Immunoreactivity of hypothalamo-neurohypophysial neurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp): immunocytochemical evidence for a correlation with their functional state in colchicine-treated rats

Summary The specific immunoreactivity of neurons containing corticotropin-releasing hormone (CRH) or vasopressin (Vp) was studied both centrally, in the parvocellular division of the paraventricular nucleus, and distally, in the external median eminence. Control rats were compared with adrenalectomized rats and with animals supplemented with corticosterone or dexamethasone, either without additional treatment, or 24, and 48 h after an intraventricular injection of colchicine. In all groups of animals, colchicine induced a progressive and parallel decrease in both CRH and Vp immunoreactivity within the axons of the external median eminence. A semi-quantitative estimation of this axonal immunostaining showed that the decrease was clearly correlated with the axons' releasing activity according to the different functional states of the adrenocorticotropic system. Increased rates of hormonal release induced by adrenalectomy could be seen in the accelerated depletion of axonal immunoreactivity whereas corticosteroid supplementation had the opposite effect. Correspondingly, the progressive intensification of the CRH and Vp immunoreactivity within the perikarya following colchicine treatment was further markedly enhanced in adrenalectomized rats and diminished after corticosteroid supplementation. Taken together, these data suggest that in these neurons, perikaryal hormone synthesis may be closely related to the releasing activity of the axon terminals. They further point to appropriate colchicine treatment as useful tool for evaluating the functional state of CRH and Vp neurons of the parvocellular paraventricular nucleus under various experimental conditions.     

Application of avidin-biotinized peroxidase (ABC) method for immunocytochemical localization of corticotropin-releasing factor (CRF) in rat hypothalamus

Studies on immunocytochemical localization of corticotropin-releasing factor (CRF) were performed in the rat hypothalamus using avidin-biotinized peroxidase (ABC) and PAP techniques. In intact and control animals CRF-immunoreactive nerve fibers were observed within outer layer of median eminence. In the adrenalectomized animals, CRF was also demonstrated in perikarya of neurocytes and in their projections in paraventricular nucleus of the hypothalamus. In both immunocytochemical techniques identical localization of CRF was obtained. However, reaction intensity was greater with the ABC technique than with the PAP one. In bilateral adrenalectomized animals, a greater number of CRF-immunoreactive neural fibers were observed in the median eminence than in control rats and rats subjected to sham operation.     

Electron microscopic immunohistochemical localization of vasopressin and neurophysin in the median eminence of normal and adrenalectomized rats

Using an immunoperoxidase technique at the ultrastructural level, vasopressin (VP) and neurophysin (NP) were localized in the axons of the median eminence of normal and adrenalectomized rats. Whereas VP and NP were generally found in 150-nm granules in the internal zone of the normal rat median eminence, in the adrenalectomized rat, many granules of about 80 to 100 nm in diameter were found to contain NP and VP in the external zone of the median eminence.     

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: Morphological evidence for neuroendocrine γ -aminobutyrate (GABA)

GABAergic cells and axon terminals were localized in the basal hypothalamus of different species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Lightmicroscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure.At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostral part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion.At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space.Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites.These findings show that glutamate decarboxylase-containing endings are localized in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.     

Subpopulations of corticotropin-releasing hormone neurosecretory cells distinguished by presence or absence of vasopressin: confirmation with multiple corticotropin-releasing hormone antisera

Parvocellular corticotropin-releasing hormone neurosecretory cells in the hypothalamic paraventricular nucleus project axons to the portal capillary plexus in the external zone of the median eminence. Immunocytochemical studies have identified two approximately equal subpopulations of these corticotropin-releasing hormone neurons in normal rats, distinguished by the presence or absence of co-existent vasopressin, and different responses to stress. However, it was recently proposed that the vasopressin deficient cells do not contain corticotropin-releasing hormone, but have been misidentified due to cross-reactivity of the corticotropin-releasing hormone antiserum to peptide histidine-isoleucineamide. It is shown here that the same set of corticotropin-releasing hormone neurons (including both vasopressin expressing and vasopressin deficient subtypes) was labeled with multiple corticotropin-releasing hormone antisera. These included two antisera that did not cross-react with peptide histidine-isoleucineamide: one against ovine corticotropin-releasing hormone, and one rat corticotropin-releasing hormone antiserum absorbed with peptide histidine-isoleucineamide. The results provide further support for the hypothesis of functionally distinct compartments of the corticotropin-releasing hormone neurosecretory system that can modulate the ratio of vasopressin to corticotropin-releasing hormone in portal blood.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Immunocytochemical study of Met-enkephalin-like cell bodies in the cat hypothalamus

The distribution of methionine-enkephalin-like cell bodies in the hypothalamus of the cat was studied using an indirect immunoperoxidase technique. Previously, intraventricular or intratissular injections of colchicine were carried out. Met-enkephalin-like neurons were located in all hypothalamic nuclei. The densest clusters of immunoreactive perikarya were observed in the nucleus arcuatus, median eminence, regio praeoptica and perifornical region, whereas the nucleus supraopticus and hypothalamus anterior had the lowest density. In other hypothalamic nuclei the density of methionine-enkephalin (Met-enkephalin) cell bodies was intermediate.     

正中隆起内NT能神经元的SP能神经支配的免疫电镜双标研究

应用包埋前免疫电镜PAP双标技术,对大鼠下丘脑正中隆起内的神经紧张素(neurotensin,NT)和P物质(substance P,SP)的分布进行了超微结构研究。先用DAB法显示SP免疫反应,然后用钼酸-TMB法显示NT免疫反应,再经DAB-氯化钴稳定后作免疫电镜包埋。电镜观察发现:在正中隆起内SP免疫反应呈电子密度高的颗粒状或絮状沉淀,广泛分布于轴突内的小清亮囊泡周围和基质内;NT免疫反应产物则为电子密度高的针状或块状,散在分布于胞体、树突和轴突内。含SP的轴突可接受免疫反应阴性轴突的传入性突触,也可与阴性树突形成传出性轴-树突触;含NT的树突和胞体均可接受阴性轴突的传入性突触。此外,SP阳性轴突末梢还可与NT阳性神经元的树突棘形成对称性轴-棘突触及与NT阳性轴突形成对称性轴-轴突触。实验结果表明:大鼠正中隆起内的NT能神经元接受SP能神经的支配,为下丘脑神经内分泌的突触调控提供了新的超微结构依据。     

Axovascular relationships in developing median eminence of perinatal rats with special reference to luteinizing hormone-releasing hormone projections

Topographical relationships of neurosecretory axons with the capillaries of the primary portal plexus were studied in the median eminence of rats from the 14th fetal till the 9th postnatal day by means of electron microscopy combined with morphometric analysis. Special attention was given to the light and electron microscopic immunocytochemical examination of luteinizing hormone-releasing hormone projections to the median eminence. Neurosecretory axons possessing secretory granules and clear microvesicles were first observed in the median eminence at the 14th fetal day. However, all of them were situated at a distance from the primary portal plexus. By the 20th fetal day, neurosecretory axons reached the external basal lamina of the primary portal plexus giving rise to so-called axovascular contacts. Some axons even penetrated into the perivascular space, apparently facilitating the neurohormone delivery into the hypophysial portal circulation. From that time on, both the number of the axons abutting on the external basal lamina and the entire area of axovascular contacts increased gradually. As to luteinizing hormone-releasing hormone axons, they grew into the median eminence from the 18th fetal day concentrating in older fetuses and neonates either over the primary portal plexus, or around the infundibular recess of the 3rd ventricle. After birth, the concentration and distribution of luteinizing hormone-releasing hormone axons within the median eminence became similar to those of adults. Luteinizing hormone-releasing hormone axons were found to arise from the neurons of septopreoptic area including the diagonal band of Broca. These data suggest the onset of neurohormone release in the median eminence from the 14th fetal day followed by the establishment of the hypothalamic control over the pituitary functions during the perinatal period in rats.     

Electron microscopic immunocytochemical study of somatostatin neurons in the periventricular nucleus of the rat hypothalamus with special reference to their relationships with homologous neuronal processes

The neurons containing somatostatin in the rat periventricular nucleus were studied by using a modified electron microscopic immunocytochemical technique that improves both the penetration of immunoreagents into unembedded immunostained tissues and the preservation of ultrastructural morphology. Inside perikarya and dendrites, immunostaining was not only associated with neurosecretory granules but also with ribosomes and saccules of the cis face of the Golgi apparatus. In the axonal profiles found in this region the labeling was observed both on neurosecretory granule cores and on the limiting membrane of small synaptic-like vesicles. Throughout the periventricular nucleus, both non-synaptic and synaptic relationships were shown between labeled neurons. Non-synaptic relationships mainly consisted of direct apposition of the membranes of neighboring neurons by dendrosomatic, somasomatic or dendrodendritic contacts. These labeled perikarya and dendrites were also synaptically contacted by labeled axonal endings containing numerous aggregated synaptic-like vesicles. The physiological significance of the synaptic and non-synaptic relationships between somatostatinergic neurons is discussed in terms of possible synchronization between homologous neurons of the somatostatin neuroendocrine system and control of these neurons by a central ultra-short loop feedback mechanism.     

Adrenalectomy increases sprouting in a peptidergic neurosecretory system

The neurons of the paraventricular nucleus of the hypothalamus have a substantial vasopressin-containing projection to the zona externa of the median eminence. Several parameters of neuronal function are increased in these cells following adrenalectomy; these increases can be prevented by glucocorticoid treatment. We have now used this system to study the effect of adrenal removal on the sprouting of axons in response to a unilateral lesion of the paraventricular nucleus. Results obtained from both immunocytochemical and anterograde tracing experiments indicate that the capacity of the paraventricular nucleus axons to sprout is enhanced in adrenalectomized animals. Anterograde tracing experiments indicate that the projection from the paraventricular nucleus to the zona externa is predominately ipsilateral confirming the findings of Alonso & Assenmacher¹ and that sprouting does not occur in intact animals with a unilateral paraventricular nucleus lesion. However, the zona externa of adrenalectomized animals with a unilateral paraventricular nucleus lesion is filled bilaterally with neurophysin terminals; the volume occupied by such fibers is the same as in non-lesioned, adrenalectomized animals. These findings are interpreted to indicate that sprouting can occur in this system if the animal is adrenalectomized. To estimate the time course of sprouting, animals were first adrenalectomized for 2 weeks and then a unilateral lesion placed in the paraventricular nucleus. Animals were killed 1–26 days following lesion placement and the brains processed for immunocytochemistry. Initial denervation in the zona externa due to the lesion can be detected by day 6 post-lesions while reinnervation (sprouting) is seen by day 26.The results suggest that the increase in metabolic activity induced by adrenalectomy in the vasopressin neurons under study may influence the ability of the axons of these cells to produce new terminals.     

Electron microscope immunohistochemical localization of vasopressin in the hypothalamus and neurohypophysis of the normal and brattleboro rat

Using an immunoperoxidase technique at the ultrastructural level, vasopressin was localized in the axons of both the supraoptic and paraventricular nuclei, in the internal zone of the median eminence and the posterior pituitary but not in the perikarya of the neurosecretory neurons. A complete absence of histochemical reaction was found in the hypothalamo-neurohypophyseal tract of the rat with hereditary hypothalamic diabetes insipidus (Brattleboro strain).     

Immunocytochemical localization of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase, in the hypothalamus of cattle

Immunocytochemical staining for the presence of catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine β-hydroxylase, was used to characterize the regional distribution of catecholaminergic neurons in the hypothalamus and adjacent areas of domestic cattle, Bos taurus. In steers, heifers and cows, tyrosine hydroxylase-immunoreactive perikarya was located throughout periventricular regions of the third cerebral ventricle, in both anterior and retrochiasmatic divisions of the supraoptic nucleus, suprachiasmatic nucleus, and ventral and dorsolateral regions of the paraventricular nucleus, dorsal hypothalamus, ventrolateral aspects of the arcuate nucleus, along the ventral hypothalamic surface between the median eminence and optic tract, and in the posterior hypothalamus. Immunostained perikarya ranged from small (10–20 μm, parvicellular) to large (30–50 μm, magnocellular) and were of multiple shapes: round, triangular, fusiform or multipolar, often with 2–5 processes of branched arborization. There were no dopamine-β-hydroxylase immunoreactive perikarya observed within the hypothalamus and adjacent structures. However, both tyrosine hydroxylase and dopamine-β-hydroxylase immunoreactive fibers and punctate varicosities were observed throughout regions of tyrosine hydroxylase immunoreactivity perikarya. Generally, the location and pattern of hypothalamic tyrosine hydroxylase immunoreactivity and dopamine-β-hydroxylase immunoreactive were similar to those reported for most other large brain mammalian species, however, there were several differences with commonly used small laboratory animals. These included intense tyrosine hydroxylase immunoreactivity of perikarya within the retrochiasmatic division of the supraoptic nucleus (ventral A15 region), the absence of tyrosine hydroxylase immunoreactive perikarya below the anterior commissure or within the bed nucleus of stria terminalis (absence of the dorsal A15 region), an abundance of tyrosine hydroxylase immunoreactive perikarya within the ependymal layer of the median eminence, heavy innervation of the arcuate nucleus with dopamine-β-hydroxylase immunoreactive fibers and varicosities, and the paucity of dopamine-β-hydroxylase immunoreactive throughout the median eminence.     

GABAergic innervation of corticotropin-releasing hormone (CRH)-secreting parvocellular neurons and its plasticity as demonstrated by quantitative immunoelectron microscopy

GABA has been identified as an important neurotransmitter in stress-related circuitry mediating inhibitory effects on neurosecretory neurons that comprise the central limb of the hypothalamo-pituitary-adrenocortical axis. Using combinations of pre-embedding immunostaining and postembedding immunogold methods at the ultrastructural level, direct synaptic contacts were revealed between GABA-containing terminals and neurosecretory cells that were immunoreactive for corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN). The vast majority of axo-dendritic GABA synapses was symmetric (inhibitory) type, and 46% of all synaptic boutons in the medial parvocellular subdivision of the PVN were immunoreactive to GABA. Using the disector method, an unbiased stereological method on serial ultrathin sections, the total calculated number of synaptic contacts within the medial parvocellular subdivision of the PVN was 55.4 x 10(6)/mm(3). On CRH-positive profiles 20.1 x 10(6) GABAergic synaptic boutons were detected per mm(3) in control, colchicine-treated rats. In the medial parvocellular subdivision, 79% of GABAergic boutons terminated on CRH neurons. Following adrenalectomy, which increases the synthetic and secretory activities of CRH neurons, the number of GABAergic synapses that terminate on CRH-positive profiles was increased by 55%. GABA-containing boutons appeared to be swollen, while the contact surfaces of cellular membranes between GABAergic boutons and CRH-positive profiles were shorter in adrenalectomized animals than in controls.Our data provide ultrastructural evidence for direct inhibitory GABAergic control of stress-related CRH neurons and suggest a pivotal role of GABA-containing inputs in the functional plasticity of parvocellular neurosecretory neurons seen in response to adrenalectomy.     

Differential distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat brain.

Urocortin, a novel 40 amino acid neuropeptide, is a member of the corticotropin-releasing factor family. With 45% homology to corticotropin-releasing factor, urocortin binds with similar affinity to the corticotropin-releasing factor- and corticotropin-releasing factor-2 receptors and may play a role in modulating many of the same systems as corticotropin-releasing factor. To assess whether urocortin and corticotropin-releasing factor are localized in the same regions of the brain, we compared the distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat central nervous system. Polyclonal antibodies to rat corticotropin-releasing factor and rat urocortin were generated and utilized to map the distribution of corticotropin-releasing factor- and urocortin-like immunoreactivities throughout the rat forebrain and brainstem. Characterization of the antibodies by radioimmunoassay showed no cross-reactivity with related peptides. Male Sprague-Dawley rats were treated with colchicine for 18-24 h. Following colchicine treatment, the rats were perfused with paraformaldehyde-lysine-periodate fixative and their brains removed. Serial coronal sections were taken throughout the rat brain and processed for either corticotropin-releasing factor- or urocortin-like immunoreactivity. Urocortin-like immunoreactivity shows a discrete localization within several regions including the supraoptic nucleus, the median eminence, Edinger-Westphal nucleus and the sphenoid nucleus. This is in contrast to the more abundant corticotropin-releasing factor-like immunoreactivity. Regions containing high levels of corticotropin-releasing factor immunoreactivity include the lateral septum, paraventricular nucleus of the hypothalamus, median eminence and locus coeruleus. There are a few regions that contain both urocortin-immunoreactive and corticotropin-releasing factor-immunoreactive cells, such as the supraoptic nucleus and the hippocampus. Therefore, urocortin and corticotropin-releasing factor appear to have different distribution patterns which may be indicative of their respective physiological functions.     

大鼠弓状核内神经紧张素能和P物质能阳性结构的免疫电镜双标记研究

用包埋前免疫电镜双标记技术研究大鼠下丘脑内的神经紧张素(NT)和P物质(SP)分布的超微结构。证实在弓状核内存在NT样和SP样免疫反应阳性的胞体、树突和轴突。NT样和SP样免疫反应阳性的树突和轴突均可接受免疫反应阴性结构的非对称性传入突触。SP样免疫反应阳性轴突终末除可与免疫反应阴性的树突和胞体形成对称性传出突触外, 还可与NT样免疫反应阳性胞体形成对称性轴-体突触。本研究首次直接证实大鼠弓状核内的NT能神经元接受SP能神经的支配。     

针刺“足三里”穴下丘脑血管加压素(Vp)样细胞的形态学变化——免疫组织化学法

采用免疫组化PAP技术,在电针大鼠“足三里”穴位后,观察丘脑下部Vp样神经元的变化。发现视上核与室旁核Vp样大神经元数目增多、胞体胀大,并伸出细长突起。室旁核少数串珠状神经纤维可插入第三脑室壁的细胞之间。在视交叉上核腹内侧Vp小神经元密集,而背外侧反应阳性神经元较少。视上核与室旁核间的神经元岛的细胞体积增大、轴索变长伸向视上区。该区的串珠状纤维集合成束达正中隆起的外层。以上实验结果提示,电针“足三里”可促进下丘脑有关核团Vp样物质的合成与分泌。     

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.     

Immunocytochemical localization of corticotropin-releasing factor in the brain of the turtle, Mauremys caspica

Brain sections of the turtle, Mauremys caspica were studied by means of an antiserum against rat corticotropin-releasing factor. Immunoreactive neurons were identified in telencephalic, diencephalic and mesencephalic areas such as the cortex, nucleus caudatus, nucleus accumbens, amygdala, subfornical organ, paraventricular nucleus, hypothalamic dorsolateral aggregation, nucleus of the paraventricular organ, infundibular nucleus, pretectal nucleus, periventricular grey, reticular formation and nucleus of the raphe. Many immunoreactive cells located near the ependyma were bipolar, having an apical dendrite that contacted the cerebrospinal fluid. Immunoreactive fibers were seen in these locations and in the lamina terminalis, lateral forebrain bundle, supraoptic nucleus, median eminence, neurohypophysis, tectum opticum, torus semicircularis and deep mesencephalic nucleus. Parvocellular bipolar immunoreactive neurons from the paraventricular and infundibular nuclei projected axons that joined the hypothalamo-hypophysial tract and reached the outer zone of median eminence, and the neural lobe of the hypophysis where immunoreactive fibers terminated close to intermediate lobe cells. From these results it can be concluded that, as in other vertebrates, corticotropin-releasing factor in the turtle may act as a releasing factor and, centrally, as a neurotransmitter or neuromodulator.     

Immunocytochemical localization of corticotropin-releasing factor in the brain of the turtle,Mauremys caspica

Brain sections of the turtle, Mauremys caspica were studied by means of an antiserum against rat corticotropin-releasing factor. Immunoreactive neurons were identified in telencephalic, diencephalic and mesencephalic areas such as the cortex, nucleus caudatus, nucleus accumbens, amygdala, subfornical organ, paraventricular nucleus, hypothalamic dorsolateral aggregation, nucleus of the paraventricular organ, infundibular nucleus, pretectal nucleus, periventricular grey, reticular formation and nucleus of the raphe. Many immunoreactive cells located near the ependyma were bipolar, having an apical dendrite that contacted the cerebrospinal fluid. Immunoreactive fibers were seen in these locations and in the lamina terminalis, lateral forebrain bundle, supraoptic nucleus, median eminence, neurohypophysis, tectum opticum, torus semicircularis and deep mesencephalic nucleus. Parvocellular bipolar immunoreactive neurons from the paraventricular and infundibular nuclei projected axons that joined the hypothalamo-hypophysial tract and reached the outer zone of median eminence, and the neural lobe of the hypophysis where immunoreactive fibers terminated close to intermediate lobe cells. From these results it can be concluded that, as in other vertebrates, corticotropin-releasing factor in the turtle may act as a releasing factor and, centrally, as a neurotransmitter or neuromodulator.