Chronic Physiological Stimulation Research Articles

Advances in Understanding of Structural Reorganization in the Hypothalamic Neurosecretory System.

The hypothalamic neurosecretory system synthesizes neuropeptides in hypothalamic nuclei and releases them from axonal terminals into the circulation in the neurohypophysis (NH) and median eminence (ME). This system plays a crucial role in regulating body fluid homeostasis and social behaviors as well as reproduction, growth, metabolism, and stress responses, and activity-dependent structural reorganization has been reported. Current knowledge on dynamic structural reorganization in the NH and ME, in which the axonal terminals of neurosecretory neurons directly contact the basement membrane (BM) of a fenestrated vasculature, is discussed herein. Glial cells, pituicytes in the NH and tanycytes in the ME, engulf axonal terminals and interpose their cellular processes between axonal terminals and the BM when hormonal demands are low. Increasing demands for neurosecretion result in the retraction of the cellular processes of glial cells from axonal terminals and the BM, permitting increased neurovascular contact. The shape conversion of pituicytes and tanycytes is mediated by neurotransmitters and sex steroid hormones, respectively. The NH and ME have a rough vascular BM profile of wide perivascular spaces and specialized extension structures called “perivascular protrusions.” Perivascular protrusions, the insides of which are occupied by the cellular processes of vascular mural cells pericytes, contribute to increasing neurovascular contact and, thus, the efficient diffusion of hypothalamic neuropeptides. A chronic physiological stimulation has been shown to increase perivascular protrusions via the shape conversion of pericytes and the profile of the vascular surface. Continuous angiogenesis occurs in the NH and ME of healthy normal adult rodents depending on the signaling of vascular endothelial growth factor (VEGF). The inhibition of VEGF signaling suppresses the proliferation of endothelial cells (ECs) and promotes their apoptosis, which results in decreases in the population of ECs and axonal terminals. Pituicytes and tanycytes are continuously replaced by the proliferation and differentiation of stem/progenitor cells, which may be regulated by matching those of ECs and axonal terminals. In conclusion, structural reorganization in the NH and ME is caused by the activity-dependent shape conversion of glial cells and vascular mural cells as well as the proliferation of endothelial and glial cells by angiogenesis and gliogenesis, respectively.

Activity-dependent Notch signalling in the hypothalamic-neurohypophysial system of adult mouse brains.

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double-labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)- and oxytocin (OXT)-containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT- but not AVP-containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4-immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity-dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.

Activity-dependent remodeling of chondroitin sulfate proteoglycans extracellular matrix in the hypothalamo–neurohypophysial system

The hypothalamo–neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.

Matrix-degrading enzymes tissue plasminogen activator and matrix metalloprotease-3 in the hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system (HNS), synthesizing arginine vasopressin (AVP) and oxytocin (OXT), is well known to show structural plasticity during chronic physiological stimulation such as salt loading and lactation. In the present study, we undertook in the HNS to study localization and activity-dependent changes in the expression of matrix-degrading enzymes such as tissue plasminogen activator (tPA) and matrix metalloprotease-3 (MMP-3). Double labeling confocal microscopy demonstrated that the immunoreactivity of tPA was localized at AVP-positive dendrites in the supraoptic nucleus (SON) and AVP-positive terminals in the neurohypophysis (NH). The immunoreactivity of tPA was also seen at astrocytic processes in the HNS. Likewise, the immunoreactivity of MMP-3 was observed at AVP-positive dendrites and terminals. High magnification observation further revealed punctate distribution of tPA and MMP-3 immunoreactivity at dendrites and terminals, suggesting that they are localized at neurosecretory granules. Salt loading, known as the chronic stimulation to cause the structural plasticity, increased protein and mRNA levels of tPA in the SON but reduced protein levels of it in the NH. The chronic stimulation also increased protein levels of urokinase plasminogen activator in the SON, but the stimulation did not change protein levels of MMP-3 in the SON and NH. Depolarizing agent KCl released tPA from isolated neurosecretosomes, and this depolarization-dependent release was abolished by verapamil, a Ca 2+ channel blocker. These results demonstrate that tPA and MMP-3 are localized mainly at dendrites and terminals of AVP-expressing magnocellular neurons and tPA is released in an activity-dependent manner, suggesting that matrix-degrading proteases are candidate molecules to be concerned with the structural plasticity in the HNS.

Activity-dependent regulation of a chondroitin sulfate proteoglycan 6B4 phosphacan/RPTPβ in the hypothalamic supraoptic nucleus

The hypothalamic magnocellular neurons, synthesizing arginine vasopressin (AVP) and oxytocin, are well known to show structural plasticity during chronic physiological stimulation. We have previously reported that 6B4 phosphacan/receptor-type protein-tyrosine phosphataseβ (RPTPβ), a chondroitin sulfate proteoglycan is highly expressed in the supraoptic nucleus (SON) of adult hypothalamus. Here, we undertook to study the activity-dependent regulation of 6B4 phosphacan/RPTPβ in this system. Double labeling confocal microscopy demonstrated in the SON that 6B4 phosphacan/RPTPβ-immunoreactive perineuronal nets were seen around AVP-containing somata and dendrites and its distribution pattern was well coincided with that of TAG-1. Quantitative immunohistochemical and Western analyses showed that 1-week salt loading, known as the chronic physiological stimulation for inducing the structural changes such as synaptic remodeling and direct neuronal membrane apposition, decreased 6B4 phosphacan/RPTPβ levels in the SON, but did not alter TAG-1 levels. The 6B4 phosphacan/RPTPβ levels were returned to control basal values within 3 weeks after the cessation of the chronic stimulation. Activity-dependent decreases in 6B4 phosphacan/RPTPβ levels of the SON were confirmed when Western and immunohistochemical samples were digested with chondroitinase ABC, indicating that the decrease in 6B4 phosphacan/RPTPβ levels was due to disappearance of 6B4 phosphacan/RPTPβ core protein rather than increase in chondroitin sulfate glycosaminoglycans. With electron microscopy, the electron-dense immunoproducts for 6B4 phosphacan/RPTPβ were found on the membrane surface of axons and glial processes, but not at synaptic junctions in control SON, and its immunoreactivity was eliminated with the chronic salt loading. The present results indicate that the levels of 6B4 phosphacan/RPTPβ are regulated with activity-dependent manner and may be concerned with the structural plasticity seen in the SON.

Activity-related, dynamic neuron-glial interactions in the hypothalamo-neurohypophysial system.

Magnocellular neurons located in the supraoptic nucleus send their principal axons to terminate in the neurohypophysis, where they release vasopressin and oxytocin into the blood circulation. This magnocellular hypothalamo-neurohypophysial system is known to undergo dramatic activity-dependent structural plasticity during chronic physiological stimulation, such as dehydration and lactation. This structural plasticity is accompanied not only by synaptic remodeling, increased direct neuronal membrane apposition, and dendritic bundling in the supraoptic nucleus, but also organization of neurovascular contacts in the neurohypophysis. The adjacent glial cells actively participate in these plastic changes in addition to magnocellular neurons themselves. Many molecules that are possibly concerned with dynamic structural remodeling are highly expressed in the hypothalamo-neurohypophysial system, although they are generally at low expression levels in other regions of adult brains. Interestingly, some of them are highly expressed only in embryonic brains. On the basis of function, these molecules are classified mainly into two categories. Cytoskeletal proteins, such as tubulin, microtubule-associated proteins, and intermediate filament proteins, are responsible for changing both glial and neuronal morphology and location. Cell adhesion molecules, belonging to immunoglobulin superfamily proteins and extracellular matrix glycoproteins, also participate in neuronal-glial, neuronal-neuronal, and glial-glial recognition and guidance. Thus, the hypothalamo-neurohypophysial system is an interesting model for elucidating physiological significance and molecular mechanisms of activity-dependent structural plasticity in adult brains.

Expression of high levels of tubulin and microtubule-associated protein 2d in the neurohypophysial astrocytes of adult rat

The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, is well known to show reversible morphological reorganization for both neurons and glial cells during chronic physiological stimulation. To determine the molecular background for these morphological changes, we investigated the expression of tubulin and microtubule-associated protein (MAP) 2d in the neurohypophysial astrocytes, pituicytes of adult rats by using reverse transcription-polymerase chain reaction, western blot, and immunohistochemistry. The mRNA of MAP2d was expressed at higher levels than that of MAP2c in the neurohypophysis, cerebral cortex, and cerebellum. In contrast, predominant expression of mRNA of MAP2c was detected in the olfactory bulb. Western blot analysis showed the presence of MAP2d in the neurohypophysis, however the amount was below the detection level in the cerebral cortex and cerebellum. A double labeling study using a confocal laser scanning microscope showed intense tubulin immunoreactivity in the glial fibrillary acidic protein (GFAP)-positive pituicytes of the intact neurohypophysis. Almost no tubulin immunoreactivity was observed in the astrocytes of the intact cerebral cortex, cerebellum, and supraoptic nucleus, in contrast to strong tubulin immunoreactivity in neuronal dendrites and somata. Interestingly, intense tubulin immunoreactivity was also observed in the GFAP-positive reactive astrocytes in the immediate vicinity of the artificial lesion of the cerebral cortex. Electron microscopic observation further demonstrated the presence of a lot of microtubules in the pituicytes of intact rats. The present results demonstrate that pituicytes in the adult rat neurohypophysis expresses high levels of tubulin and MAP2d compared with normal brain astrocytes, and suggest that the ability of astrocytic morphological alteration may be at least partly ascribed to this high expression of microtubule proteins.

Plasticity of neurohypophysial terminals with increased hormonal release during dehydration: ultrastructural and biochemical analyses.

Arginine vasopressin- (AVP) and oxytocin- (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy-1 together with those of AVP- and OXT-neurophysin, but the levels of synaptophysin, SNAP-25, and GAP-43 were unchanged. Both F3 and Thy-1 were recovered in the buffer-insoluble pellet, and phosphatidyl inositol-specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy-1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca(2+) binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal-astrocyte and terminal-vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down-regulation of F3 and Thy-1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration.

Expression of the IgLON cell adhesion molecules Kilon and OBCAM in hypothalamic magnocellular neurons.

The vasopressin (AVP) and oxytocin (OXT) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) display reversible structural plasticity of neurons and glial cells under different conditions of neuropeptide secretion. In the present study, we investigated the expression of two immunoglobulin superfamily (IgSF) proteins, Kilon and OBCAM, in the magnocellular neurons by using monoclonal antibodies. Anti-Kilon antibody reacted specifically with the bacterially expressed recombinant Kilon but not with the recombinant OBCAM, and similarly anti-OBCAM antibody specifically recognized the recombinant OBCAM. Western blotting analysis revealed the specific expression of Kilon and OBCAM in the SON homogenates. Although Kilon and OBCAM of the SON homogenates were present as the insoluble form, most Kilon was present in the Triton-insoluble fraction, and OBCAM was localized mainly in the Triton-soluble fraction. Immunocytochemistry revealed Kilon and OBCAM immunoreactivity in the magnocellular neurons of the SON and PVN of the rat hypothalamus compared with outside of the SON and PVN in the hypothalamus. The double-labeling study with confocal microscopy further demonstrated that Kilon immunoreactivity was observed mainly in the dendrites of AVP-secreting neurons and also occasionally OXT-secreting neurons. However, OBCAM immunoreactivity was exclusively seen in the dendrites of AVP-secreting magnocellular neurons. Chronic physiological stimulation by 2% NaCl had no effect on the expression levels of either IgLON protein in the SON. Our study thus demonstrated specific expression of Kilon and OBCAM in the hypothalamic magnocellular neurons, particularly in dendrites, suggesting that they confer on magnocellular neurons the ability to rearrange dendritic connectivity.

Morphological plasticity and rearrangement of cytoskeletons in pituicytes cultured from adult rat neurohypophysis.

The adult rat neurohypophysis reveals drastic morphological plasticity of neuron-glial organization during chronic physiological stimulation. Pituicytes are modified astrocytes in the neurohypophysis, and shape conversion of them largely contributes to the morphological plasticity. The present study aimed to investigate the receptor-mediated mechanism for shape conversion of the pituicyte morphology, particularly in relation with changes of cytoskeletal organization. The cultured pituicytes from adult rat neurohypophysis were mostly flat amorphous shape in normal salt solution. Histochemical experiments showed that thick bundle of microfilament (stress fibers) and fine fibers of microtubule distributed evenly within the pituicyte. When pituicytes were treated with adenosine (more than 1 microM), isoproterenol (IPR); beta-agonist, more than 10 nM), and dibutyryl cyclic AMP (dBcAMP, 1 mM), the pituicyte morphology changed from flat to stellate shape. Upon treatment with dBcAMP, stress fibers within pituicyte cytoplasm disappeared, and microtubule assembled in the cellular processes and cytoplasm surrounding the nucleus. Pretreatment with colchicine (microtubule-disrupting agent, 25 microM) and orthovanadate (tyrosine phosphatase inhibitor, 1 mM) prevented dBcAMP-induced stellation of the pituicyte morphology. Treatment with sphingosine (protein kinase C inhibitor, 10 microM), W-7 (calmodulin dependent protein kinase inhibitor, 40 microM), ML-9 (myosin light chain kinase inhibitor, 20 microM), and cytochalasinB (CytB; microfilament disrupting agent, 5 microM), induced stellation of the pituicyte morphology. Treatment of endothelin-1 (more than 0.1 nM) and endotheline-3 (more than 0.1 nM) reverted dBcAMP-induced stellation of the pituicyte morphology to original flat one and also reverted arrangement of cytoskeletons of stress fiber and microtubules as seen in control one. The present results reveal that pituicyte shape conversion is mediated via beta-adrenergic, adenosine and endotheline and depend on rearrangement of stress fibers and microtubules. In addition, the mechanism of shape conversion of pituicytes cultured from adult neurohypophysis is quite similar to that of astrocytes cultured from neonatal brains and possibly is useful for understanding morphological plasticity of adult brains.

Changes in Tyrosine Hydroxylase and Substance P Immunoreactivity in the Cat Carotid Body Following Chronic Hypoxia and Denervation

Long-term hypoxia elicits functional changes in the cat carotid body which are manifest as altered chemosensitivity in response to hypoxia. Previous studies have suggested that these functional adjustments may be mediated by changes in neurotransmitter levels in chemosensory type I cells. Neurotransmitter metabolism in the carotid body has also been shown to be regulated by the neural innervation to the organ. The present study using the cat carotid body demonstrates profound changes in the levels of immunoreactivity of the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and the neuropeptide, substance P, in response to a two-week exposure to hypoxia (10% O 2 in 90% N 2). Furthermore, these changes were modulated both by sensory and sympathetic denervation of the organ. For TH, the intensity of immunostaining in type I cells was markedly increased by long-term hypoxia in both normal and chronic carotid sinus nerve-denervated carotid bodies, but this effect was blocked following chronic sympathectomy. Substance P immunoreactivity in type I cells was dramatically attenuated by hypoxia in both intact and chronic carotid sinus nerve-denervated preparations, but this effect was reduced following chronic sympathectomy. Tyrosine hydroxylase- and substance P-positive axon terminals were observed to innervate type I cells. These axons were also present in chronically sympathectomized preparations, but they disappeared following chronic carotid sinus nerve-denervation suggesting that they very likely arise from sensory neurons in the petrosat ganglion. Our data indicate that chronic chemoreceptor stimulation by hypoxia elicits multiple neurochemical adjustments in the cat carotid body. These changes suggest that catecholaminergic enzymes and neuropeptides play a significant role in the adaptive mechanisms of chemoreceptor function which occur in response to chronic physiological stimulation. Furthermore, the data suggest that neurotrophic mechanisms may influence neurotransmitter metabolism in chemosensory type I cells.

Differential withdrawal of retinal axons induced by a secreted factor

The hypothalamo–neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.