL of mice lacking Dp71 than in wt mice. This finding points at a crucial role played by Dp71 in retinal function.     

Dystrophin 71 and α1syntrophin in morpho-functional plasticity of rat supraoptic nuclei: Effect of saline surcharge and reversibly normal hydration

Dystrophin (Dp) is a multidomain protein that links the actin cytoskeleton to the extracellular matrix through the dystrophin associated proteins complex (DAPC). Dp of 71?kDa (Dp71), corresponding to the COOH-terminal domain of dystrophin, and α1-syntrophin (α1Syn) as the principal component of the DAPC, are strongly expressed in the brain. To clarify their involvement in the central control of osmotic homeostasis, we investigated the effect of 14?days of salt loading (with drinking water containing 2% NaCl) and then reversibly to 30?days of normal hydration (with drinking water without salt), first on the expression by western-blotting and the distribution by immunochemistry of Dp71 and α1Syn in the SON of the rat and, second, on the level of some physiological parameters, as the plasma osmolality, natremia and hematocrit.Dp71 is the most abundant form of dystrophin revealed in the supraoptic nucleu (SON) of control rat. Dp71 was localized in magnocellular neurons (MCNs) and astrocytes, when α1Syn was observed essentially in astrocytes end feet. After 14?days of salt-loading, Dp71 and α1Syn signals decreased and a dual signal for these two proteins was revealed in the astrocytes processes SON surrounding blood capillaries. In addition, salt loading leads to an increase in plasma osmolality, natremia and hematocrit. Reversibly, after 30?days of normal hydration, the intensity of the signal for the two proteins, Dp71 and α1Syn, increased and approached that of control. Furtheremore, the levels of the physiological parameters decreased and approximated those of control.This suggests that Dp71 and α1Syn may be involved in the functional activity of the SON. Their localization in astrocyte end feet emphasizes their importance in neuronal–vascular–astrocyte interactions for the central detection of osmolality. In the SON, Dp71 and α1Syn may be involved in osmosensitivity.     

The role of utrophin and Dp71 for assembly of different dystrophin-associated protein complexes (DPCs) in the choroid plexus and microvasculature of the brain

In the brain, utrophin is present in the choroid plexus epithelium and vascular endothelial cells, whereas the short C-terminal isoform of dystrophin (Dp71) is localized in the glial end-feet surrounding blood vessels. Both proteins serve as anchors for the so-called dystrophin-associated protein complex (DPC), composed of isoforms of syntrophin, dystroglycan and dystrobrevin. Numerous transporter proteins and channels have a polarized distribution in vascular endothelial cells and in glial end-feet, suggesting an association with the DPC. We investigated the composition and localization of the DPC in dependence on the anchoring proteins in mice lacking either utrophin (utrophin0/0) or dystrophin isoforms (mdx3Cv). Three distinct complexes were identified: (i) associated with utrophin in the basolateral membrane of the choroid plexus epithelium, (ii) associated with utrophin in vascular endothelial cells, and (iii) associated with Dp71 in the glial end-feet. Upon ablation of utrophin or Dp71, the corresponding DPCs were disrupted and no compensation of the missing protein by its homologue was observed. Association of the water channel aquaporin 4 with the glial DPC likewise was disrupted in mdx3Cv mice. These results demonstrate the essential role of utrophin and Dp71 for assembly of the DPC and suggest that these proteins contribute to the proper functioning of the cerebrospinal fluid and blood-brain barriers.     

Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina

Dp260 is a C-terminal isoform of dystrophin and is expressed specifically in the retina. Abnormal electroretinograms (ERG) in some Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients are likely linked to a disruption of Dp260. To clarify the importance of Dp260 in the retina, we examined dystrophin exon 52 knock- out mice, whose expression of Dp260 is impaired. We also confirmed the localization of Dp260 in the outer plexiform layer (OPL) of the retina. Disruption of Dp260 causes a change in the localization of beta- dystroglycan, which is normally found in the OPL of the retina. This suggests a requirement for Dp260 for normal formation of the dystrophin- dystroglycan complex in the retina. Dp71, also expressed in the retina, was, however, not detected in the OPL. The difference in localization of Dp260 and Dp71 implies that the two isoforms have different functions. The dystrophin exon 52 knock-out mice had a prolonged implicit time of the b-wave in ERG, although no significant change was observed in amplitude. These ERG findings differed from those of DMD and BMD patients, especially with regard to amplitude of the b-wave, but make it clear that Dp260 is required for normal electrophysiology.      

腹腔注射2-脱氧-D-葡萄糖可诱发大鼠视上核和室旁核神经元表达Fos

目的 探讨腹腔注射2-脱氧-D-葡萄糖（2-DG）能否激活大鼠下丘脑视上核（SON）和室旁核（PVN）神经元而表达Fos。方法 健康雄性SD大鼠12只,随机分为腹腔注射2-DG组（6只）、生理盐水对照组（3只）及正常对照组（3只）。各自处理后,应用免疫组织化学方法,观察各组下丘脑SON和PVN内Fos表达及其与催产素（OT）和加压素（VP）的双标情况,同时采用ELISA方法对血清中OT和VP的含量进行检测。 结果与生理盐水对照组和正常对照组相比,2-DG引发的特异性Fos免疫阳性产物主要集中分布于下丘脑外侧区和穹隆周区,在SON、PVN也有密集表达。SON和PVN内的Fos表达与该区的特异性神经活性物质OT和VP有共存。OT/Fos双标细胞率（双标细胞占OT阳性细胞的百分率）在SON和PVN分别为87.10%、90.57%,明显高于VP/Fos在这两个核团的双标率（双标细胞占VP阳性细胞的百分率,68.42%、76.92%）,两者比较差异有统计学意义（P<0.05）。ELISA检测结果显示,2-DG组动物血清中OT和VP水平与对照组相比无明显变化。 结论 腹腔注射2-DG可激活大鼠下丘脑SON和PVN内OT和VP神经元表达Fos,SON和PVN可能参与2-DG诱导的急性应激反应。     

Colocalization of oxytocin and phosphorylated form of elongation factor 2 in the rat hypothalamus

Oxytocin (OT) is one of the neuropituitary hormones and is synthesized in the neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Previous studies have shown that the mRNAs encoding OT are delivered from the soma to both dendrites and axons of the neurons in the PVN and SON. However, it has not been elucidated whether a translational regulation mechanism to enable local synthesis of the hormone exists in the axons of the neurons of PVN and SON. Elongation factor 2 (EF2) is essential for polypeptide synthesis during protein translation. Moreover, phosphorylation of EF2 by EF2 kinase enhances the translation of certain mRNA species. In the present study, in order to shed light on the mechanisms involved in the translational regulation of OT synthesis, we investigated the localization of phosphorylated EF2. Phospho-EF2 was localized in the soma of the neurons in PVN and SON, and in the swellings of the median eminence where axonal tracts of the neurons in the PVN and SON exist. The phosphorylated form was also observed in the rat hypophysis. Moreover, phospho-EF2 and OT were colocalized in a part of the neurons in the PVN and SON. These results suggest that OT may be partially translated in the axons of neurons in the PVN and SON, and then secreted from the pituitary.     

Localization of dystrophin isoform Dp71 to the inner limiting membrane of the retina suggests a unique functional contribution of Dp71 in the retina

The electroretinograms (ERGs) of patients with Duchenne muscular dystrophy and an allelic variant of the mdx mouse (mdxCv3) have been shown to be abnormal. Analysis of five allelic variants of the mdx mouse with mutations in the dystrophin gene has shown that there is a correlation between the position of the mutation and the severity of the ERG abnormality. Three isoforms are expressed in the retina: Dp427, Dp260 and Dp71. Using indirect immunofluorescence and isoform-specific antibodies on retinal sections from three allelic mdx mouse strains, we have examined the localization of each of the isoforms. We show that Dp71 expression does not overlap with Dp427 and Dp260 expression at the outer plexiform layer (OPL). Instead, Dp71 is localized to the inner limiting membrane (ILM) and to retinal blood vessels. Moreover, we show that Dp260 and Dp71 differ structurally at their respective C-termini. In addition, we find that the proper localization of the beta- dystroglycan is dependent upon both Dp260 at the OPL and Dp71 expression at the ILM. Thus, Dp260 and Dp71 are non-redundant isoforms that are located at different sites within the retina yet have a common interaction with beta-dystroglycan. Our data suggest that both Dp71 and Dp260 contribute distinct but essential roles to retinal electrophysiology.      

Meal parameters and vagal gastrointestinal afferents in mice that experienced early postnatal overnutrition

Early postnatal overnutrition results in a predisposition to develop obesity due in part to hypothalamic and sympathetic dysfunction. Potential involvement of another major regulatory system component — the vagus nerve — has not been examined. Moreover, feeding disturbances have rarely been investigated prior to development of obesity when confounds due to obesity are minimized. To examine these issues, litters were culled on the day of birth to create small litters (SL; overnutrition), or normal size litters (NL; normal nutrition). Body weight, fat pad weight, meal patterns, and vagal sensory duodenal innervation were compared between SL and NL adult mice prior to development of obesity. Meal patterns were studied 18 h/day for 3 weeks using a balanced diet. Then vagal mechanoreceptors were labeled using anterograde transport of wheatgerm agglutinin-horseradish peroxidase injected into the nodose ganglion and their density and morphology were examined. Between postnatal day 1 and weaning, body weight of SL mice was greater than for NL mice. By young adulthood it was similar in both groups, whereas SL fat pad weight was greater in males, suggesting postnatal overnutrition produced a predisposition to obesity. SL mice exhibited increased food intake, decreased satiety ratio, and increased first meal rate (following mild food deprivation) compared to NL mice, suggesting postnatal overnutrition disrupted satiety. The density and structure of intestinal IGLEs appeared similar in SL and NL mice. Thus, although a vagal role cannot be excluded, our meal parameter and anatomical findings provided no evidence for significant postnatal overnutrition effects on vagal gastrointestinal afferents.     

Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development

The dystrophin gene, which is defective in Duchenne muscular dystrophy (DMD), also encodes a number of smaller products controlled by internal promoters. Dp71, which consists of the two C-terminal domains of dystrophin, is the most abundant product of the gene in non-muscle tissues and is the major product in adult brain. To study the possible function of Dp71 and its expression during development, we specifically inactivated the expression of Dp71 by replacing its first and unique exon and a part of the concomitant intron with a beta-galactosidase reporter gene. X-Gal staining of Dp71-null mouse embryos and tissues revealed a very stage- and cell type-specific activity of the Dp71 promoter during development and during differentiation of various tissues, including the nervous system, eyes, limb buds, lungs, blood vessels, vibrissae and hair follicles. High activity of the Dp71 promoter often seemed to be associated with morphogenic events and terminal differentiation. In some tissues the activity greatly increased towards birth.      

Detailed organization of nitric oxide synthase, vasopressin and oxytocin immunoreactive cell bodies in the supraoptic nucleus of the female rat

The anatomical distribution and quantitative relations of cell bodies containing neuronal nitric oxide synthase (nNOS), 8-arginine vasopressin (AVP) and oxytocin (OT) were examined throughout the supraoptic nucleus (SON) of the female rat by means of immunocytochemical and NADPH-diaphorase (NADPH-d) histochemical techniques using a triple labelling methodology. Seven chemically defined populations of neurons containing all combinations of either nNOS, AVP or OT were identified. nNOS-containing (NADPH-d positive) neurons, amounting to about 40% of all neurons counted, were most frequent in central and dorsal regions, and were evenly distributed along the rostro-caudal axis. Two small nNOS-positive neuronal populations were preferentially located dorso-centrally in the nucleus: nNOS-positive neurons containing both AVP- and OT-immunoreactivity, and neurons only containing nNOS. Slightly less than half of all nNOS-positive neurons contained AVP, and a similar share of nNOS-positive neurons contained OT. The occurrence of nNOS-positive/ AVP-containing neurons was highest in the caudal half, whereas that of nNOS-positive/OT-neurons was highest in the rostral half of SON. The data demonstrate new findings concerning the anatomical organization and co-localization patterns of nNOS-, AVP- and OT-containing neuronal populations in SON. We conclude that the absolute and relative occurrence of the identified neuronal populations vary markedly in different parts of SON. This is important to take into consideration when performing, and evaluating experimental investigations concerned with neurochemical changes in SON. Accepted: 29 December 2000     

大鼠双侧海马伞离断对脂多糖诱导的下丘脑催产素能神经元功能活动的影响

为观察去海马传入对免疫应激时下丘脑室旁核、视上核催产素能神经元功能活动的影响 ,以探索海马调控外周免疫反应的脑内环路 ,本研究预先切断大鼠双侧海马伞 ,2 8d后腹腔内注射细菌内毒素脂多糖 (lipopolysaccharide,LPS,75 0μg/kg) ,用免疫组织化学双标记方法 ,以 F os蛋白作为神经元功能活动的标记物 ,观察下丘脑催产素能神经元中即刻早期基因的表达变化。结果显示 :双侧海马伞离断后 ,下丘脑室旁核中因腹腔脂多糖刺激而发生活化的催产素能神经元活化百分率显著下降 ;而视上核中催产素能神经元的活化未受明显影响。上述结果表明 ,海马对下丘脑 -垂体 -肾上腺轴以及免疫系统的抑制作用部分是通过其支配的下丘脑室旁核的催产素能神经元来完成的。     

Effects of inhibitory feedback in a network model of avian brain stem

The avian auditory brain stem consists of a network of specialized nuclei, including nucleus laminaris (NL) and superior olivary nucleus (SON). NL cells show sensitivity to interaural time difference (ITD), a critical cue that underlies spatial hearing. SON cells provide inhibitory feedback to the rest of the network. Empirical data suggest that feedback inhibition from SON could increase the ITD sensitivity of NL across sound level. Using a bilateral network model, we assess the effects of SON feedback inhibition. Individual cells are specified as modified leaky-integrate-and-fire neurons with time constants and thresholds that vary with inhibitory input. Acoustic sound level is reflected in the discharge rates of the model auditory-nerve fibers, which innervate the network. Simulations show that with SON inhibitory feedback, ITD sensitivity is maintained in model NL cells over a threefold range in auditory-nerve discharge rate. In contrast, without SON feedback inhibition, ITD sensitivity is significantly reduced as input rates are increased. Feedback inhibition is most beneficial in maintaining ITD sensitivity at high-input rates (simulating high sound levels). With SON inhibition, ITD sensitivity is maintained for both interaurally balanced inputs (simulating an on-center sound source) and interaurally imbalanced inputs (simulating a lateralized source). Further, the empirically observed temporal build-up of SON inhibition and the presence of reciprocal inhibitory connections between the ipsi- and contralateral SON both improve ITD sensitivity. In sum, our network model shows that inhibitory feedback can substantially increase the sensitivity and dynamic range of ITD coding in the avian auditory brain stem.     

Developmental changes of nitric oxide synthase expression in the rat hypothalamoneurohypophyseal system

The present study investigated the immunohistochemical localization of neuronal nitric oxide synthase (nNOS) in the hypothalamoneurohypophyseal system (HNS) of the developing rats on postnatal day 1 (PN1), 7 (PN7), 14 (PN14), 21 (PN21), and the adult rats. The nNOS-positive neurons were not discernable in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the median eminence (ME) at PN1 and PN7. A few neurons positive for nNOS were first detected at PN14. At PN21, the nNOS-positive cells in SON and PVN rapidly increased in number. The pattern of nNOS expression at this stage approached that of the adult. Moreover, the increase of nNOS expression in the SON and PVN during the postnatal period was accompanied by the maturation of arginine vasopressin (AVP) and oxytocin (OT) neurons as indicated by the number and size of OT or AVP neurons in the SON and PVN. The patterns of AVP versus OT expression also reached that of the adult by the end of the third postnatal week. The time course of the change in nNOS expression coincided with the maturation of AVP and OT neurons in the HNS and suggested that NO synthesized by conversion of NOS is involved in the modulation of activity of neurons in the SON and PVN of the HNS.     

An N-terminal variant of Trpv1 channel is required for osmosensory transduction

Body fluid homeostasis requires the release of arginine-vasopressin (AVP, an antidiuretic hormone) from the neurohypophysis. This release is controlled by specific and highly sensitive 'osmoreceptors' in the hypothalamus. Indeed, AVP-releasing neurons in the supraoptic nucleus (SON) are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. However, the molecular nature of these channels remains unknown. Here we show that SON neurons express an N-terminal splice variant of the transient receptor potential vanilloid type-1 (Trpv1), also known as the capsaicin receptor, but not full-length Trpv1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1(-/-)) mice could not generate ruthenium red-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1(-/-) mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised AVP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor.     

Neurohypophyseal peptides in aging and Alzheimer's disease

The neurohypophyseal hormones arginine-vasopressin (AVP) and oxytocin (OT) are produced in the neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nucleus and in the much smaller cells of the suprachiasmatic (SCN) nucleus. The SON is the main source of plasma AVP. Part of the AVP and OT neurons of the PVN join the hypothalamo-neurohypophyseal tract, whereas others send projections to the median eminence or various brain areas, where AVP and OT are involved in a number of central functions as neurotransmitters/neuromodulators. AVP and OT from the PVN can also regulate via the autonomous innervation endocrine glands and fat tissue. OT is produced for a major part in the PVN but some OT neurons are present in the SON. Moreover, both AVP and OT containing neurons are observed in the "accessory nuclei", i.e. islands situated between the SON and PVN. The SCN is the biological clock, and the number of AVP expressing neurons in the SCN shows both diurnal and seasonal rhythms. In addition to these hypothalamic areas, AVP and OT may be found to a lesser extent in some other brain areas, such as the bed nucleus of the stria terminalis, diagonal band of Broca, nucleus basalis of Meynert, lateral septal nucleus, globus pallidus and the anterior amygdaloid nucleus, as well as in the peripheral tissues. The AVP and OT containing neurons should not be considered as one system. Prominent functional differences exist between the different nuclei. The heterogeneity also becomes clear from the marked differences in the neurohypophyseal peptides containing neurons of the SON, PVN and SCN during aging, and in the most prevalent age-related neurodegenerative diseases, i.e. Alzheimer's disease (AD). For those reasons, we will discuss the SON, PVN and SCN separately.     

Mechanisms underlying oxytocin-induced excitation of supraoptic neurons: prostaglandin mediation of actin polymerization

In nonneuronal tissues, activation of oxytocin receptors (OTRs), like other Galpha(q/11) type G-protein-coupled receptors (Galpha(q/11)/GPCRs), increase prostaglandin (PG) expression. This is not known for the OTRs expressed by central OT neurons. We examined mechanisms underlying OT's effects on supraoptic nucleus (SON) OT and vasopressin (VP) neurons in hypothalamic slices from lactating rats. OT application (10 pM, 10 min) significantly increased firing rates of OT and VP neurons, both of which expressed OTRs. Indomethacin, an inhibitor of PG synthetases, blocked these increases. OTR (but not a V1 receptor) antagonist blocked OT effects without blocking the excitatory effect of PGE2. Tetanus toxin blocked OT effects on fast synaptic inputs and firing activity of SON neurons but not OT-evoked depolarization, suggesting involvement of both pre- and postsynaptic neurons. Indomethacin also blocked the excitatory effects of phenylephrine, another Galpha(q/11)/GPCR activating agent but not those of PGE2, a non-Galpha(q/11)/GPCR activating agent in the SON. OT or phenylephrine, but not glutamate or KCl, enhanced cyclooxygenase 2 expression at cytosolic loci in SON neurons and nearby astrocytes, as revealed by immunocytochemistry. This OT effect was not blocked by TTX. Western blot analyses showed that OT significantly increased cyclooxygenase 2 but not actin expression. OT promoted the formation of filamentous actin (F-actin) networks at membrane subcortical areas of both OT and VP neurons. Indomethacin blocked enhancement of F-actin networks by OT but not by PGE2. These results indicate that PGs serve as a common mediator of Galpha(q/11)/GPCR-activating agents in neuronal function.