Decline and recovery of olfactory receptor expression following unilateral bulbectomy

Zinc absorption, mineral balance, and blood lipid concentrations were measured in 21 women aged 33 +/- 7 y (range: 20-42 y) consuming controlled lactoovovegetarian and nonvegetarian diets for 8 wk each in a crossover design. The lactoovovegetarian and nonvegetarian diets, respectively, provided (by analysis) 973 and 995 mg Ca, 1.8 and 1.3 mg Cu, 367 and 260 mg Mg, 5.9 and 2.5 mg Mn, 1457 and 1667 mg P, 9.1 and 11.1 mg Zn, and (by calculation) 40 and 16 g dietary fiber, 2.5 and 0.8 mmol phytic acid, molar ratios of phytate to Zn of 14 and 5, and millimolar ratios of (phytate x Ca) to Zn of 344 and 111. Dietary zinc absorption was measured by extrinsic isotopic labeling and whole-body counting. Plasma cholesterol, cholesterol fractions, and lipoproteins were reduced 7-12% with the lactoovovegetarian diet, consistent with predictions based on dietary cholesterol and fat. Blood pressure was unaffected. Calcium, copper, magnesium, and phosphorus balances were not different between diets; manganese balance tended to be greater with the lactoovovegetarian diet (P < 0.07). The lactoovovegetarian diet was associated with a 21% reduction in absorptive efficiency that, together with a 14% reduction in dietary zinc, reduced the amount of zinc absorbed by 35% (2.4 compared with 3.7 mg/d) and reduced plasma zinc by 5% within the normal range. Zinc balance was maintained with both diets. Although there is a greater risk of zinc deficiency in persons consuming lactoovovegetarian compared with omnivorous diets, with inclusion of whole grains and legumes zinc requirements can be met and zinc balance maintained.     

Expression of Fos in the rat forebrain following experimental tooth movement

Orthodontic tooth movement is known to cause pain and discomfort to patients. Mechanically induced inflammatory responses in the periodontium are assumed to be related to the mechanism of pain sensation. An immediate-early gene, c-fos, that is expressed within some neurons following synaptic activation, is widely used as a marker for neuronal activity following noxious or innocuous stimulation. We have recently demonstrated that experimental tooth movement produced Fos induction in the ipsilateral trigeminal subnucleus caudalis and in the bilateral lateral parabrachial nucleus, which is known to be involved in the transmission of nociceptive information. As a further step, we investigated the distribution of Fos-like immunoreactive neurons in the upper brain regions. Twenty-four hours after the commencement of the experimental tooth movement, the Fos-like immunoreactive neurons appeared in the central nucleus of the amygdala (Ce), paraventricular nucleus of the hypothalamus (PVH), and paraventricular nucleus of the thalamus (PV) of the experimental rats. The numbers of the labeled neurons were significantly increased by 639% (P < 0.001) and 644% (P < 0.001) in the ipsilateral and contralateral sides of the Ce, respectively, by 292% (P < 0.001) and 307% (P < 0.001) in the ipsilateral and contralateral sides of the PVH, and by 264% (P < 0.0001) in the PV with respect to sham control rats. These results suggest that nociceptive information caused by experimental tooth movement might be transmitted and modulated in several regions of the forebrain.     

Association of the p75 neurotrophin receptor with TRAF6

In addition to the Trk tyrosine kinase receptors, neurotrophins also bind to a second receptor, p75, a member of the tumor necrosis factor receptor superfamily. Several signaling pathways have been implicated for p75 in the absence of Trk receptors, including induction of NF-kappaB and c-Jun kinase activities and increased production of ceramide. However, to date, the mechanisms by which the p75 receptor initiates intracellular signal transduction have not been defined. Here we report a specific interaction between p75 and TRAF6 (tumor necrosis factor receptor-associated factor-6) after transient transfection in HEK293T cells. The interaction was ligand-dependent and maximal at 100 ng/ml of nerve growth factor (NGF). Other neurotrophins also promoted the association of TRAF6 with p75 but to a lesser extent. The binding of TRAF6 was localized to the juxtamembrane region of p75 by co-immunoprecipitation and Western blotting. To assess the functional significance of this interaction, we have tested responses in cultured Schwann cells that express p75 and TRAF6. An NGF-mediated increase in the nuclear localization of the p65 subunit of NF-kappaB could be blocked by the introduction of a dominant negative form of TRAF6 in Schwann cells. These results indicate that TRAF6 can potentially function as a signal transducer for NGF actions through the p75 receptor.     

GAP-43 and p75NGFR immunoreactivity in presynaptic cells following neuromuscular blockade by botulinum toxin in rat

Peripheral nerve lesion results in changes in protein expression by neurons and denervated Schwann cells. In the present study we have addressed the question whether similar changes take place following functional denervation. Using immunohistochemistry and immunoelectron microscopy we examined changes in growth-associated protein (GAP-43) and low-affinity nerve growth factor receptor (p75NGFR) in rat gastrocnemius muscle following botulinum toxin-induced paralysis. GAP-43 and p75NGFR were selected because they are not expressed by mature intact motor neurons or Schwann cells, but are expressed following nerve lesion in both motor neurons and denervated Schwann cells. In control muscle, GAP-43 and p75NGFR immunoreactivity was seen only in nerve fibres near blood vessels. Two weeks after toxin injection, GAP-43 immunoreactivity could be seen at the motor endplates and in axons. Intensity of staining increased with longer survival and reached a peak between 4 and 8 weeks post-injection. Ultrastructurally, GAP-43 immunoreactivity was confined to nerve terminals and axons, whereas Schwann cells remained negative. Immunostaining for p75NGFR also increased following toxin injection and was detected in some terminal Schwann cells and in perineurial cells of small nerve fascicles near the paralyzed target cells, but not in axons. These results show that changes in expression of GAP-43 in motor neurons following functional denervation closely resemble the changes following anatomical interruption of nerve-muscle contact. GAP-43 was not expressed in Schwann cells, indicating that its upregulation in these cells is induced by loss of axonal contact or nerve degeneration products. There is no support for a role of p75NGFR in incorporation of neurotrophins in axons. The restriction of p75NGFR expression to terminal Schwann cells and perineurial cells in close proximity to the paralyzed target suggests a role for a target-derived signal or, alternatively, macrophages in eliciting this expression.     

Focal cerebral ischemia in rats induces expression of P75 neurotrophin receptor in resistant striatal cholinergic neurons

Expression of p75 neurotrophin receptor and survival of medium-sized spiny projection neurons and cholinergic interneurons in the rat striatum were studied using immunocytochemistry at different times after transient, unilateral middle cerebral artery occlusion. Thirty minutes of middle cerebral artery occlusion caused a major loss of projection neurons, identified by their immunoreactivity to dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein with a molecular weight of 32,000, in the lateral part of the striatum, as observed at 48 h following the insult with no further change at one week. In contrast, no reduction of the number of choline acetyltransferase-positive, cholinergic interneurons, which also expressed TrkA, was detected at either time-point. At 48 h following middle cerebral artery occlusion, expression of p75 neurotrophin receptor was observed in striatal cells which, by the use of double-label immunostaining, were identified as the cholinergic interneurons. No p75 neurotrophin receptor immunoreactivity remained in cholinergic cells after one week of reperfusion. Based on current hypotheses regarding the function of the p75 neurotrophin receptor, the transient expression of this receptor in striatal cholinergic interneurons might contribute to their high resistance to ischemic neuronal death. However, the expression of p75 neurotrophin receptor could also be a first step in a pathway leading to apoptosis, which is inhibited after the present insult due to concomitant activation of TrkA.     

Expression of low affinity NGF (p75) receptors in rat superior colliculus: studies in vivo, in vitro, and in fetal tectal grafts

The distribution and level of expression of the low affinity nerve growth factor receptor (LNGFR) in the rat visual system has been investigated under a number of experimental conditions. The aim was to determine the cellular location of the receptor and to study the factors which influence its expression. The monoclonal antibody 192-Ig and immunohistochemical techniques were used to examine LNGFR expression in (i) developing and adult rat superior colliculus (SC), (ii) fetal collicular tissue transplanted to the midbrain of newborn host rats, (iii) the SC of rats which had been unilaterally enucleated at birth, and (iv) mixed glial cell cultures from the neonatal SC. The effect of eye removal on LNGFR immunoreactivity in other normally retino-recipient areas was also assessed. Postnatal maturation of the rat SC was associated with an increase in LNGFR immunoreactivity. At birth, weak staining was seen ventral to the superficial gray layer. Staining gradually became located more dorsally until in the adult there was a dense band of immunoreactivity that extended 50-100 microns from the surface. Immunoreactive processes and cellular-like profiles were seen. Compared to adult host SC there was considerably more LNGFR immunoreactivity in transplanted tectal tissue, irrespective of whether the grafts were connected to the host. LNGFR expression in transplants was patchy and usually contiguous with the graft surface. Staining was not obviously related to the distribution of astrocytes or microglia and very few cells were LNGFR positive in tectal glial cell cultures. In SC and in tectal grafts it is probable that LNGFR immunoreactivity was primarily associated with intrinsic neurons. In support of this, LNGFR expression in the superficial SC was unaffected by neonatal eye removal; however, LNGFR staining in the pretectum and diencephalon was reduced or absent on the side contralateral to visual deafferentation. These conflicting sets of data suggest that (i) LNGFRs in central visual pathways are sometimes, but by no means always, associated with retinal innervation and (ii) LNGFR expression in visual target areas originates from diverse sources and is influenced and regulated by a variety of factors.     

The distribution of p75 neurotrophin receptor-immunoreactive cells in the forebrain of the common marmoset (Callithrix jacchus)

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In spite of long-standing speculation, definitive roles of PGs in various events of early pregnancy remain elusive. We demonstrate herein that the targeted disruption of COX-2, but not COX-1, in mice produces multiple failures in female reproductive processes that include ovulation, fertilization, implantation, and decidualization. Using multiple approaches, we conclude that these defects are the direct result of target organ-specific COX-2 deficiency but are not the result of deficiency of pituitary gonadotropins or ovarian steroid hormones, or reduced responsiveness of the target organs to their respective hormones.     

Chronic loss of glucocorticoids following adrenalectomy down-regulates the expression of glucocorticoid receptor mRNA in the rat forebrain

In the negative feedback model, loss of endogenous glucocorticoids up-regulates the expression of glucocorticoid receptor mRNA. To elucidate further the effect of chronic lack of glucocorticoids on the expression of glucocorticoid receptor mRNA and protein, in situ hybridization and immunohistochemical methods were used to examine the long-term alteration of glucocorticoid receptor mRNA and its immunoreactivity in the forebrain of adrenalectomized rats. Constant lack of glucocorticoids resulted in marked decrease in the expression of glucocorticoid receptor mRNA and disappearance of glucocorticoid receptor immunoreactivity in many forebrain structures. In particular, in the suprapyramidal blade of the hippocampal granule cell layer and cerebral cortex, many cells showed almost no glucocorticoid receptor mRNA signals. These results suggest that long-term loss of endogenous glucocorticoids down-regulates the levels of glucocorticoid receptor mRNA, leading to reduction in the synthesis of glucocorticoid receptors in the rat forebrain. Therefore, the presence of endogenous glucocorticoids is vital to the continued expression of glucocorticoid receptor mRNA.     

Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis

We have asked whether p75(NTR) may play a role in neuronal apoptosis by producing transgenic mice that express the p75(NTR) intracellular domain within peripheral and central neurons. These animals showed profound reductions in numbers of sympathetic and peripheral sensory neurons as well as cell loss in the neocortex, where there is normally little or no p75(NTR) expression. Developmental loss of facial motor neurons was not observed, but induced expression of the p75(NTR) intracellular domain within adult animals led to increased motor neuron death after axotomy. Biochemical analyses suggest that these effects were not attributable to a p75(NTR)-dependent reduction in trk activation but instead indicate that the p75(NTR) intracellular domain may act as a constitutive activator of signaling cascades that regulate apoptosis in both peripheral and central neurons.     

Expression of the low-affinity p75 nerve growth factor receptor in the developing rat pituitary gland

We investigated, by means of in situ hybridization with a digoxigenin-labelled RNA probe, the expression of the low-affinity p75 nerve growth factor receptor (NGFR) in the developing pituitary primordium of the rat. In 13-day pc embryos, intense staining of p75 NGFR mRNA was present in the cytoplasm of all cells of Rathke's pouch. In day-17 pc embryos p75 NGFR expression was present primarily in the cells of the intermediate lobe. In the newborn rat pituitary only very weak staining was observed, predominantly in the intermediate lobe. In neural structures the staining at day 13 pc was comparable to that of day 17 pc. Since p75 expression is seen very early during pituitary development and declines during the time the expression of pituitary hormonal phenotypes are steadily increasing, we suggest that the p75 NGFR expression in Rathke's pouch may play a temporally defined role in the commitment rather than in the differentiation of the various pituitary cell types.     

NGF induces apoptosis in a human neuroblastoma cell line expressing the neurotrophin receptor p75NTR

Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R-mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this "postembedding glycolipid-labeling technique" holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level.     

Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat

PURPOSE: There is considerable evidence that vascular endothelial growth factor (VEGF) mediates ocular neovascularization in retinal vascular diseases. We investigated the time-dependent changes in the expression of VEGF and its receptor KDR/ Flk in a transient retinal ischemia-reperfusion injury model. METHODS: Transient retinal ischemia was induced by increasing the intraocular pressure in albino rats eyes for 45 min. In situ hybridization was used to identify the retinal cells synthesizing VEGF mRNA and KDR mRNA at various times following reperfusion. Immunohistochemical analysis was also carried out to detect VEGF immunoreactivity. RESULTS: In the control, non-ischemic retinas, signals for VEGF mRNA and KDR mRNA were observed in the cells of the ganglion cell layer. Immunoreactivity to VEGF was also found in the nerve fiber layer, the ganglion cell layer, and the retinal pigment epithelial (RPE) cell layer. Immediately and 6 h after reperfusion, VEGF and KDR mRNA expression was markedly decreased, but recovered by 24 h to the levels observed in normal retinas. Immunoreactivity for VEGF was also decreased immediately and 6 h after reperfusion, and was detected in the endothelial cells of the retinal vessels after 24 h. Immunoreactivity to VEGF recovered by 48 h after reperfusion. CONCLUSIONS: The hybridization pattern of VEGF and KDR mRNA in the ganglion cell layer strongly suggests that the ganglion cells are the major source of this growth factor. The decrease of VEGF mRNA, KDR/Flk mRNA and VEGF protein levels after ischemia and recovery after reperfusion suggest that transient hypoxia might mediate short-term down-regulation of VEGF and KDR mRNA.     

Neurotrophins inhibit major histocompatibility class II inducibility of microglia: involvement of the p75 neurotrophin receptor

Major histocompatibility complex (MHC) molecules are rare in the healthy brain tissue, but are heavily expressed on microglial cells after inflammatory or neurodegenerative processes. We studied the conditions leading to the induction of MHC class II molecules in microglia by using explant cultures of neonatal rat hippocampus, a model of interacting neuronal networks. Interferon-gamma (IFN-gamma)-dependent MHC class II inducibility in microglia cells was very low, but strongly increased in the hippocampal slices after the blockade of neuronal activity by neurotoxins [tetrodotoxin (TTX), omega-conotoxin] or glutamate antagonists. None of these agents acted directly on isolated microglia cells. We found that neurotrophins modulate microglial MHC class II expression. MHC class II inducibility was enhanced by neutralization of neurotrophins produced locally within the cultured tissues and was inhibited by the addition of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT3). NGF and, to a lower extent, NT3 acted directly on isolated microglia via the p75 neurotrophin receptor and inhibited MHC class II inducibility as shown by blockade of the p75 neurotrophin receptor with antibodies. Our data suggest that neurotrophins secreted by electrically active neurons control the antigen-presenting potential of microglia cells, and indicate that this effect is mediated partly via the p75 neurotrophin receptor.     

Effects of fluoxetine administration on mu-opoid receptor immunostaining in the rat forebrain

Fluoxetine is a selective serotonin reuptake inhibitor. Analysis of mu-opioid receptor immunostaining after chronic fluoxetine administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic fluoxetine treatment.     

Progesterone receptor isoforms are differentially regulated by sex steroids in the rat forebrain

We studied the effects of estradiol (E2) and progesterone (P4) on expression of genes coding for PR isoforms in the forebrain of ovariectomized rats by RT-PCR analysis. In the hypothalamus the expression of both PR isoforms was induced by E2 and down-regulated by P4. In the preoptic area these changes were only observed in the PR-B isoform. In contrast, in the hippocampus PR induction by E2 was only observed for PR-A. In this region P4 did not modify the expression of any PR isoform. These results indicate that PR isoforms expression is differentially regulated by sex steroid hormones in distinct forebrain regions and suggest that the tissue-specific regulation of either PR-A or PR-B may be involved in the physiological actions of P4 upon the rat brain.     

Widespread expression in adult rat forebrain of mRNA encoding high-affinity neurotensin receptor

The peptides neurotensin (NT) and neuromedin N exert effects on neurons by means of a high-affinity NT receptor (NTRH) belonging to the superfamily of G-protein-coupled receptors. In the present study, we used in situ hybridization histochemistry with sensitive riboprobe methodology to investigate the distribution of NTRH mRNA in the forebrain of adult rats. Labeled cells were abundant in the hypothalamus, epithalamus, ventral thalamus, septum, amygdala, and pallidum, including many regions where NTRH mRNA had not been detected previously. In the hypothalamus, novel sites of NTRH mRNA expression included the arcuate, periventricular, paraventricular, supraoptic, medial preoptic, anterior, ventromedial, and posterior nuclei, as well as the lateral hypothalamic area. In the thalamus, novel sites of expression included the anterodorsal nucleus, lateral habenula, and zona incerta, where labeling was much more extensive than previously reported. Novel telencephalic sites of expression included most bed nuclei of the stria terminalis, most divisions of the amygdala, the main olfactory bulb, the endopiriform nucleus, the claustrum, many parts of retrohippocampal allocortex, and limited parts of most isocortical areas. Novel sites of expression were also observed in the midbrain and pons. Taking into account expected differences in the subcellular locations of receptor mRNA and protein, the regional distribution of NTRH mRNA agrees well with that of NTRH determined previously. Our results identify many novel sites of NTRH mRNA expression in adult brain and provide a basis for investigating involvement of NT and related peptides in regulating the activity of these diverse cells, whose phenotypes remain largely undetermined.     

Absence of the p75 neurotrophin receptor alters the pattern of sympathosensory sprouting in the trigeminal ganglia of mice overexpressing nerve growth factor

Sympathetic axons invade the trigeminal ganglia of mice overexpressing nerve growth factor (NGF) (NGF/p75(+/+) mice) and surround sensory neurons having intense NGF immunolabeling; the growth of these axons appears to be directional and specific (). In this investigation, we provide new insight into the neurochemical features and receptor requirements of this sympathosensory sprouting. Using double-antigen immunohistochemistry, we demonstrate that virtually all (98%) trigeminal neurons that exhibit a sympathetic plexus are trk tyrosine kinase receptor (trkA)-positive. In addition, the majority (86%) of those neurons enveloped by sympathetic fibers is also calcitonin gene-related peptide (CGRP)-positive; a smaller number of plexuses (14%) surrounded other somata lacking this neuropeptide. Our results show that sympathosensory interactions form primarily between noradrenergic sympathetic efferents and the trkA/CGRP-expressing sensory somata. To assess the contribution of the p75 neurotrophin receptor (p75(NTR)) in sympathosensory sprouting, a hybrid strain of mice was used that overexpresses NGF but lacks p75(NTR) expression (NGF/p75(-/-) mice). The trigeminal ganglia of NGF/p75(-/-) mice, like those of NGF/p75(+/+) mice, have increased levels of NGF protein and display a concomitant ingrowth of sympathetic axons. In contrast to the precise pattern of sprouting seen in the ganglia of NGF/p75(+/+) mice, sympathetic axons course randomly throughout the ganglionic neuropil of NGF/p75(-/-) mice, forming few perineuronal plexuses. Our results indicate that p75(NTR) is not required to initiate or sustain the growth of sympathetic axons into the NGF-rich trigeminal ganglia but rather plays a role in regulating the directional patterns of axon growth.     

The acetylcholine, GABA, glutamate triangle in the rat forebrain

The present overview demonstrates that stress, fear, novelty, and learning processes are associated with arousal and increases of extracellular levels of cortical and hippocampal ACh, independently of increases of motor activity. Forebrain cholinergic systems appears to be regulated by GABAergic and glutamatergic inputs. However, several other neurotransmitter systems play a role.     

Expression of D1 receptor mRNA in projections from the forebrain to the ventral tegmental area

The presence of subunit proteins, 1H9 for the alpha-subunit and 2B6 for the beta-subunit, of H(+)-K+ ATPase and its activity in tubulovesicles and intracellular canaliculi of gastric parietal cells were immunocytochemically and enzyme cytochemically examined. Specimens were taken from healthy human volunteers by endoscopic biopsy in resting, tetragastrin-stimulated and omeprazole-inhibited conditions. H(+)-K+ ATPase was present in both intracellular canaliculi and tubulovesicles in these three conditions. Gold particles of the alpha-subunit decreased in number, and those showing the beta-subunit increased under both gastrin-stimulating and omeprazole-inhibiting conditions compared with parietal cells in the resting state. We suggest that the administration of tetragastrin and omeprazole alter the turnover rate of each subunit of H(+)-K+ ATPase, resulting in the difference of the proportions of alpha- and beta-subunits. Moreover, the activity of H(+)-K+ ATPase was detected strongly beneath the membrane of microvilli and weakly in that of tubulovesicles under these three conditions. After 7 days of daily oral omeprazole intake, H(+)-K+ ATPase in parietal cells were detected in intracellular canaliculi and tubulovesicles. However, the H(+)-K+ ATPase activity in tubulovesicles was diminished 1 h after omeprazole intake, and the activity in intracellular canaliculi was completely inhibited even 3 h after omeprazole administration. These results show that omeprazole inhibited the H(+)-K+ ATPase activity in both intracellular canaliculi and tubulovesicles.