Polymorphism analysis of the VNTR locus D17S5 in Central Spain

The fragment length polymorphism YNZ22 (D17S5) was analysed for a sample of 207 unrelated individuals living in Madrid (Spanish Caucasians) using PCR-methodology and high resolution separation. Hardy-Weinberg expectations (HWE) were calculated after pooling alleles into four groups. No deviations from HWE were detectable using the conventional ²-test. The power of discrimination was estimated as 0.96 and the mean paternity exclusion chance as 0.7587. A comparison of the allele frequency distribution with those of other Caucasian groups revealed no major differences.     

Amacrine cells,bipolar cells and ganglion cells of the cat retina: A Golgi study

Neuronal types contributing to the inner plexiform layer of the cat retina are described based primarily on light microscopy of Golgi-impregnated retinal whole-mounts. Cells have been characterized on morphological criteria that include dendritic branching patterns, dendritic tree sizes, cell body sizes and stratification of processes in the inner plexiform layer. Nine different types of bipolar cell, 22 different types of amacrine cell and 23 different types of ganglion cell can be distinguished using one or more of these morphological criteria. The significance of the different morphological types of cells is discussed, particularly in relationship to the functional bisublamination of the cat inner plexiform layer.     

Specific staining of the axon membrane at nodes of Ranvier with ferric ion and ferrocyanide

Ferric ion and ferrocyanide were used as stains for light and electron microscopy of peripheral nerves. In rat sciatic nerves, it was found that ferric ion preferentially binds to the cytoplasmic surface of the axon membrane at nodes of Ranvier but not at internodal regions. In myelinated axons in the electric organ of the gymnotid fish, Sternarchus albifrons, the small excitable nodes are similarly stained, but the larger inexcitable nodes are not stained by ferric ion. Staining of the inner surface of the nodal membrane appears to be related to a structural specialization of this membrane, rather than accessibility to stain. Our data thus show a chemical differentiation of the inner surface of the axon membrane between nodes and internodes in normal peripheral nerve fibers and between the inner surface of the axon membrane at active nodes, inactive nodes, and the internodes in the Sternarchus electrocyte axons.     

Comparative surface thrombogenicity of implanted vascular grafts.

In order to study the comparative thrombogenicity of neointimal surfaces that develop with three types of vascular graft materials (ultralightweight knitted dacron, knitted dacron external velour, and expanded Teflon), 36 female mongrel dogs had their infrarenal aortas alternately replaced with one of the three grafts. At the end of 3 or 6 months, the grafts were removed and the surface thrombogenicity of the neointimal surface was determined. Each graft was examined visually and microscopically for evidence of “healing.” At 6 months the external velour graft is lined more frequently than the other two grafts. The external velour graft has a markedly better incidence of cellular healing noted microscopically than the other two grafts at both time intervals. While the expanded Teflon has an initially lower surface thrombogenicity (probably due to the characteristics of Teflon surface), at 6 months, the velour graft has the lowest surface thrombogenicity. This is most likely due to cellular healing. Of all the completely lined grafts at both time intervals, the surface thrombogenicity of the velour grafts was most like that of the normal aorta. The velour graft appears to develop the least thrombogenic neointimal surface while becoming most frequently healed with a cellular neointimal surface.     

Widespread Horizontal Connections Arising from Layer 5/6 Border Inverted Cells in Rabbit Visual Cortex

Herein we describe the inverted cells [defined as those projection neurons having a major dendritic shaft abpially oriented (Bueno-López et al., Eur. J. Neurosci. , 3, 415, 1991)] originating a unique set of cortical connections characterized by extraordinarily widespread horizontal distribution. Single and multiple injections of wheatgerm agglutinin - horseradish peroxidase were made in areas 17 and 18 and the resulting retrograde labelling in the cortex was analysed. The findings were assessed in independent control experiments in which Fluoro-Gold was used as retrograde tracer. Following single injections in area 17 several separate patches of labelled cells comprising layers 2–6 were consistently found in area 18. In addition to these associational cells a number of labelled cells appeared at the layer 5/6 border but were distributed over most of the tangential extent of the visual occipital cortex. This widespread pattern was particularly striking in brains after multiple injections. In these brains a conspicuous band of labelled cells at the 5/6 border radiated from the injection sites, making up an apparently continuous horizontal sheet that intersected the striate - extrastriate boundary and merged with the patches of labelled cells in area 18 and beyond. Most of the cells in the 5/6 border band were inverted cells (82%; n = 2081). Injections in area 18 failed to produce such a widespread set of labelled cells in area 17. The functional significance of these connections furnished by the 5/6 border inverted cells remains to be determined, but their distribution would allow for convergent/divergent binding interactions both intra-areally (within area 17) and inter-areally (from area 18 to area 17).     

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in ascending spinal projections to the brainstem (nucleus of the solitary tract, lateral reticular nucleus area), pontine (parabrachial area) and mesencephalic (periaqueductal grey) structures. All these central structures are important in the processing of visceroception and visceronociception and all are targets for spinal efferents from similar areas. CaBP28k controls the excitability of cells by acting on intrinsic calcium metabolism. Results refer to the caudal spinal areas where the visceroceptive regions are concentrated. Experiments were performed through a double labelling approach that combined the retrograde transport of a protein - gold complex to identify the projection cells and immunohistochemistry to identify the CaBP28k-positive cells. The caudal spinal cord is rich in both CaBP28k-containing and projection cells. Cells colocalizing the protein and the retrograde tracer were quite numerous, with a particularly high concentration in the superficial layers of the dorsal horn (laminae I and outer II) and the lateral spinal nucleus. The other spinal areas containing immunoreactive projection cells were the reticular part of the neck of the dorsal horn, the medial laminae VII and VIII, lamina X and the sacral parasympathetic nucleus. The superficial layers and the neck of the dorsal horn are targets for nociceptive, visceroceptive and thermal inputs; the sacral parasympathetic column and lamina X are involved in visceroceptive integration. A functional role for the lateral spinal nucleus has not yet been established. Quite similar results were obtained for each of the ascending pathways under study. The high incidence of CaBP28k in spinal pathways suggests that calbindin has a major role in controlling the excitability of spinal cells subserving the processing of visceroception and/or visceronociception information to supraspinal levels. The participation of CaBP28k-immunoreactive cells in spinal ascending tract cells largely outnumbers those previously reported for various neuropeptides (Leah et al., Neuroscience, 24, 195 - 207, 1988)     

Progesterone Receptor-Containing Neurons in the Guinea-Pig Mediobasal Hypothalamus have Axonal Projections to the Medial Preoptic Area

The location and number of progesterone receptor-containing neurons in the mediobasal hypothalamus that project to the medial preoptic area were determined by combining retrograde fluorescent tract tracing with progesterone receptor immunocytochemistry. Injections of the retrograde tract tracer Fluoro-gold were made in the preoptic area of female guinea-pigs ovariectomized and primed with estradiol. After 5 days survival to allow for retrograde transport, tissue sections were incubated with monoclonal antibodies to the progesterone receptor to detect the presence of progesterone receptor-immunoreactive neurons. Cell bodies were labelled with Fluoro-gold throughout the arcuate nucleus. These neurons were not concentrated in any particular area of the nucleus but were diffusely distributed bilaterally. Retrogradely-labelled neurons were also observed in the ventrolateral and ventromedial nuclei mainly contralateral to the injection site. Progesterone receptor immunofluorescence labelled a subpopulation (7% to 10%) of these retrogradely-labelled cells particularly in the arcuate nucleus, including the median eminence. The double-labelled cells were more numerous in the anterior two-thirds of the arcuate nucleus. Although our estimates of the proportion of hypothalamic progesterone receptor-immunoreactive neurons that sent axons directly to the medial preoptic area were low, (about 0.35%), these neurons may be part of a neural circuit involved in the regulation of reproductive processes.     

Further Evidence that Most Luteinizing Hormone-Releasing Hormone Neurons are not Directly Estrogen-Responsive: Simultaneous Localization of Luteinizing Hormone-Releasing Hormone and Estrogen Receptor Immunoreactivity in the Guinea-Pig Brain

Gonadotropin secretion from the pituitary is regulated in large part by steroid action on the brain. An important question concerns whether luteinizing hormone-releasing hormone (LHRH) neurons themselves transduce steroid signals, or whether, alternatively, steroid-sensitive interneuronal populations regulate their activity. A previous study in the rat employing steroid autoradiography combined with LHRH immunocytochemistry revealed that only an exceedingly small percentage of LHRH-immunoreactive (ir) neurons was estrogen concentrating. This study has examined the relationship of estrogen receptive and LHRH-ir cells in the male and female guinea-pig brain with double label immunocytochemistry. Since estrogen receptor-ir, as demonstrated with antibody H222, is known to be confined predominantly to the cell nucleus, whereas LHRH-ir is localized mainly in the cytoplasm, single cells can be double-labeled. Diaminobenzidine tetrahydrochloride was used for localization of LHRH-ir while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of estrogen receptor-ir. The results revealed that there were many brain nuclei that contained both LHRH and estrogen receptor-positive cells, including the preventricular periventricular nucleus, the anterior subcompact nucleus of the medial preoptic nucleus (MPNa), the remainder of the medial preoptic nucleus, the retrochiasmatic area, and the anterior, dorsomedial, ventrolateral and arcuate nuclei. However, of a total of 2,604 LHRH-ir cells that were examined, we observed only 5 double-labeled cells (<0.2%). The double-labeled cells were not restricted to a single nucleus; they were present in the MPNa, the retrochiasmatic area and the arcuate nucleus. Moreover, at the light microscopic level, LHRH cells quite frequently appeared to be apposed to estrogen receptor-positive cells (8.8% in the female), especially in the MPNa. These results lend further support to the notion that estrogen-mediated regulation of the LHRH system is achieved primarily through estrogen receptive interneurons. However, due to the existence of LHRH-LHRH synaptic interactions, the possibility also exists that a small population of estrogen-sensitive LHRH neurons could contribute to generalized activation of the LHRH system.     

Brain Angiotensin II Receptor Subtypes and the Control of Luteinizing Hormone and Prolactin Secretion in Female Rats

The present experiments examined the role of the two recently identified angiotensin II (Ang II) receptor subtypes, AT, and AT(2) , in the central nervous system regulation of luteinizing hormone (LH) and prolactin secretion in estrogen- and progesterone-treated ovariectomized rats. In this animal model, intracerebroventricular (icv) injection of Ang II stimulates LH and inhibits prolactin release. The specific Ang II receptor subtype antagonists losartan (AT(1) ) or PD123177 (AT(2) ) were administered (icv) in various doses (10 ng to 1,000 ng) 10 min prior to icv injection of Ang II (100 ng). Control animals were pretreated with artificial cerebrospinal fluid prior to Ang II administration. Blood samples for LH and prolactin determinations were taken from conscious, freely-moving rats prior to and following injection of the antagonists and Ang II. Water intake was measured. Ang ll-induced water intake was attenuated 62% by 1,000 ng losartan; water intake was not affected by lower doses of losartan or by any dose of PD123177. Ang ll-induced stimulation of LH release was abolished by the 1,000 ng doses of losartan and PD123177 and attenuated by the 500 ng doses of both drugs. Lower doses did not affect Ang ll-induced LH secretion. Ang ll-induced inhibition of prolactin release was significantly reduced by the 1,000 ng doses of both losartan and PD123177. Lower doses of either drug did not affect the Ang II inhibition of prolactin release. Previous studies had shown that Ang II administration into the anterior hypothalamus-medial preoptic (AHPO) area stimulated LH release. This brain area contains AT(1) receptors. To investigate the potential brain site where the AT(2) receptor may influence LH release, Ang II was injected into the locus ceruleus, a brain nucleus which contains predominately the AT(2) receptor subtype. Ang II administration into the locus ceruleus was paired with an injection of artificial cerebrospinal fluid or Ang II into the AHPO area. Injection of Ang II into the AHPO area stimulated LH release. Injection into the locus ceruleus did not affect LH secretion, nor did it modify the rise in LH elicited by administration of Ang II into the AHPO area. Plasma levels of prolactin were not altered by any of these injections. Taken together, these data demonstrate that, in estrogen- and progesterone-treated female rats, icv Ang ll-induced water intake is mediated by the AT, receptor subtype, while Ang ll-induced changes in LH and prolactin secretion appear to be mediated by both the AT(2) and AT(2) receptor subtypes. The latter observations are one of the first suggesting a potential function for the AT(2) subtype in vivo, although the physiological relevance of this observation, as well as the site of action for the effects on LH and prolactin, remain to be established.