Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone.     

Activity-dependent dynamics of coexisting brain-derived neurotrophic factor, pro-opiomelanocortin and alpha-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.     

Distribution of corticotropin-releasing factor-immunoreactive neurons in the central nervous system of the domestic chicken and Japanese quail

In birds, as in mammals, corticotropin-releasing factor (CRF) is present in a number of extrahypothalamic brain regions, indicating that CRF may play a role in physiological and behavioral responses other than the control of adrenocorticotropin hormone release by the pituitary. To provide a foundation for investigation of the roles of CRF in the control of avian behavior, the distribution of CRF immunoreactivity was determined throughout the central nervous system of the domestic chicken (Gallus domesticus) and Japanese quail (Coturnix japonica). The distribution of CRF-immunoreactive (-ir) perikarya and fibers in the chicken and quail brain was found to be more extensive than previously reported, notably in the telencephalon. Numerous CRF-ir perikarya and fibers were present in the hyperstriatum, hippocampus, neostriatum, lobus parolfactorius, and archistriatum, as well as in the nucleus taeniae, nucleus accumbens, and bed nucleus of the stria terminalis, which exhibited the strongest immunolabeling in the telencephalon. The presence of dense populations of CRF-ir perikarya in the medial lobus parolfactorius, nucleus of the stria terminalis, and paleostriatum ventrale, apparently giving rise to CRF-ir projections to the mesencephalic reticular formation, the parabrachial/pericerulear region, and the dorsal vagal complex, suggests that these telencephalic areas may constitute part of the avian "central extended amygdala." These results have important implications for understanding the role of extrahypothalamic CRF systems in emotional responses in birds.     

High-pressure freezing followed by cryosubstitution as a tool for preserving high-quality ultrastructure and immunoreactivity in the Xenopus laevis pituitary gland

Subcellular localisation of proteins and peptides yields fundamental information about cell functioning. Immunoelectron microscopy is a powerful tool to achieve this goal, but combining good tissue preservation with strong immunoreactivity is a great challenge in electron microscopy. We have applied a novel approach, using high-pressure freezing (HPF) followed by cryosubstitution, to prepare the pituitary gland of the amphibian Xenopus laevis for immunogold-electron microscopy. In this way, we investigated the subcellular distribution of brain-derived neurotrophic factor and the amphibian neurohormone mesotocin in the pituitary neural lobe, and the peptide hormone alpha-melanophore-stimulating hormone and its protein precursor proopiomelanocortin in melanotrope cells of the pituitary intermediate lobe. In contrast to conventional chemical fixation (followed by cryosubstitution), HPF not only revealed strong immunoreactivity of the secretory products, but also provided excellent ultrastructural preservation of cell organelles, including secretory granule subtypes. We conclude that HPF followed by cryosubstitution provides a preparation technique of choice when both optimal tissue ultrastructure and strong immunoreactivity are required.     

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.     

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.     

Estradiol or ovariectomy decreases CRF synthesis in hypothalamus

Gonadectomy and gonadectomy plus chronic estradiol administration decrease the content of hypothalamic corticotropin-releasing factor-like immunoreactivity (CRF-ir). This investigation was conducted to determine whether this was a result of chronic inhibition of CRF synthesis or stimulation of release. Administration of the protein synthesis inhibitor anisomycin one hour prior to sacrifice in otherwise untreated controls resulted in a 21% decrease in median eminence CRF-ir (p less than 0.01). Rats were ovariectomized and then administered either estradiol or vehicle daily for 3 weeks. Compared to vehicle-treated controls, estradiol treatment decreased CRF-ir in median eminence (p less than 0.01). In contrast to controls, administration of anisomycin to these ovariectomized rats did not significantly decrease median eminence CRF-ir in either the vehicle or estradiol-treated groups, implying that CRF synthesis was already depressed. Plasma adrenocorticotropin (ACTH) was increased by anisomycin treatment in all groups whether intact, ovariectomized or estradiol-treated (p less than 0.0005). Adrenal weights did not differ between these groups, or in comparison to sham-ovariectomized rats, indirectly implying lack of chronically elevated plasma ACTH. These data suggest that the mechanism for both the ovariectomy and chronic estradiol-induced decrease in CRF-ir content is an inhibition of CRF synthesis as opposed to a stimulation of CRF release.     

Single or repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor

The effect of a specific mild stress on the levels of corticotropin-releasing factor immunoreactivity (CRF-ir) in the hypothalamus of adult male rats was determined using a radioimmunoassay specific for rat CRF. A single 5 min restraint significantly increased CRF-ir in the median eminence 24 h later compared to appropriate controls (P less than 0.025), with no change detected earlier. Plasma ACTH, an indirect index of CRF release, was significantly elevated within 15 min (P less than 0.025). Repetition of a mild stress daily for 9 days (P less than 0.01), or a single episode of handling (P less than 0.05), both resulted in significantly increased CRF-ir in the whole hypothalamus 24 h later. Blockade of axonal transport by intracisternal colchicine decreased CRF-ir in the median eminence 24 h later (P less than 0.005). Inhibition of protein synthesis by anisomycin during a single 5 minute restraint resulted in significantly decreased CRF-ir in the median eminence 24 h later compared to vehicle-injected stressed rats (P less than 0.005) or to anisomycin-injected unstressed controls (P less than 0.025). These data show that mild stress increased net hypothalamic CRF content as a result of the balance between augmented synthesis and augmented release.     

Gonadal regulation of corticotropin-releasing factor immunoreactivity in hypothalamus

The effect of gonadectomy and subsequent gonadal steroid administration on hypothalamic corticotropin-releasing factor immunoreactivity (CRF-ir) in male and female rats was determined using a radioimmunoassay specific for rat CRF. When compared to appropriate controls, gonadectomy resulted in significantly decreased CRF-ir in both males and females (p less than 0.025). Short-term replacement with testosterone or estradiol did not significantly alter hypothalamic CRF-ir. Acute IC administration of gonadotropin-releasing hormone to male rats significantly decreased CRF-ir in the median eminence 15 minutes later. Chronic administration of estradiol for 3 weeks significantly reduced CRF-ir in ovariectomized rats (p less than 0.005). This decrease was shown not to correlate to the marked increase in anterior pituitary weight (r2 = 0.063, not significant). Chronic administration of estradiol to hypophysectomized female rats significantly decreased hypothalamic CRF-ir (p less than 0.01), an effect localized to the median eminence (p less than 0.005). These data imply that the gonadectomy-induced reduction in hypothalamic CRF-ir was not directly mediated by the loss of gonadal steroids. Chronic estradiol treatment decreased hypothalamic CRF-ir, by a non-pituitary mechanism. These data show significant effects of the gonadal axis in the regulation of CRF in the hypothalamus in both sexes.     

Distribution of corticotropin-releasing factor binding protein-immunoreactivity in the rat hypothalamus: association with corticotropin-releasing factor-, urocortin 1- and vimentin-immunoreactive fibres

Corticotropin-releasing factor binding protein (CRF-BP) is a 37-kDa protein with high affinity binding sites for both corticotropin-releasing factor (CRF) and urocortin 1. Previous studies have examined the distribution of CRF-BP mRNA and peptide within the central nervous system. Due to the predominant cortical localisation, very little is known about CRF-BP in subcortical structures including the hypothalamus. The present study employed immunohistochemistry to characterise the distribution of CRF-BP-like-immunoreactive (-ir) cells and fibres in the rat hypothalamus. Bipolar and multipolar CRF-BP-ir neurones were scattered throughout the rostro-caudal extent of the hypothalamus. Distinct clusters of CRF-BP-ir neurones were identified in the anterior and posterior parvocellular and dorsal cap subdivisions of the paraventricular nucleus (PVN), as well as in the dorsal hypothalamic area, dorsomedial hypothalamic nucleus (DMN), ventral premammillary nucleus and zona incerta. CRF-BP-ir fibres extending from the third ventricle were found in the mediobasal hypothalamus and within the arcuate nucleus-median eminence region. Double immunostaining together with confocal microscopy demonstrated that the CRF-BP-immunostained fibres within the mediobasal hypothalamus coincided with vimentin immunostaining indicating that CRF-BP-ir is present within tanycytes. To define the relationship between CRF-BP-ir cells and endogenous ligands for CRF-BP, double immunohistochemistry was performed to examine possible sites within the hypothalamus where CRF- or urocortin 1-ir fibres innervate regions that contain CRF-BP-ir cell bodies. CRF-BP-ir cell bodies typically coincided with dense CRF-ir, but not urocortin 1-ir fibre innervation. CRF-ir fibre innervation was moderate to high within the anterior and posterior parvocellular subdivisions of the PVN, the dorsal cap of the PVN, DMN and the zona incerta; all regions that contained CRF-BP-ir cell populations. These studies demonstrate that, within the hypothalamus, CRF-BP-ir cells and fibres are concentrated within a circuitry known to be involved in mediating neuroendocrine and autonomic responses to stress.     

Localisation and physiological regulation of corticotrophin-releasing factor receptor 1 mRNA in the Xenopus laevis brain and pituitary gland

In Xenopus laevis , corticotrophin-releasing factor (CRF) and urocortin 1 are present in the brain and they both are potent stimulators of α-melanophore stimulating hormone (MSH) secretion by melanotroph cells in the pituitary gland. Because both CRF and urocortin 1 bind with high affinity to CRF receptor type 1 (CRF₁) in mammals and Xenopus laevis , one of the purposes of the present study was to identify the sites of action of CRF and urocortin 1 in the Xenopus brain and pituitary gland. Moreover, we raised the hypothesis that the external light intensity is a physiological condition controlling CRF₁ expression in the pituitary melanotroph cells. By in situ hybridisation, the presence of CRF₁ mRNA is demonstrated in the olfactory bulb, amygdala, nucleus accumbens, preoptic area, ventral habenular nuclei, ventromedial thalamic area, suprachiasmatic nucleus, ventral hypothalamic area, posterior tuberculum, tectum mesencephali and cerebellum. In the pituitary gland, CRF₁ mRNA occurs in the intermediate and distal lobe. The optical density of the CRF₁ mRNA hybridisation signal in the intermediate lobe of the pituitary gland is 59.4% stronger in white-adapted animals than in black-adapted ones, supporting the hypothesis that the environmental light condition controls CRF₁ mRNA expression in melanotroph cells of X. laevis , a mechanism likely to be responsible for CRF- and/or urocortin 1-stimulated secretion of α-MSH.     

Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.     

Reciprocal synaptic relations between CRF-immunoreactive- and TRH-immunoreactive neurons in the paraventricular nucleus of the rat hypothalamus

By double immunoelectron microscopy, we studied synaptic relations between corticotropin-releasing factor (CRF)-immunoreactive (ir) and thyrotropin-releasing hormone (TRH)-ir neurons in the paraventricular nucleus (PVN) of the rat hypothalamus. CRF-ir and TRH-ir neurons made reciprocal synaptic connections in the medial and periventricular parvocellular regions. These results may suggest that both the parvocellular neurons interplay on their hypophysiotropic functions within the PVN.     

Neuropeptide Y-induced effects on hypothalamic corticotropin-releasing factor content and release are dependent on noradrenergic/adrenergic neurotransmission

Neuropeptide Y (NPY) administration increases both hypothalamic corticotropin-releasing factor-like immunoreactivity (CRF-ir) and plasma adrenocorticotropin (ACTH). The dependence of these effects on noradrenaline and adrenaline was investigated by selectively depleting these neurotransmitters with 6-hydroxydopamine (6-OHDA) prior to administration of NPY. This combined treatment decreased hypothalamic CRF-ir (P less than 0.025), an effect isolated to the median eminence (P less than 0.025), whereas plasma ACTH increased greatly compared to 6-OHDA treatment alone (P less than 0.0005). In order to further investigate the potential mechanism of this NPY effect, the alpha 2-adrenergic agonist clonidine was administered to normal rats. This treatment increased plasma ACTH (P less than 0.005) and decreased hypothalamic CRF-ir (P less than 0.025), an effect localized to the median eminence (P less than 0.01). The results from both of these treatments are consistent with increased release of hypothalamic CRF. These data imply that the NPY-induced effects are dependent on normal noradrenergic/adrenergic neurotransmission. Depletion of these neurotransmitters allowed NPY to profoundly stimulate CRF release with no evidence for alteration in synthesis, a result common to alpha 2 stimulation.     

Localization of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Hypothalamus-Pituitary System in Rats: Light and Electron Microscopic Immunocytochemical Studies

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1–38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.     

Interaction of corticotropin-releasing factor (CRF) and alpha-helical CRF on rat neurointermediate lobes: in vitro studies

Neurointermediate lobes (NILS) of the pituitary glands of adult male Sprague-Dawley rats were incubated in media in the presence of corticotropin-releasing factor (CRF), a stimulator of proopiomelanocortin (POMC) peptide release. Alpha-helical CRF, a peptide known to inhibit CRF induced POMC peptide release from the anterior pituitary, was incubated with NILS for a period of 90 min, to study its potential ability to modulate peptide release from the intermediate lobe. The alpha-helical peptide reduced beta-endorphin release from NILS, as measured by radioimmunoassay (RIA), when added for the entire incubation, or when added 30 min after start of the incubation period, with CRF present. Alpha-helical CRF alone reduced beta-endorphin release, as compared to control or CRF-treated lobes. Ultrastructural examination of intermediate lobes fixed at the end of incubations revealed a reduction in the numbers of Golgi-associated dense granules, an indicator of new peptide synthesis, in intermediate lobe tissue treated with alpha-helical CRF alone, both peptides together, or with CRF followed by alpha-helical peptide. The in vitro studies demonstrate the effectiveness of the antagonist peptide on intermediate lobe peptide secretion, thereby extending its effects to both POMC-secreting areas of the pituitary gland.     

Functional organization of the suprachiasmatic nucleus of Xenopus laevis in relation to background adaptation

The process of background adaptation in the toad Xenopus laevis is controlled by neurons in the suprachiasmatic nucleus (SC) that inhibit the release of alpha-melanophore-stimulating hormone from the neuroendocrine melanotrope cells in the pituitary gland. We have identified the structural and functional organization of different neuropeptide Y (NPY)-containing cell groups in the Xenopus SC in relation to background adaptation. A ventrolateral, a dorsomedial, and a caudal group were distinguished, differing in location as well as in number, size, and shape of their cells. They also show different degrees of NPY immunoreactivity in response to different background adaptation conditions. In situ hybridization using a Xenopus mRNA probe for the exocytosis protein DOC2 revealed that melanotrope cells of black-adapted animals have a much higher expression of DOC2-mRNA than white-adapted ones. This establishes that the degree of DOC2-mRNA expression is a good parameter to measure cellular secretory activity in Xenopus. We show that in the ventrolateral SC group, more NPY-positive neurons express DOC2-mRNA in white- than in black-adapted animals. In contrast, NPY-positive neurons in the dorsomedial group have a high secretory activity under the black-adaptation condition. We propose that in black-adapted animals, NPY-positive neurons in the ventrolateral group, known to inhibit the melanotrope cells in white-adapted animals synaptically, are inhibited by NPY-containing interneurons in the dorsmedial group. NPY-positive neurons in the caudal group have similar secretory dynamics as the dorsomedial NPY neurons, indicating that they also play a role in background adaptation, distinct from that exerted by the ventrolateral and dorsomedial group.     

Corticoliberin (CRF) activity in selected hypothalamic regions in rats with anterolateral hypothalamic cuts

The hypothalamic path of the corticoliberin (CRF) containing fibers was investigated in male rats with an anterolateral cut around the medial basal hypothalamus (MBH). Frozen samples of different brain regions were taken from cryostat sections and they were extracted and bioassayed using immunoreactive ACTH release from cell cultures of anterior pituitary as an index of CRF activity. Three days after placing an anterolateral cut there was no change in CRF activity of tissue surrounding a thalamic cut, but a significant rise was observed in the strip of tissue surrounding the hypothalamic cut at the level of the retrochiasmatic area. Seven days after the operation CRF activity decreased in the samples of the stalk-median eminence as well as the arcuate nucleus but it was maintained in the samples taken from that portion of the lateral retrochiasmatic area, which was outside the hypothalamic cut.We suggest that CRF containing fibers traverse the basallateral part of the retrochiasmatic area and the arcuate-ventro-medial region of the MBH en route to the stalk-median eminence and neural lobe. Within 3 days following partial isolation of the MBH neurosecretory material including CRF accumulates near the hypothalamic cut by redistribution in the damaged neurons, while by 7 days after transection the distal part of the axons in the MBH degenerates and is removed.