ISSN:0003-276X    

DOI:10.1002/ar.1092310402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn this review, we show how some of the recent developments in quantitative morphology (QM) are creating exciting new opportunities for studying the structure of the nervous system. We begin with a brief overview of QM, focusing on the problems neurobiologists are likely to encounter when collecting and interpreting data from tissue sections. Many of these problems, which range from selecting a sampling method to learning the latest methods, are being solved by creating a new generation of research tools. We describe several of these new tools and show how they can be used to assemble new quantitative methods for in situ hybridization, immunocytochemistry, and camera lucida drawings. The review includes examples of how QM is being used to study the brain and concludes with a brief discussion of diagnostic pathology and its need for new quantitative approaches.

ISSN:0003-276X    

DOI:10.1002/ar.1092310403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractCompetition between neurons for limited amounts of trophic factors is believed to be the basis for large‐scale neuronal death during the normal development of the vertebrate nervous system. In this study, an unbiased stereological counting method, an optical disector/Cavalieri combination, was used to estimate the total number of motor neurons in the lateral motor column of the developing chick and to assess the effects of four growth factors on neuronal numbers. The total number of neurons in lateral motor columns at embryonic day 6 (E6), E8, E10 and E12 were 18,747 ± 1,369 (mean ± SD), 15,037 ± 1,816, 10,245 ± 940, and 8,802 ± 797, respectively. Daily exposure from E6 to E9 to three of the growth factors (basic fibroblast growth factor, bFGF; leukemia inhibitory factor, LIF; nerve growth factor, NGF) had no effect on total neuron number at E10. However, exposure to ciliary neurotrophic factor (CNTF) from E6 to E9 significantly increased (P<0.05) the number of neurons in the lateral motor column (13,610 ± 725, compared with 10,058 ± 204 in normal saline controls). These results are in agreement with previous reports of large scale neuronal death in the developing chick lumbar lateral motor column between E6 and E12 and confirm that exposure to growth factors such as CNTF can mitigate the course of normal ontogenetic cell death. The optical disector/Cavalieri combination is an efficient method for counting neurons: on average, following sectioning and staining, less than 30 min was required to estimate the total number of motor neurons in a lateral motor column with a coefficient of error of approxima

ISSN:0003-276X    

DOI:10.1002/ar.1092310404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe origin of the driving forces for neural tube formation remains uncertain but is currently thought to involve the participation of microfilament bundles situated in the apical ends of neuroepithelial cells. In the work presented here, we show how morphometric measurements that map local variations in the apical geometry of neuroepithelial cells (especially apical constriction) can provide information on the distribution of motive forces within the neuroepithelium during neural tube formation. When used in combination with computer‐assisted, three‐dimensional reconstruction, it becomes possible to analyze the morphometric data from a dynamic, three‐dimensional perspective. As an example application of this method, we have used morphometry to evaluate the effects of ionomycin on the developing neuroepithelium. Treatment of early (stages 6‐8) chick embryos with 5 μM ionomycin was found to cause rapid bending of the neuroepithelium within 1 min of exposure and a dramatic acceleration of the normal sequence of neural tube formation. Electron microscopy and morphometry revealed that this acceleration was coincident with a marked increase in the local degree of apical constriction of neuroepithelial cells, presumably a consequence of enhanced contractile activity of apical microfilament bundles. This work shows that transient elevation of free calcium levels can accelerate the usualal sequential phases of NT formation. The rapidity of the response (hours of normal development reduced to minutes), increased prominence of apical microfilament bundles, and the enhanced degree of apical constriction strongly support a direct causal role for apical microfilament bundles and apical constriction of neuroepithelial cells in bending of the neuroep

ISSN:0003-276X    

DOI:10.1002/ar.1092310405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractTannic acid methods have been applied to capture the exocytosis of peptide‐containing granules from peptidergic neurons. The captured exocytoses have been quantitated to assess the proportion and amount of peptide released at different parts of the neuronal membrane. Examination of hypothalamic synaptic boutons shows that only about one‐half of the peptidergic vesicles is exocytosed into the synaptic cleft and also that exocytosis also occurs from undilated peptidergic axons. Study of the magnocellular neurosecretory system reveals that all parts of their extensive terminal arborization appear to be equally capable to exocytose peptide. Only about one‐half of their peptide is released from their nerve endings, which abut the capillaries. The remainder is released much deeper in the lobules of secretory tissue where its principal target(s) could be the pituicytes and/or neurosecretory axons. Dendrites of magnocellular neurons are also capable of releasing peptide by exocytosis and dendrites could release sufficient oxytocin and vasopressin to account for the peptide known to be released into the hypothalamus.We conclude that peptidergic neurons release substantial amounts of peptides from all of their processes and that this must be taken into account when considering what functions those peptides might

ISSN:0003-276X    

DOI:10.1002/ar.1092310406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractSpontaneous dwarf mice, in which both growth hormone (GH) and prolactin (PRL) are undetectable, are severely deficient in the PRL‐inhibiting catecholamine dopamine (DA), as well as its synthetic enzyme, tyrosine hydroxy‐lase (TH), in the basal hypothalamus (Phelps et al., Cell Tissue Res.,240:19–25, 1985; Phelps, Brain Res.,416:354–358, 1987). In contrast, transgenically constructed dwarf mice (Behringer et al., Genes Dev.,2:453–461, 1988) show complete ablation of pituitary GH cells, but PRL cells are retained at a level of ≈ 10% of normal. In order to determine the feedback effect of this reduced, rather than absent, PRL on hypothalamic DA neurons, brains of transgenic dwarf mice were examined for catecholamine transmitters by histofluorescence, for the synthetic enzyme TH by immunocytochemistry, and for TH mRNA expression by in situ hybridization. DA histofluorescence in transgenic dwarfs was comparable to that of normal littermate mice in nonpituitary regulating areas (perikarya ofzona incerta[A13] of hypothalamus and in midbrainsubstantia nigraareas [A9]). Arcuate nucleus (A12) DA neurons that inhibit PRL secretion, however, showed dim to absent fluorescence in perikarya and in external median eminence terminals in dwarfs. There were reduced (P<0.05) numbers of A12 TH‐immunoreactive neurons in transgenic dwarfs, to approximately 60% of those in normal mice. In contrast, TH‐positive neurons in other hypothalamic areas (A13, A14) had average populations equivalent to those in normal mice. Quantification of TH mRNA abundance by in situ hybridization using both image analysis of hybridization over the arcuate nucleus, and grain counts per individual A12 cell in this nucleus, indicated that relative mRNA levels were the same in normal and transgenic dwarfs. The observations indicate that reduction in pituitary PRL is accompanied by defective expression in hypothalamic tuberoinfundibular neurons, which is severe at the DA neurotransmitter level, significant regarding observable TH immunoreactivity, and undetectable with regard to TH mRNA expression. Collectively, the findings suggest that posttranscriptional processes are involved with the mediation of PRL feedback upon hypothalamic neurons. Technically and quantitatively, the report presents the feasibility of simultaneous evaluation of transmitter histofluorescence, synthetic enzyme immunocytochemistry, and mRNA expression in ind

ISSN:0003-276X    

DOI:10.1002/ar.1092310407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe decapeptide gonadotropin‐releasing hormone (GnRH) stimulates release of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) from the anterior pituitary. In the present study we used a 51‐base oligonucleotide probe and in situ hybridization to study the neuronal content of GnRH mRNA at several time points in the estrous cycle and 7 days after castration of male rats. GnRH mRNA containing cells were found in the medial septum (SEPT), the vertical and horizontal limbs of the diagonal band of Broca (DBB), and throughout the preoptic area (POA) from the organum vasculosum of the lamina terminalis (OVLT) to its caudal merger with the anterior hypothalamus.The number of neurons producing detectable quantities of GnRH mRNA was not different either among females killed at 0700 h proestrus, 1000 h estrus, or 1900 h of diestrus 1 or between intact male rats and male rats killed 1 week after castration. We did, however, detect a significant difference in the number of GnRH mRNA producing neurons between males and females (P<0.05), where females had 20% more labeled cells.We detected no significant difference in the relative copy number of GnRH mRNA molecules (grains per labeled cell) either over the estrous cycle or between intact and castrate males. However, females overall had 24% more grains per labeled cell than males (P<0.05). These results suggest that gonadal steroid regulation of GnRH both over the estrous cycle and after short‐term castration of males is mediated primarily by cellular processes subsequent to GnRH gene regulation. Furthermore, these results suggest that biosynthetic activity of GnRH is higher in females than i

ISSN:0003-276X    

DOI:10.1002/ar.1092310408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn order to design an approach for localizing IGF‐I receptors within the intact CNS, the effect of paraformaldehyde (PAF) fixation on receptor binding was examined. Cryostat sections of rat brains, which were perfused with O to 10% PAF, were incubated in125I‐IGF‐I and assayed for binding under equilibrium conditions. Binding was quantified from 10 brain regions, involving laminae and nuclei from median eminence, thalamus, hippocampus, choroid plexus, pyriform cortex, and cerebral cortex, by computer densitometry of film autoradiographs. The specific binding, saturation curves, Bmaxand Ka, ligand specificity, and binding reversibility of IGF‐I binding sites were not significantly affected by 1% or 2% PAF. However, 4% PAF elevated IGF‐I receptor total binding, nonspecific binding, and Ka, and decreased Bmax, presumably by increasing the number of tissue‐receptor interconnections, Only nonspecific125I‐IGF‐I binding persisted when 10% PAF was used. These results indicate that tissue perfused with 2% PAF can be used for localizing IGF‐I receptors by autoradiographi

ISSN:0003-276X    

DOI:10.1002/ar.1092310409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100–200 μm) sections of the deep pineal gland of the Mongolian gerbil stained immunohis‐tochemically using antisera to S‐antigen and tyrosine hydroxylase (TH). Both dual‐stained and single‐stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1–3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH‐immunopositive fibers were distributed throughout the deep pineal gland, often forming “baskets” of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi‐stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi‐stained images were visualized using the reflected image mode of the confocal system. A summary of the advantages of CLSM include the following: (1) the optical sectioning capabilities allow for true three‐dimensional imaging thus aiding in the determination of the three‐dimensional structure of a cell plus cell‐cell associations, (2) intact tissue as well as thick sections can be used without mechanical sectioning artifacts, (3) higher resolution can be obtained due to elimination of out‐of‐focus fluorescence, (4) CLSM requires significantly less time than other three‐dimensional reconstruction techniques, and (5) once computerized, the data can be analyzed

ISSN:0003-276X    

DOI:10.1002/ar.1092310410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA stereological method for obtaining estimates of the total number of neurons in five major subdivisions of the rat hippocampus is described. The new method, the optical fractionator, combines two recent developments in stereology: a three‐dimensional probe for counting neuronal nuclei, the optical disector, and a systematic uniform sampling scheme, the fractionator. The optical disector results in unbiased estimates of neuron number, i.e., estimates that are free of assumptions about neuron size and shape, are unaffected by lost caps and over‐projection, and approach the true number of neurons in an unlimited manner as the number of samples is increased. The fractionator involves sampling a known fraction of a structural component. In the case of neuron number, a zero dimensional quantity, it provides estimates that are unaffected by shrinkage before, during, and after processing of the tissue. Because the fractionator involves systematic sampling, it also results in highly efficient estimates. Typically only 100–200 neurons must be counted in an animal to obtain a precision that is compatible with experimental studies. The methodology is compared with those used in earlier works involving estimates of neuron number in the rat hippocampus and a number of new stereological methods that have particular relevance to the quantitative study of the structure of the nervous system are briefly described in an app

ISSN:0003-276X    

DOI:10.1002/ar.1092310411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY