The effects of orchidectomy and replacement therapy on the ultrastructure and gonadotropin-releasing hormone content of the median eminence of the rat

The effects of 2 weeks of orchidectomy and replacement therapy with testosterone upon the content and distribution of gonadotropin-releasing hormone (GnRH) in the median eminence were determined by means of radioimmunoassay and electron microscopy. Photographic montages were prepared from electron micrographs of the lateral median eminence at the point of deepest invagination of the tuberoinfundibular sulcus. Morphometric analysis of photographs of tissues immunohistochemically stained for GnRH was performed to determine changes in the volume density of GnRH-containing axon profiles following the experimental treatments. A decrease in GnRH content after orchidectomy was observed both by morphometric analysis of axon volume density and radioimmunoassay of total GnRH content. Testosterone treatment of orchidectomized animals prevented the postorchidectomy loss of GnRH. Morphometric analysis of conventional electron micrographs revealed an increase in the number of axons containing no dense-core vesicles following orchidectomy, but no decrease in volume density of the neuropil. The results indicate that the change in volume density of immunostained axons was related to the loss of immunostainable dense-core vesicles and not to a change in the size or number of axons. The area corresponding to the location of the highest concentration of GnRH-containing axons was observed to be largely avascular and separated from the vessels of the tuberoinfundibular sulcus by a “border zone” composed of glial foot processes. The unique morphology of the GnRH area has suggested the name “compact zone” to distinguish it from the palisade zone with which it is continuous medially. GnRH axons in this region are probably part of a tract extending farther caudally rather than a terminal field.     

Evidence for the existence of a dopamine receptor of the D-1 type in the rat median eminence

By means of receptor autoradiography using the dopamine (DA) receptor radioligands [3H]cis(z)-flupenthixol ([3H]FLU), [3H]spiperone, [3H]N-propyl-norapomorphine and amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphtalene-2-(5,8[3H]) in combination with a microdensitometrical analysis indications have been obtained for the existence of a DA receptor of the D-1 type in the median eminence of the male rat. Thus, only [3H]FLU (10-20 nM) strongly labeled both the nuc. caudatus putamen and the median eminence and the labeling was markedly prevented by (+)-butaclamol in both regions. Furthermore, a DA receptor agonist of the D-1 type preferentially displaced [3H]FLU from the median eminence. Thus, a DA receptor of the low affinity type may regulate the secretion of hypothalamic hormones from the median eminence.     

Origin of the nerve endings in the surface zone of the median eminence of the rat hypothalamus

Summary In order to study the origin of the nerve endings in the surface zone (zona palisadica) of the median eminence and the proximal part of the pituitary stalk, experiments were designed to determine the extent of the smallest region of the brain, isolation of which does not result in terminal degeneration in this zone. Various hypothalamic areas were partially or totally separated from the brain by means of a stereotaxically manipulated bayonet-shape knife and the zona palisadica was examined for secondary degeneration of axon terminals under the electron microscope. If the medial basal hypothalamus, including the suprachiasmatic area was disconnected from the rest of the brain, degenerated nerve endings do not occur in the palisade zone. Axon degeneration is observed if any part of the medio-basal area was separated from the median eminence or became necrotic. — These findings indicate that the nerve terminals in the zona palisadica arise exclusively from neurons in the medial basal hypothalamus, and that fibers of other origin do not terminate in this zone. The present observations support the assumption that the hypothalamic releasing (and inhibiting) factors are produced by, and the neural control of the anterior pituitary is exercised over a short final neuronal link, localized in the medial basal hypothalamus.     

Physiological evidence for alpha 1-adrenergic facilitatory control of the cold-induced TRH release in the rat, obtained by push-pull cannulation of the median eminence

The alpha-adrenergic antagonists phentolamine and prazosin were administered to male rats to explore their effects on cold-induced TRH release, measured by a chronic push-pull cannula stereotaxically implanted in the median eminence (ME). Phentolamine was given either i.p. (24 or 40 mg/kg), or locally (10(-5) M) in the ME, whereas prazosin was only applied locally (10(-5) M). Phentolamine significantly decreased the cold response (5 +/- 1 pg/15 min vs 21 +/- 5 pg/15 min; P less than 0.02), whatever the administration mode. Moreover, the blocking effect of prazosin directly perfused into the ME (11 +/- 3 pg/15 min vs 26 +/- 9 pg/15 min; P less than 0.05), indicates the specific involvement of alpha 1-adrenergic receptors in the cold-induced TRH response, and points to the ME as a possible site of facilitatory adrenergic control.     

Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence

The role of the neuropeptide galanin (GAL) in rat hypothalamus has been studied in different experimental models. Thus, the effect of GAL on potassium-induced dopamine release was analyzed in vitro, and the localization of GAL and GAL binding sites was studied with immunohistochemistry and receptor autoradiography, respectively. In the median eminence GAL and presumably dopamine were found to coexist in nerve endings and this area contained a high density of 125I-GAL binding sites. In vitro experiments revealed that GAL inhibited the release of [3H]dopamine in a dose-dependent manner (IC50 = 7-10 nM), possibly via a presynaptic receptor.     

Ultrastructural changes in the rat endometrium during the normal estrous cycle

Dynamic changes in the endometrial stroma during the following 5 stages of the estrous cycle in normal rats were examined by transmission electron microscopy. Diestrus: Macrophages migrated into the endometrial stroma from blood vessels. Proestrus: Eosinophils migrated into the endometrial stroma from blood vessels. They possessed specific crystalloid granules and small granules. Estrus: The endometrial stroma was swollen and stromal cells degenerated. Eosinophils contained a few or no crystalloid granules, while the number of small granules increased. Metestrus-1: Epithelial projections protruded through the basal lamina and established focal adhesions to stromal cells. Stromal cells also adhered to one another. Metestrus-2: Most eosinophils were engulfed by macrophages. In this report, we discuss the interaction of epithelial cells with endometrial stromal cells during the normal estrous cycle.     

Peripheral administration of picrotoxin and bicuculline stimulates in vivo somatostatin release from rat median eminence

The gamma-Aminobutyric acid-A (GABAA) antagonist picrotoxin and bicuculline were administered to male rats to determine their effects on somatostatin (SRIF) release, measured in unanesthetized animals stereotaxically implanted with push-pull cannula in the median eminence (ME). I.p. injection (3 mg/kg) of picrotoxin (n = 5) or bicuculline (n = 6) significantly increased (35.4 +/- 10.8 vs 13.7 +/- 4.3 pg/15 min; P less than 0.03 and 38 +/- 3.5 vs 14 +/- 1.8 pg/15 min; P less than 0.001, respectively) SRIF release from the ME compared to baseline levels measured in the same animals. In contrast, with local perfusion of picrotoxin, (10(-4) to 10(-6) M) SRIF release from the ME was not affected. These data suggest a physiological endogenous inhibitory tone of GABA on SRIF release.     

Electron microscopic immunohistochemical localization of growth hormone-release inhibiting hormone (somatostatin) in the rat median eminence

Using an immunohistochemical technique at the electron microscopic level, we have observed that growth hormone-release inhibiting hormone (somatostatin), the hormone which inhibits the release of growth hormone, is contained in the secretory granules of many nerve endings mainly located in the external zone of the median eminence.     

The distribution of orthogonal arrays in the freeze-fractured rat median eminence

Summary The distribution of orthogonal arrays of particles and their relationships to gap and tight junctions have been studied in the glia of the freeze-fractured rat median eminence (ME). These rectilinear clusters of intramembrane particles are thought to represent trans-membrane channels for ions or metabolites, and were found to be densely packed on the membranous laminations of the pial-glial limitans. Additionally, arrays were found to be present on all of the perivascular glial end-feet examined. Two classes of end-feet were distinguished by their relative densities of orthogonal arrays. End-feet displaying low densities of arrays occurred more frequently in the internal zone, while end-feet displaying high densities occurred more often in the external zone. Similar distinctions based on array density could be made in membranes from other regions of the cell as well. Cross-fractures revealing the cytoplasm underlying these membranes often exposed lipid inclusion bodies, suggesting that membranes containing few arrays belong to tanycytes (or to astrocyte-like tanycytes). The distribution of arrays appeared to be unrelated to the distribution of gap junctions in the membranes of astrocytes and tanycytes (and astrocyte-like tanycytes) of the ME, appearing near to and far from gap junctions with approximately equal frequency. Orthogonal arrays were absent from glial membranes near synaptic profiles in the ME. Arrays were also absent from the microvillous membranes of the apical surfaces of ependymal cells, from the cytoplasmic protrusions into the CSF of tanycytes, and from the vicinity of the tight and complex junctions linking the tanycyte and ependymal cell lateral membranes near their apical poles. These results suggest that there is a gradient of array density for most glia of the ME, increasing from the ventricular to the pial surface.     

Morphological characterization of relationship between gap junctions and gonadotropin releasing hormone nerve terminals in the rat median eminence

The present study aimed to reveal possible morphological relationships between gonadotropin-releasing hormone (GnRH) nerve terminals and gap junctions in the median eminence. Coronal brain sections from castrated male rats were dual immunostained with GnRH and either connexin 26, 32, or 43, and examined by confocal laser microscopy. Connexin 43-immunoreactive puncta were distributed between GnRH-immunoreactive fibers, and some of them were colocalized with GnRH-immunoreactivities. Dual immunostaining with connexin 43 and glial fibrillary acidic protein revealed that most of the puncta were located in astrocytes. At the immunoelectron microscopic level, connexin 43-immunoreactivities were mainly located on the plasma membranes of glial-like processes. Few connexin 26- or connexin 32-immunoreactivities were found in the median eminence. The present results indicate the possibility that gap junctions play a role in the GnRH release at the median eminence.     

Uptake and release of [3H]dopamine by the median eminence: evidence for presynaptic dopaminergic receptors and for dopaminergic feedback inhibition

The accumulation and release of [3H]dopamine by the median eminence in vitro was studied after treatments with different pharmacological agents, to determine whether such a procedure would be useful for measuring neuronal activity in the tuberoinfundibular dopaminergic system. The accumulation of [3H]dopamine was temperature, time, and sodium dependent, and reduced by unlabelled dopamine and by a potent dopamine uptake blocker, nomifensine. The outflow of tritium was studied after blocking the oxidative deamination of dopamine by nialamide. The outflow of tritium was elicited consistently by biphasic square wave electrical pulses and by high molarity potassium ions. The response to electrical stimulation was dependent largely on calcium and partially on sodium. The response to high molarity potassium ions was reduced in the absence of calcium ions. The response to electrical stimulation was increased by nomifensine and by a dopaminergic antagonist, haloperidol, and was reduced by dopamine and by a dopaminergic agonist, piribedil. The inhibitory action of dopamine was antagonized by haloperidol. These results indicate the existence of uptake and release mechanisms in the tuberoinfundibular dopamine neurons, and suggest that dopamine may inhibit its own release via dopaminergic receptors. This in vitro method may be useful for measuring dopamine uptake and release by tuberoinfundibular dopaminergic neurons.     

Changes in posterior pituitary oxytocin release in vitro during the estrous cycle of female rats

Summary The in vitro release of oxytocin (OXT) from posterior pituitary lobes (PPL) of adult female rats is linked to the stage of the animals' sexual cycles. After incubation in normal Locke's solution (K⁺ 5,6 mM), the basal OXT release from the PPL of rats on diestrus 1 and estrus amounted to 2.25±0.53 ng/lobe/10 min (mean±S.E.M.) and 4.71±0.61 ng/ lobe/10 min, respectively. Excess K⁺ (56 mM/l) in the presence of Ca⁺⁺ (2.2 mM/l) increased OXT liberation from the PPL of diestrous and estrous rats to 12.41±2.65 ng/lobe/10 min und 36.33±6.18ng/lobe/ 10 min, respectively. When Ca⁺⁺ was omitted from the incubation medium, the K⁺-excess induced OXT release from the PPL of estrous rats decreased to 21.54±2.65 ng/lobe/10 min whereas no change occurred in the OXT-release from the rats' PPL on diestrus 1 in the presence of Ca⁺⁺.The results indicate a cycle-dependent release and suggest that OXT plays a role in the reproductive processes.     

正中隆起室管膜细胞在高血压大鼠的形态学改变

目的:观察大鼠正中隆起室管膜细胞在高血压不同时期的形态学变化,探讨该室周器官在高血压发病过程的作用.方法:选择健康雄性Wistar大鼠60只,随机分为对照组,左旋-硝基精氨酸1、2、3、4周组.制备高血压动物模型,检测巢蛋白的表达情况.结果:用药动物组血压明显高于对照组,模型制备成功.光镜下观察到高血压初期(1、2周组)正中隆起室管膜细胞胞体增大,室管膜下细胞排列密集,室管膜下血管扩张.高血压后期(3、4周组)室管膜细胞排列稀疏,室管膜下细胞深染,软膜下血管扩张明显.巢蛋白免疫阳性细胞在高血压初期多出现在正中隆起室管膜下层,在高血压后期多出现在室管膜层.结论:正中隆起室管膜细胞与高血压发生有密切联系.     

GABAergic neurones in the rat periaqueductal grey matter express alpha4, beta1 and delta GABAA receptor subunits: plasticity of expression during the estrous cycle

Immunoreactivity for alpha4, beta1 and delta GABAA receptor subunits on neurones in the periaqueductal gray matter was investigated at different stages of the estrous cycle in Wistar rats. Immunostaining for alpha4, beta1 and delta GABAA receptor subunits was present on neurones throughout the periaqueductal gray matter. The numbers of subunit-immunoreactive neurones remained constant during the early phases of the estrous cycle (proestrus to early diestrus) but increased significantly in late diestrus. Dual immunolabeling for the GABA synthesizing enzyme glutamic acid decarboxylase revealed that almost 90% of the subunit-positive cells contained immunoreactivity for glutamic acid decarboxylase. During the early phases of the estrous cycle (proestrus to early diestrus), approximately one third of the glutamic acid decarboxylase-positive population co-localized alpha4, beta1 and delta GABAA receptor subunits. When the number of subunit positive cells increased during late diestrus, the proportion of the glutamic acid decarboxylase-containing population that expressed alpha4, beta1 and delta GABAA receptor subunits almost doubled. We propose that GABAA receptors with the alpha4beta1delta configuration are expressed by GABAergic neurones in the periaqueductal gray matter and that the numbers of cells expressing these subunits are increased in late diestrus in line with falling plasma progesterone levels. Changes in GABAA receptor expression may lead to changes in the excitability of the neural circuitry in the periaqueductal gray matter.     

Tyramine-immunoreactive neuronal structures in the rat brain: abundance in the median eminence of the mediobasal hypothalamus

Immunoreactivity to p-tyramine, one of the natural trace amines, was studied in the rat brain by an anti-p-tyramine antibody. Immunoreactivity to this amine is very weak in the nigrostriatal dopaminergic neurons and terminals, and weak in the locus coeruleus noradrenergic ones. It was intensified in these structures after monoamine oxidase inhibition. On the other hand, this amine was highly concentrated in the median eminence of the mediobasal hypothalamus, in which its physiological function on prolactin release has been demonstrated.     

Ontogenetic development of TRH-like immunoreactive nerve terminals in the median eminence of the rat

Ontogenetic development of TRH-like immunoreactive nerve terminals in the median eminence of the rat was studied immunocytochemically. By light microscopy, TRH-like immunoreactivities were first detected on the 1st day after birth in the external layer of the median eminence. By electron microscopy, TRH-like immunoreactive nerve fibers and terminals were visible on the 0.5th day after birth. The nerve terminals were first found in direct contact with the perivascular basal lamina of the portal vessel on the 2nd day. TRH-like immunoreactivities were only localized on dense granular vesicles about 105 nm in diameter in the axoplasm throughout the developmental stages. The immunoreactive nerve fibers with TRH-like immunoreactive granular vesicles gradually increased in number with development. The physiological significance of TRH as a hormone is discussed in relation to the presence of TRH-like immunoreactive nerve terminals in the median eminence of the developing rat.