Sequential activation of p75 and TrkB is involved in dendritic development of subventricular zone-derived neuronal progenitors in vitro.

Dendritic arbor development of subventricular zone-derived interneurons is a critical step in their integration into functional circuits of the postnatal olfactory bulb. However, the mechanism and molecular control of this process remain unknown. In this study, we have developed a culture model where dendritic development of purified subventricular zone cells proceeds under serum-free conditions in the absence of added growth factors and non-neural cells. We demonstrate that the large majority of these cells in culture express GABA and elaborate dendritic arbors with spine-like protrusions but they do not possess axons. These neurons expressed receptors for neurotrophins including p75, TrkB and TrkC but not TrkA. Application of exogenous neurotrophins, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF), to cultures stimulated dendritic growth and led to more complex dendritic arbors during the initial 3 days in culture. Our results suggest that these effects are independent of Trk receptors and mediated by the p75/ceramide signaling pathway. We also show that brain-derived neurotrophic factor is the only neurotrophin that is able to influence late-phase dendritic development via TrkB receptor activation. These results suggest that dendritic arbor development of subventricular zone-derived cells may be regulated by neurotrophins through the activation of p75 and the TrkB receptor signaling pathways in a sequentially defined temporal pattern.

A role of adhesion molecules in neuroglial plasticity.

Glial cells are exquisitely sensitive to changes in neuronal activity, and their capacity for structural plasticity including migration is critical for remodeling an repair of nervous tissue. Our in vitro studies suggest that isoforms of the neural cell adhesion molecule (NCAM) carrying an unconventional carbohydrate polymer, polysialic acid (PSA), are involved in these events. We have demonstrated that neurohypophyseal explants from newborn rats generate cellular outgrowth of immature astrocytes displaying the characteristics of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells previously identified in the optic nerve. Treatment of O-2A cells with the enzyme Endo N, which specifically removes PSA from the cells surface, produced a complete blockade of the dispersion of the O-2A cell population from the explant. Identical effects of Endo N were observed in migration assays using cortical O-2A cells. Neurohypophyseal O-2A cells express functional NMDA class of glutamate receptors and the pharmacological blockade of these receptors inhibit PSA-NCAM biosynthesis and dramatically diminish O-2A cell migration from neurohypophyseal explants. This suggests a potential mechanism through which neuronal activity via glutamate release may regulate PSA-NCAM expression on immature glial cells, which in turn is critical for their migration.

The relationship of endophytic fungi to the gametophyte of the fern Schizaea pusilla.

Schizaea pusilla is a rare and threatened fern restricted in North America to acidic bogs of Nova Scotia, Newfoundland, and New Jersey. The gametophyte lives in close association with two endophytic fungi. To characterize the nature of this fern's relationship with these fungi, we introduced axenic gametophytes to bog soil for colonization. Following colonization, the endophytic fungi were isolated and reintroduced to axenic gametophytes. The gametophytes introduced to bog soil were colonized by an aseptate fungus that formed vesicles and arbuscules within the gametophyte. However, culture of colonized gametophytes produced two fungal isolates: an aseptate fungus (fungus B) and a septate fungus (fungus A). Upon reintroduction of fungal isolates to axenically grown gametophytes, the aseptate fungus demonstrated a positive growth response to the presence of the gametophytes and colonized the gametophytes without harm to the host. The septate fungus did not exhibit any specific recognition but contacted the gametophytes randomly, leaving a large percentage of the host nonviable. We propose that the relationship of the septate fungus to the gametophyte of S. pusilla is nonmycorrhizal while the relationship of the aseptate fungus to the gametophyte is mycorrhizal. Furthermore, based on lack of nutrient availability in local soils, formation of specialized structures in the gametophyte for harboring fungi, and dependence of the fern on fungal presence for completion of its life cycle, we propose that S. pusilla maintains an obligatory relationship with the aseptate mycorrhizal fungus.

Extracellular proteolysis in the adult murine brain.

Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

A tissue culture model of the hypophysiotrophic CRF producing neuronal system.

To investigate functional and chemical properties of anatomically characterized corticotropin-releasing factor-41 (CRF-41) producing neurons in vitro, hypothalamic slices of 6-day-old rats were maintained in culture for up to 6 weeks using a modified roller culture technique. This technique yields thick (100 microns) slices that contained an average of 300-400 CRF-41-immunostained neurons. The majority of CRF-41-positive cells were of small size (12-15 microns in diameter), and contained CRF-41-labeled dense core vesicles of 100 nm diameter as detected by electron microscopic postembedding immunocytochemistry. These cells represented the only CRF-41-positive cell population in the culture. Light microscope double immunolabelling of colchicine-treated cultures kept in a serum-containing media (SCM) indicated that about 60% of these CRF-41-positive neurons contains detectable levels of vasopressin-associated neurophysin (VP-NP). Culturing slices in serum-free, chemically defined media (SFM) resulted in an increased VP-NP immunostaining: parvicellular neurons labeled for both CRF-41 and VP-NP could be detected without colchicine treatment, and practically all CRF-41-positive neurons expressed VP-NP immunoreactivity. At the electron microscopic level there was a significant increase in VP-NP labeling density in the dense core vesicle compartment of CRF-41-positive varicosities. Adding dexamethasone (10 nM) to the SFM restored the staining pattern originally observed in SCM. Hence, the increased VP-NP and CRF-41 immunostaining after culturing CRF-41 neurons in SFM is most likely due to the absence of inhibitory glucocorticoids. The capacity of cultured paraventricular cells to release CRF-41 was assessed using an immunoassay. Unstimulated (basal) secretion of CRF-41 was not altered by five successive samplings at 2-hour intervals and stimulation of the same culture with 56 mmol K+ significantly increased (2-3 times) the CRF-41 content in the medium. The presence of dexamethasone (10 nM) in SFM induced a 6-fold reduction of K(+)-stimulated CRF-41 release and a 5 times reduction in tissue content in relation to cultures maintained in SFM without dexamethasone. In summary, we have demonstrated that cultured CRF-41 cells display morphological and biochemical features, as well as responsiveness to glucocorticoids, that is reminiscent to the situation in vivo. Thus, the model is well suited for studies of hypophysiotrophic CRF-41 cell functions.

Structural polarity in the Chara rhizoid: a reevaluation.

The Chara rhizoid is a useful model system to study gravitropism since all phases of gravitropism occur in a single cell. Despite years of study, a complete description of the distinctive ultrastructure of Chara rhizoids is not available. Therefore, in this paper, we reevaluate the ultrastructural features of vertically grown rhizoids, which have a structural polarity consisting of seven distinct zones. We also characterize the apical vesicles and the cell wall in these rhizoids by using antibodies against pectic polysaccharides. These studies demonstrate that the cell wall consists of two pectinaceous domains and that a distinct population of apical vesicles contain methyl esterified pectin.

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold.

The distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.

Immunocytochemical study on the intracellular localization of the type 2 glucocorticoid receptor in the rat brain.

The localization of the glucocorticoid receptor (GR) (type 2) in the rat brain was studied with immunocytochemistry using a monoclonal antibody against the rat liver GR. Strong GR immunoreactivity (GR-ir) was observed in neurons of limbic and brainstem structures known to be associated with the stress-activated circuitry, which suggest that these sites are responsive to glucocorticoid feedback. The intracellular localization of GR-ir was examined in CA1 and CA2 pyramidal neurons of the hippocampus. In intact rats GR-ir is predominantly present in the cell nucleus. Adrenalectomy (ADX) caused a slow depletion of the GR-ir signal from the cell nucleus until near detection limits at two weeks postsurgery. At that time, 1 h after administration to longterm ADX rats the synthetic glucocorticoid (type 2) agonist RU 28362 as well as a moderate and high dose of corticosterone (CORT) markedly enhanced the cell nuclear GR-ir. The type 2 antagonist RU 38486 also caused an increase of GR immunostaining in cell nuclei upon acute administration to ADX rats. The mineralocorticoid aldosterone (ALDO) and a low dose of CORT, which bind almost exclusively to type 1 corticosteroid receptors, were ineffective. In conclusion, our data suggest that in the hippocampal CA1-2 neurons type 1 and type 2 corticosteroid receptors may coexist. The steroid-induced changes in cell nuclear immunoreactive GR staining intensity suggest possible cytoplasmic-cell nuclear translocation of GR and/or exposure of immunogenic GR domains.

Distribution of tyrosine-hydroxylase (TH)-immunoreactive neurons in the diencephalon of the pigeon (Columba livia domestica).

The distribution of tyrosine-hydroxylase (TH)-immunoreactive cell bodies and fibers in the diencephalon has been investigated with immunohistological techniques in the pigeon. The results suggest that TH is present in a number of morphologically distinct neuronal systems. Preoptic and hypothalamic TH neurons were subdivided into a medial periventricular and a lateral group. The medial group starts with a rostral collection of small cells in the preoptic region. A significantly larger collection of TH neurons occupies the paraventricular nucleus (PVN) (stratum cellulare internum) and mainly consists of large multipolar cells. Further caudally, the main concentration of cells is in the hypothalamic posteromedial and the periventricular regions of the tuberoinfundibular (arcuate) nucleus. No TH neuron was found in the ventral and lateral parts of the tuberoinfundibular region, suggesting that the prominent tuberoinfundibular dopaminergic system described in mammals is absent in the pigeon. This further substantiated by the relative scarcity of TH immunoreactive fibers and varicosities in the neurohemal zone of the median eminence (ME). The caudalmost components of the medial group appear to be continuous with the large population of TH neurons distributed in the midline of the mesencephalon. Tyrosine-hydroxylase-immunopositive cells have not been found in the paraventricular organ. The lateral group consists of TH neurons loosely arranged in the lateral hypothalamus, including regions of the supraoptic nucleus and hypothalamic posterolateral nucleus. Tyrosine-hydroxylase containing neurons vary widely in size, shape, and dendritic arborization in each diencephalic region. However, it is possible to distinguish two main cell types. Small bipolar neurons with two simple arborizing dendrites were concentrated in the medial periventricular system. The second type of cell is large, multipolar with four to five branching dendrites. This latter cell type occurs mainly in the lateral system and in the PVN. Major fiber bundles containing TH immunoreactivity were identified in the lateral and periventricular hypothalamus. The paraventricular organ and the organum vasculosum laminae terminalis contained the densest arborization of fibers and varicosities. In the ME, dense innervation was found in the subependymal layer. Dense arborizations of TH positive fibers and varicosities were located in the septal nuclei and the paleostriatum augmentatum.

Neuronal architecture in the rat central nucleus of the amygdala: a cytological, hodological, and immunocytochemical study.

The organization of neurons in the rat central nucleus of the amygdala (CNA) has been examined by using Nissl stain and immunocytochemical and retrograde tracing techniques. Four main subdivisions were identified on the basis of quantitative analyses of Nissl-stained material: medial (CM), lateral (CL), lateral capsular (CLC), and ventral (CV). An intermediate subdivision (CI), previously described by McDonald ('82), was apparent only in animals that had HRP-WGA injected into the bed nucleus of the stria terminalis. Large populations of neurotensin-, corticotropin-releasing factor (CRF)-, and enkephalin-immunoreactive neurons were present within the lateral divisions (mainly CL), although they were also seen within CM. Somatostatin-immunoreactive neurons were distributed mainly within CL and CM. Within CL, neurotensin- and enkephalin-immunoreactive neurons were more numerous laterally whereas CRF- and somatostatin-immunoreactive neurons were more numerous medially. Substance P-immunoreactive neurons were almost exclusively confined to CM. Only a few cholecystokinin- and vasoactive-polypeptide-immunoreactive neurons were seen in the CNA, and they were observed within CL, CV, and CM. The majority of neurons projecting to the dorsal medulla, hypothalamus, and ventral tegmental area were located within CM, although a significant number of cells were also seen within CL. Efferent projections to the bed nucleus of the stria terminalis were found to arise from neurons located within all subdivisions of the CNA. Thus, the distributional patterns of peptidergic and efferent neurons were not confined to individual cytoarchitectonically- defined subdivisions of the CNA. Rather, the results suggest overlapping medial to the lateral trends. Comparisons with the results of previous studies indicate that peptidergic and afferent terminal distribution patterns are more restricted to individual cytoarchitectonically defined subregions of the CNA. These observations suggest that the detailed cytoarchitecture of the CNA more likely reflects the functional integration of afferents rather than the organization of the CNA output neurons.

Distribution of the pro-opiomelanocortin derived peptides, adrenocorticotrope hormone, alpha-melanocyte-stimulating hormone and beta-endorphin (ACTH, alpha-MSH, beta-END) in the rat hypothalamus.

Rat hypothalamic nuclei were removed and assayed for adrenocorticotropic hormone (ACTH), beta-endorphin (beta-END) and alpha-melanocyte-stimulating hormone (alpha-MSH) content by radioimmunoassay, from the same samples. We also performed immunostaining for these 3 pro-opiomelanocortin (POMC) derived peptides on paraffin embedded serial sections of the hypothalamus. Areas known to project to the external zone of the median eminence receive a dense POMC innervation while those projecting to the posterior pituitary are not innervated. In addition, hypothalamic areas previously suggested to project to medullary autonomic centers are densely innervated. This innervation pattern may provide the morphological basis for the involvement of POMC peptides in neuroendocrine and autonomic functions. The biochemical data raise the possibility that the POMC precursor is processed differently in various brain regions.

Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline.

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.

Topography of the somatostatin-immunoreactive fibers to the stalk-median eminence of the rat.

The course of the hypothalamo-infundibular somatostatin containing pathway was visualized by immunocytochemistry (the unlabeled antibody peroxidase-antiperoxidase complex method) combined with various knife cuts in the hypothalamus. Somatostatin immunoreactive fibers left the perikarya in the anterior hypothalamic-preoptic periventricular cell mass in a lateral direction. After a loop-like path through the lateral hypothalamus, fibers enter the medial basal hypothalamus mainly in the lateral retrochiasmatic area near the ventral surface and project to the ipsilateral half of the median eminence and the pituitary stalk. Somatostatin immunoreactivity almost completely disappeared from the stalk-median eminence 3-7 days following a complete anterolateral transection while transections dorsal or posterior to the lateral retrochiasmatic area failed to influence it. 7 weeks after anterolateral deafferentation an increased density of somatostatin containing terminals and immunopositive perikarya were seen in the arcuate and ventromedial nuclei but these neurones failed to reinnervate the stalk-median eminence. Thus there appears to be a neuron system containing somatostatin-like immunoreactivity within the medial basal hypothalamus, cell bodies being most abundant in the posterior divisions of the ventromedial and arcuate nuclei. However, most if not all of the somatostatin immunoreactivity in the stalk median eminence originates from outside of the medial-basal hypothalamus. Further, we have demonstrated the importance of the careful histological evaluation of each individual operation if functional studies are to be carried out after hypothalamic deafferentation.

Lack of correlation between hypothalamic serotonin and the ether-induced ACTH secretion in adrenalectomized rats.

The central serotonergic system was manipulated using a serotonin receptor antagonist (cyproheptadine), electrolytic lesioning of the raphe nuclei and neurochemical destruction of the serotonergic terminals in the hypothalamus. The effects of these interventions on ether-induced ACTH secretion were studied in adrenalectomized rats. Serotonin, norepinephrine and dopamine concentrations were measured in the medial basal hypothalamus (MBH) or in individual nuclei of the hypothalamus and of certain midbrain regions. Cyproheptadine pretreatment inhibited ether-induced ACTH hypersecretion in adrenalectomized animals. Neither the electrolytic lesions of the midbrain raphe nuclei, nor the neurotoxic destruction of the hypothalamic serotonergic terminals (by intraventricular administration of 5,6-dihydroxytryptamine) caused any alteration of stimulated ACTH secretion after ether inhalation and/or long-term corticoid deficiency. These results suggest a lack of correlation between the activity of the central serotonergic system and the ACTH releasing effect of ether-stress in adrenalectomized rats.