Changes in oestrogen receptor alpha expression in the nucleus of the solitary tract of the rat over the oestrous cycle and following ovariectomy.

Oestrogen is capable of modulating autonomic outflow and baroreflex function via actions on groups of neurones in the brainstem. We investigated the presence of oestrogen receptor (ER) alpha in a part of the nucleus of the solitary tract (NTS) associated with central cardiovascular control, aiming to determine whether ERalpha mRNA and protein expression is correlated with levels of circulating oestrogen during the oestrous cycle. Polymerase chain reaction (PCR) detected ERalpha mRNA in the NTS at each stage of the oestrous cycle, from ovariectomised, sham-operated and male rats. Real-time PCR showed variations in ERalpha mRNA expression during the oestrous cycle, with the highest levels seen in oestrus, and lowest levels in metoestrus (P < 0.05 versus oestrus) and proestrus (P < 0.05 versus oestrus). Expression in males was lower than in dioestrus and oestrus females (P < 0.05). After ovariectomy, ERalpha mRNA levels were decreased compared to sham-operated animals (P < 0.01). Confocal fluorescence immunohistochemistry with stereological analysis showed that numbers of ERalpha immunoreactive cell nuclei per mm(3) of tissue in the caudal NTS were significantly greater in proestrus than in other groups of rats (P < 0.05). There were also differences among the groups in the extent of colocalisation of ERalpha in neurones immunoreactive for tyrosine hydroxylase and nitric oxide synthase. These results imply a complex pattern of region-specific oestrogen signalling in the NTS and suggest that ERalpha expression in this important autonomic nucleus may be related to circulating oestrogen levels. This may have consequences for the regulation of autonomic tone and baroreflex sensitivity when oestrogen levels decline, for example following menopause.

Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous systems.

The ionotropic ATP receptor subunits P2X(1-6) receptors play important roles in synaptic transmission, yet the P2X(7) receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X(7) receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X(7) receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X(7) receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP; 30 microm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X(7) receptor antagonists oxidized ATP (100 microm) and Brilliant Blue G (2 microm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X(7) receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1-43 in isolated NMJ preparations destained after application of BzATP (30 microm). This BzATP evoked destaining is blocked by oxidized ATP (100 microm) and Brilliant Blue G (1 microm). This indicates that activation of the P2X(7) receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

Neurochemistry of superficial spinal neurones projecting to nucleus of the solitary tract that express c-fos on chemical somatic and visceral nociceptive input in the rat.

We have investigated the presence of three neurochemical markers, glutamate, calbindin-D28k, and nitric oxide synthase, in spinal neurones that transmit chemical noxious inputs from both the skin and the viscera, by combining retrograde labelling with the fluorescent tracer Fluorogold with dual labelling immunohistochemistry. Neurones projecting to the nucleus of the solitary tract (NTS) that expressed Fos protein in response to cutaneous or visceral noxious stimulation were concentrated in lamina I of the cervical and lumbosacral segments, respectively. Although both labelled neuronal populations were numerous, the spino-solitary cells that transmit visceral nociceptive input predominated over those transmitting cutaneous nociceptive input. Calbindin-D28k-immunoreactivity was observed in neurones of three morphological types (fusiform, flattened, and pyramidal) projecting to the NTS that were activated by somatic or visceral nociceptive neurones. Nitric oxide synthase and glutamate immunoreactivities were present only in viscerally activated nociceptive neurones projecting to the NTS. Glutamate-immunopositive NTS-projecting cells were exclusively of the flattened type, and the nitric oxide synthase-immunolabelled NTS-projecting cells comprised 75%/fusiform cells and 25% flattened cells. These data suggest that the involvement of excitatory spinal lamina I projection neurones in the transmission of peripheral chemical nociceptive inputs to the NTS may be restricted to information of visceral origin.

A GABAergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study.

The central nucleus of the amygdala is involved in the modulation of autonomic, somatic and endocrine functions, as well as behavioural responses to stressful stimuli. Anatomical and physiological studies have suggested that this nucleus sends projections to the nucleus of the solitary tract, the primary site of termination of vagal and glossopharyngeal afferent fibres in the brain stem. To determine the neurochemical nature of the amygdaloid input to the nucleus of the solitary tract, anterograde tracing with biotinylated dextran amine was combined with post-embedding immunogold labelling for GABA and glutamate immunoreactivities and with pre-embedding labelling for the vesicular GABA transporter. Following injection of biotin dextran amine into the central nucleus of the amygdala, anterogradely labelled axons and varicosities were found throughout the rostrocaudal extent of the nucleus of the solitary tract, particularly in the medial, ventral and ventrolateral subnuclei. The anterogradely labelled terminals were found to make predominantly symmetrical synaptic contacts with dendrites, and occasionally onto cell bodies and dendritic spines, and to contain immunoreactivity for GABA and for the vesicular GABA transporter. Immunolabelling of serial sections with antibodies to glutamate showed that none of these axon terminals contained high enough densities of gold particle labelling to suggest that they contained other than low metabolic levels of glutamate immunoreactivity. These results provide conclusive evidence for a GABAergic pathway from the central nucleus of the amygdala to the nucleus of the solitary tract. This GABAergic projection may provide a substrate for inhibition of lower brain stem visceral reflexes, including baroreflex inhibition, through which the central nucleus of the amygdala could participate in cardiovascular regulation related to emotional behaviour and the defence reaction.

P2X(2) receptor immunoreactivity in the dorsal vagal complex and area postrema of the rat.

Adenosine 5'-triphosphate (ATP) can function as a fast synaptic transmitter through its actions on ionotropic (P2X) and metabotropic (P2Y) receptors in neuronal tissue. The ionotropic receptors have been classified into seven subtypes (P2X(1)-P2X(7)) by molecular cloning. However, they are difficult to distinguish pharmacologically owing to an absence of specific agonists and antagonists. In this study we used neuroanatomical methods to determine the origin and neurochemical phenotype of the P2X(2) subtype of purinoceptor in the dorsal medulla of the rat. Using immunohistochemistry we observed dense networks of P2X(2) receptor immunoreactive labelled fibres and terminals in the dorsal vagal complex and area postrema, as well as labelled cell bodies in the dorsal vagal nucleus and the area postrema. The P2X(2) receptor was localized presynaptically in vagal afferent fibres and terminals in the nucleus tractus solitarius at the ultrastructural level by combining injections of an anterograde tracer (biotin dextran amine) into the nodose ganglion with pre-embedding immunogold visualization of P2X(2) immunoreactivity. Terminals immunoreactive for the P2X(2) receptor in the nucleus tractus solitarius were found to contain glutamate, but not GABA immunoreactivity by post-embedding immunogold-labelling techniques. In cell bodies in the area postrema, dual immunofluorescence also indicated that P2X(2) receptor immunoreactive cells are glutamatergic but not GABAergic. The P2X(2) receptor was localized to vagal preganglionic neurons in the dorsal vagal nucleus that were identified by prior intraperitoneal injections of the retrograde tracer FluoroGold. No cells immunoreactive for the P2X(2) receptor were observed in the nucleus tractus solitarius. The localization of P2X(2) receptor immunoreactivity presynaptically in vagal afferent terminals indicates that the receptor may be involved in modulating transmitter release from vagal afferent fibres. Furthermore, the presence of the P2X(2) receptor in vagal preganglionic cells and in glutamatergic cells of the area postrema implies that it may, respectively, play a role in regulation of vagal efferent cell activity and modulation of excitatory outputs from the area postrema to other brain regions.

Hypoxic enhancement of quantal catecholamine secretion. Evidence for the involvement of amyloid beta-peptides.

Prolonged exposure to hypoxia (10% O(2)) enhanced quantal catecholamine release evoked from O(2)-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca(2+) influx pathway that persisted during complete blockade of known voltage-gated Ca(2+) channels. Secretion triggered by this Ca(2+) influx was severely reduced by known inhibitors of Alzheimer's amyloid beta-peptides (AbetaPs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AbetaP to cells under normoxic conditions, although the effects of AbetaP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca(2+)-permeable AbetaP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AbetaP pathophysiology following cerebral ischemia.

Redistribution of F-actin and large dense-cored vesicles in the human neuroblastoma SH-SY5Y in response to secretagogues and protein kinase Calpha activation.

Our previous studies have shown that noradrenaline release is enhanced by activation of protein kinase Calpha in SH-SY5Y cells. In the present study, we report that activation of protein kinase Calpha leads to (a) partial redistribution of the F-actin cytoskeleton and (b) a 2.5-fold increase in the number of large dense-cored vesicles within 100 nm of the plasma membrane. This redistribution can be prevented by down-regulation of protein kinase Calpha by up to 48 h exposure to phorbol dibutyrate. Treatment with the secretagogues 100 mM KCl, the Ca2+ ionophore A23187 (20 microM) and 1 mM carbachol also leads to a partial disassembly of the F-actin cytoskeleton. This is accompanied by an increase in the number of large dense cored vesicles at the plasma membrane following exposure to KCl and A23187 but not following exposure to carbachol. These results are discussed in relation to the hypothesis that a key step in the enhancement of noradrenaline release following activation of protein kinase Calpha and elevation of intracellular calcium is the movement of large dense cored vesicles to the plasma membrane following partial disassembly of the F-actin cytoskeleton.

Innervation and control of the adenohypophysis by hypothalamic peptidergic neurons in teleost fishes: EM immunohistochemical evidence.

Previous light microscopic studies have revealed neuropeptide-immunoreactive neurosecretory fibers in the teleostean neurohypophysis, and ultrastructural work has reported direct innervation of endocrine cells by the terminals of fibers penetrating the adenohypophysis. This paper reviews our recent data from ultrastructural, immunohistochemical, receptor localization, and superfusion studies, which suggest a role for neuropeptides in the control of teleost pituitary secretion. We have used a combination of pre- and post-embedding electron microscopic immunolabeling methods to determine which neuropeptides are present in fibers innervating the pituitaries of three species: Poecilia latipinna, Dicentrarchus labrax, and Clarias gariepinus. Numerous axon profiles with immunoreactivity for the neurosecretory peptides vasotocin and isotocin formed large Herring bodies and terminal-like boutons in contact with corticotropic, growth hormone, thyrotropic, and pars intermedia cells. Numerous melanin-concentrating hormone-immunoreactive fibers and scarcer neurotensin and corticotropin-releasing factor-immunoreactive fibers showed similar distributions, terminating close to pars intermedia and corticotropic cells. Somatostatin, cholecystokinin, galanin, substance P, neuropeptide Y, growth hormone-releasing factor, thyrotropin-releasing hormone, and gonadotropin-releasing hormone-immunoreactivities were found in small calibre fibers penetrating among growth hormone, thyrotropic, and gonadotropic cells. These morphological findings have been supplemented by autoradiographic studies, which showed the distribution of binding sites for vasotocin, isotocin, galanin, and neuropeptide Y ligands over specific groups of pituitary cells, and superfusion studies that showed growth hormone release was stimulated by growth hormone-releasing factor and thyrotropin-releasing hormone, but inhibited by somatostatin. The implications of these results for neuropeptidergic control of teleostean pituitary secretions are discussed.

Co-localization of c-Fos and neurotransmitter immunoreactivities in the cat brain stem after carotid sinus nerve stimulation.

To reveal neurones in the cat medulla oblongata involved in carotid baroreceptor/chemoreceptor reflexes, the distribution of c-Fos oncoprotein immunoreactivity was studied following electrical stimulation of the right carotid sinus nerve. The neurochemistry of the activated neurones was investigated using antisera to tyrosine hydroxylase, neuropeptide Y, somatostatin, and glutamate. Nitric oxide containing neurones were identified using antiserum to nitric oxide synthase (NOS) and by the histochemical localization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Following sinus nerve stimulation numerous c-Fos-IR cells were detected both ipsilaterally and contralaterally in the nucleus tractus solitarii, the area postrema and throughout the ventrolateral medulla. Dual labelling studies revealed that 3.3% of c-Fos-immunoreactive cells in the nucleus tractus solitarii were also immunoreactive for tyrosine hydroxylase. The double labelled cells were scattered within the medial and ventrolateral subnuclei, predominantly rostral to obex. A higher proportion (10.3%) of c-Fos-IR cells in the ventrolateral medulla also showed tyrosine hydroxylase immunoreactivity. Caudal to obex, these were scattered in the reticular formation between the spinal trigeminal nucleus and the lateral reticular nucleus, while more rostrally they were found within the lateral reticular nucleus, the nucleus ambiguus and the lateral tegmental field. Cells expressing c-fos and reactive for glutamate, neuropeptide Y or NADPH-diaphorase (or NOS) were only rarely seen, and co-localization of c-Fos and somatostatin immunoreactivities was not seen. These results suggest that of the neurones forming pathways within the medulla activated on carotid sinus nerve stimulation, presumably mediating baro- and chemoreceptor reflexes, relatively few utilize catecholamines, glutamate, neuropeptide Y or nitric oxide as their transmitter substance.

Immunolocalization of putative neurotransmitters innervating autonomic regulating neurons (correction of neurones) of cat ventral medulla.

This study investigated possible sites of contact of nerve fibers containing a range of putative neurotransmitter substances onto neurons in the cat ventral medulla oblongata concerned with autonomic, particularly cardiovascular, regulation. The parasympathetic preganglionic neurons of the nucleus ambiguous (correction of ambiguus) were identified by retrograde horseradish peroxidase tracing from the vagus nerve, and the groups of neurons in the A1 and C1 cell areas and the raphe nucleus by catecholamine enzyme or 5-hydroxytryptamine (5-HT) immunohistochemistry, respectively. Immunoreactive (-ir)nerve fibers and terminals in the vicinity if these neurons were visualized by subjecting the sections to a dual-staining technique using a brown peroxidase-diaminobenzidine reaction product and a blue alkaline phosphatase-Fast blue reaction product. By employing monochrome photography with combinations of blue and orange-red filters, it was possible to discriminate neural elements displaying one or the other reaction product, or colocalization of reaction products. The results revealed the presence of calcitonin gene-related peptide (CGRP) and galanin (GAL)-ir in some motoneurons of the nucleus ambiguus, but not in those innervating the heart via the cardiac vagus nerve. The latter group of parasympathetic efferent neurons were found to be densely innervated by fibers immunoreactive for dopamine beta-hydroxylase (DBH, indicating noradrenaline), glycine (GLY), gamma-aminobutyric acid (GABA), 5-HT, enkephalin (ENK), neuropeptide Y (NPY), substance P (SP), and thyrotropin-releasing hormone (TRH), and, to a lesser extent, by other neuropeptide-ir fibers. The catecholamine cells of the rostral C1 and caudal A1 groups showed a broadly similar pattern of innervation, most noticeably by fibers immunoreactive for DBH, GABA, 5-HT, cholecystokinin (CCK), CGRP, ENK, GAL, NPY, and SP. The 5-HT-ir neurons of the raphe nucleus, some also containing SP, TRH, ENK, or corticotropin-releasing factor (CRF)-ir, were most prominently innervated by terminals containing DBH, GABA, CCK, ENK, NPY, TRH, somatostatin (SRIF), and vasoactive intestinal polypeptide (VIP)-ir. Although the proof that these groups of neurons receive functional synaptic contacts from the immunoreactive fibers awaits further ultrastructural studies, the results do suggest that a wide range of putative transmitters may influence the activity of efferent neurons in the cat medulla controlling autonomic functions.

Autoradiographic distribution of galanin binding sites in the brain and pituitary of the sea bass (Dicentrarchus labrax).

Specific binding sites for galanin (GAL) were detected in brain and pituitary of a marine teleost fish, the sea bass, after in vitro incubation of tissue sections with [125I]GAL and light microscopic autoradiography. Binding conditions were optimized and as a result the binding was saturable and specific. In the brain, [125I]GAL binding was found to occur in all parts of the dorsal and ventral telencephalon, in the anterior, tuberal and posterior hypothalamus, in the thalamus and in the tectum opticum, in the inferior lobe and in the ventral medulla oblongata. In the pituitary dense [125I]GAL binding was confined to the area occupied by the prolactin cells in the rostral part of the adenohypophysis. These findings provide the first anatomical evidence for the presence of GAL specific binding sites in the teleost brain and pituitary.

Anatomical distribution of galanin-like immunoreactivity in the brain and pituitary of teleost fishes.

Immunohistochemical study of brains of five teleost fishes (molly, sea bass, killifish, flounder, tilapia) revealed similar extensive systems of galanin immunoreactive (GAL-ir) neurons. Cell bodies were located in the anterior preoptic recess (where coexistence with corticotrophin-releasing factor-like-ir was found), posterior tuberal hypothalamus and vagal lobe of the medulla oblongata. Fibres in the fingers of neurohypophysial tissue penetrating the pituitary pars distalis suggested an anatomical relationship between GAL-ir terminals and the hormone secreting cells. Electron microscopic studies on sea bass pituitary revealed contacts of GAL-ir fibres with growth hormone cells and gonadotrophs. Thus a GAL-like peptide may be released from nerve terminals in the teleost pituitary, where it may act locally to modulate the secretion of one or more pituitary hormones.

Peptidergic innervation of the adrenocorticotropic hormone (ACTH)- and growth hormone (GH)-producing cells in the pars distalis of the sea bass (Dicentrarchus labrax).

Due to its unique organization, the teleost pituitary is an ideal model in which to investigate the relationship of the nervous system with the pituitary endocrine cells. A light microscope immunocytochemical study of the sea bass pituitary revealed six different neuropeptides in nerve fibers which projected into the pituitary neurohypophysis and bordered the adenohypophysial cells. Double staining showed separate nerve fibers immunoreactive for corticotropin-releasing factor (CRF), vasotocin (VT), somatostatin (SRIF), growth hormone-releasing factor (GRF), and neurotensin (NT) in the vicinity of the adrenocorticotropic hormone-releasing cells (ACTH-cells) in the rostral pars distalis (PD). In the proximal PD cholecystokinin (CCK)-, SRIF-, GRF-, and VT-immunoreactive fibers penetrated between the growth hormone-releasing cells (GH-cells). These results suggest a possible role for CCK, GRF, SRIF, and VT in the modulation of GH-cell activity, while the synthesis and/or secretion of the ACTH-cells might be affected by the release of VT, CRF, SRIF, GRF, and NT.

Neurotensin-like immunoreactivity in the pituitary and hypothalamus of bony fishes.

Using an antiserum raised against synthetic neurotensin (NT), the distribution of immunoreactivity in the pituitary and hypothalamus has been examined by immunocytochemistry at light and electron microscope level in a number of species of bony fishes. In most species immunoreactive perikarya were found in the preoptic region of the hypothalamus, with fibres throughout the tuberal hypothalamus and neurohypophysis (neural lobe and median eminence). In the neurohypophysis of teleosts NT-like immunoreactivity was seen in a dense band of fibres bordering the ACTH cells of the rostral pars distalis: absorption controls showed that this was due to the presence of an NT(8-13)-like or xenopsin-like sequence, which, according to electron microscopic observations, was contained in small dense cored vesicles. The antiserum also stained the pituitary ACTH cells of some species, apparently due to cross-reaction with the 17-19 sequence of ACTH. These results suggest that an NT-like peptide may have a role in control of the adenohypophysis in fishes.

Immunocytochemical demonstration of pituitary cell types in the teleost Poecilia latipinna, by light and electron microscopy.

Using the unlabelled antibody method at the light microscope level, and the immunogold method at the electron microscope level, the distribution of the different adenohypophysial cells was demonstrated in the teleost Poecilia latipinna, by means of antisera to both teleostean and mammalian pituitary hormones and their subunits. Anti-salmon prolactin, but not anti-rat or -ovine prolactin, gave a specific staining of the acidophils of the rostral pars distalis (RPD), while anti-trout growth hormone (GH), but not anti-rat GH, stained similar but always separate cells in the proximal pars distalis (PPD). Antisera to the whole molecules of mammalian glycoprotein hormones stained the entire population of basophils in the PPD, but separate populations of gonadotrophs and thyrotrophs could be discriminated using anti-salmon gonadotrophin and anti-human thyrotrophin beta subunit. Antisera to ACTH (1-24) and (11-24) sequences, as well as beta-endorphin and met-enkephalin, stained the lead haematoxylin-positive cells of the RPD and pars intermedia (PI), whereas anti-alpha-MSH stained only the PI cells. Ultrastructural examination showed that these immunoreactivities were present in the same secretory granules, and were always greater in pale granules rather than electron dense granules. In the RPD, blebs of ACTH-immunoreactive cytoplasm were found to protrude through the gaps in the basement membrane into the neurohypophysis. The second "PAS-positive" cell type of the PI showed a strong cross-reaction with anti-salmon gonadotrophin, suggesting that it may produce a glycoprotein chemically related to the gonadotrophin(s).

Direct control of the gonadotroph in a teleost, Poecilia latipinna. II. Neurohormones and neurotransmitters.

Pituitaries from male and female mollies were incubated with varying amounts of mammalian LH-RH, arginine vasotocin, dopamine, or serotonin for 18 hr. Ultrastructural differences between control and experimentally treated glands were used to define the direct effects of these neurohormones and neurotransmitters on the gonadotrophic cells of the adenohypophysis. The effects varied in intensity according to the sex and reproductive state of the donor animal. LH-RH stimulated gonadotrophin secretion by the gonadotrophs, as did vasotocin, although to a much lesser extent and with noticeable differences between the sexes. Dopamine inhibited secretion by basally active gonadotrophs and probably from active cells also, although to a lesser extent. Serotonin mildly stimulated secretion at all stages in both sexes. The results of this study indicate the possible involvement of neurohypophysial octapeptides and of monoamines in the direct control of the gonadotroph of Poecilia latipinna.

Immunocytochemical identification and localization of the different cell types in the pituitary of the seabass (Dicentrarchus labrax).

Antisera raised against chum salmon prolactin (PRL), trout growth hormone (GH), mammalian adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and alpha-melanophore-stimulating hormone (alpha-MSH) were used to localize PRL, GH, ACTH, gonadotropic, TSH, and MSH cells in the hypophysis of the teleost Dicentrarchus labrax using the unlabeled peroxidase anti-peroxidase method. In the rostral pars distalis, ACTH cells stained very intensively with anti-ACTH; so did the MSH cells in the pars intermedia. The prolactin cells stained very specifically with anti-prolactin without staining the growth hormone cells. In the proximal pars distalis anti-GH, anti-TSH beta, and anti-LH stained selectively the corresponding cells; with these antisera no cross-reaction with any other cell type was observed. Anti-alpha-MSH only stained cells in the pars intermedia. Some cells in the pars intermedia did not react at all; these could correspond to the PAS-positive cells. A characteristic feature was positive staining with anti-LH in some cell groups encircling the pars intermedia, indicating the fact that in the seabass some cells of the proximal pars distalis surround the pars intermedia.

Direct control of the gonadotroph in a teleost, Poecilia latipinna: gonadal steroids.

Pituitaries from male and female fish were incubated with 1, 5, and 10 micrograms/ml of oestradiol-17 beta, testosterone, 17 alpha OH-progesterone, and 17 alpha,20 beta-dihydroxy-4-pregnan-3-one for 18 hr. Ultrastructural differences between control and experimentally treated glands were used to define the direct effects of these steroids on the gonadotropic cells of the adenohypophysis. The effects of the steroids differed according to sex and reproductive state of the donor animal. Oestradiol and testosterone stimulated gonadotropin secretion by active gonadotrophs, but inhibited it in inactive cells. Both the progesterones generally inhibited gonadotropin secretion although 17 alpha,20 beta-dihydroxy-4-pregnan-3-one had no effect on active gonadotrophs. The four steroids investigated all show potential for direct control of gonadotropin secretion in Poecilia latipinna although factors affecting the balance of these actions, and their relative importance in vivo, remain to be elucidated.

Ultrastructural characterization of neurosecretory fibres immunoreactive for vasotocin, isotocin, somatostatin, LHRH and CRF in the pituitary of a teleost fish, Poecilia latipinna.

Using the electron-microscopic immunogold method, vasotocin, isotocin, somatostatin (SRIF), gonadotrophin-releasing hormone (LHRH) and corticotrophin-releasing factor (CRF)-like immunoreactivities were localized in separate neurosecretory fibres in the pituitary of a teleost fish Poecilia latipinna. Antigenicities were preserved in sections of conventionally fixed tissue, except in the case of LHRH and CRF-like substances which were sensitive to osmium postfixation. Under the same fixation conditions, ultrastructural differences were observed between the 5 fibre types, and morphometric analysis of their granule sizes revealed significant differences in mean diameter except between vasotocin and isotocin fibres. Terminal-like regions of each type were identified on blood vessels, glial cells or other fibres in the neurohypophysis, on the basement lamina of the adenohypophysis, or directly on adenohypophysial endocrine cells. The fibres containing the two neurohypophysial hormones, originating from separate preoptic perikarya, were intermingled with, and may form endings near all the adenohypophysial cell types except those secreting prolactin. Although both types had similar mean granule diameters, the granules in the vasotocin fibres (mean 135 nm) were markedly less electron dense than those in the isotocin fibres (mean 140 nm). SRIF-immunoreactive fibres (mean 101 nm) appeared to form synapse-like endings on the somatotrophs, and a few thyrotrophs in the proximal pars distalis, and near the pars intermedia cells. An LHRH-positive type (mean 103 nm) contacted only the gonadotrophs of the proximal pars distalis. The rarer CRF-like fibres (mean 116 nm) appeared to project mainly towards the pars intermedia, but a few appeared to terminate rostrally near the adrenocorticotrophic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory activity of Poecilia latipinna (Teleostei) pituitary in vitro: rostral pars distalis and proximal pars distalis.

The ultrastructure of each adenohypophysial secretory cell type was examined in pituitaries of adult female mollies (Poecilia latipinna) after various periods in vitro, and with varied medium osmotic pressure (OP), Na+, and Ca2+ concentrations. Prolactin (PRL) cells were markedly activated by 18 hr, and after 7 or 14 days were almost totally degranulated, with massive arrays of Golgi and RER. Reduction in OP, but not Na+ or Ca2+, caused an additional activation of PRL cells after periods of 18 hr or longer. Corticotroph (ACTH) cells became noticeably activated by 4 hr, and were possibly affected by OP, but not Na+ or Ca2+. Growth hormone (GH) cells were activated by 6 hr, and after 18 hr were quite degranulated with extensive arrays of RER. OP had no effect on GH cells before 3 days, when reduced OP appeared to cause an additional activation, with the appearance of large irregular secretory granule (SG)-like inclusions. Na+ and Ca2+ again had no effect. Gonadotrophic (GtH) cells appeared to be little affected by in vitro incubation; however, the very active cells from vitellogenic fish underwent a reduction in dilated RER after prolonged culture. Thyrotrophic (TSH) cells gradually became activated in vitro, but the response again varied with the sexual condition of the fish. Neither GtH nor TSH cells were affected by OP, Na+, or Ca2+. The findings are discussed in relation to hypothalamic control, via releasing/inhibiting factors, of adenohypophysial cell activity.