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 (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.     

Ganglionic character of mesencephalic trigeminal neurons in the turtle Mauremys caspica

A study of the morphology of the mesencephalic trigeminal neurons and their arrangement in the optic tectum has been made in the turtle Mauremys caspica employing the Cajal reduced silver method. The ganglionic features of its neurons suggest that the mesencephalic trigeminal nucleus (MTN) is a diffuse sensory ganglion located in the central nervous system. The different distribution of two cell populations, probably as a result of a distinct embryonic origin of their elements, and the atypical pattern of the ependyma lying underneath the MTN are discussed.     

Ultrastructural peculiarities of the supraoptic nucleus of Mauremys caspica (turtle): its evolutionary interest in reptiles

The hypothalamic Supraoptic Nucleus (SON) supposes an evolutionary achievement in the reptiles, zoological group in which this nucleus is first differentiated. Its scarce development in the chelonian, subject of the present study, is related to their primitive brain pattern. The peculiar location and the topographical configuration of the SON in the hypothalamus of the turtle Mauremys caspica allows to evidence morphologically the successive steps in the evolutionary process from lower to higher vertebrates. The marked neurosecretory character of the chelonian SON is the unequivocal proof of its philogenetic origin derived from the magnocellular preoptic nucleus of lower vertebrates.     

Corticotropin releasing factor: Immunocytochemical localization in rat brain

Using antiserum generated against the synthetic CRF_1–41 we have immunocytochemically localized perikarya and processes in rat brain. Areas observed to have particularly dense accumulation of CRF-ir cells were in the extra-hypothalamic areas of nucleus accumbens septi, nucleus of the stria terminalis, the medial preoptic region, and the central amygdaloid nucleus. Within the hypothalamus cell bodies were scattered throughout the anterior hypothalamic region and densely packed in the paraventricular nucleus. Fibers appear most dense in the lateral septal region and throughout the external layer of the median eminence.     

Changes in the thymus and spleen of the turtle Mauremys caspica after testosterone injection: a morphometric study.

To confirm a possible role of sex hormones in governing the seasonal variations affecting the reptilian lymphoid organs, a morphometric analysis was carried out on the thymus and spleen of turtles, Mauremys caspica, intraperitoneally injected with a single dose of testosterone propionate (TP) at the third week of June when physiological levels of testosterone are low. At 4 and 6 weeks, control turtles show an apparent lymphocyte mobilization both from thymus and spleen with a decrease in the percentage of thymic cortex, numbers of cortical lymphocytes, and mitotic index, but increased numbers of medullary lymphocytes. In the thymic cortex of treated turtles, there is a decrease in the same parameters but they occur in the first 2 weeks, whereas the medullary lymphocytes also undergo reduction at 4 and 6 weeks. In addition, the number of reticuloepithelial cells per area unit decreases at 2 weeks in the thymic cortex of treated turtles. These results are discussed from the point of view of a biphasic effect of testosterone on turtle lymphoid organs: In the first weeks, the changes observed could be attributed to the high levels of testosterone; after 4 and 6 weeks, variations are dependent both on long-term effects of testosterone and on those induced by the high values of corticosterone occurring in summer.     

Immunocytochemical localization of angiotensinogen in the rat brain

The distribution of angiotensinogen-like immunoreactivity in the rat brain was investigated using specific antisera against pure rat plasma angiotensinogen in conjunction with the sensitive streptavidin-biotin peroxidase method. Angiotensinogen antisera were shown by radioimmunoassay and Western blotting to recognize angiotensinogen from both rat plasma and cerebrospinal fluid, and to cross-react with des-AI-angiotensinogen (100%) but not with angiotensin I and II, tetradecapeptide, luteinizing hormone-releasing hormone, rat albumin and angiotensinogen from eight other species. Angiotensinogen-like immunoreactivity was detected throughout the rat brain in both neuroglia and neurons. The highest concentration of neuroglial angiotensinogen-like immunoreactivity was in the hypothalamus and preoptic areas, with moderate to heavy concentrations in the mesencephalon and myelencephalon. The cerebellum demonstrated neuroglial staining in the granular layer and fibre tracts. Very little neuroglial staining was noted in the cerebral cortex or olfactory bulbs. Neuronal immunostaining was observed throughout the globus pallidus and the caudate putamen, in various parts of the thalamus and the supraoptic nucleus of the hypothalamus. In the midbrain moderate immunostaining was observed in periaquaductal central gray, the deep mesencephalic nucleus, the inferior colliculus and in scattered cells in the anterior mesencephalon. In the medulla, neuronal staining was localized to the vestibular nuclei and to other cell bodies mainly in the dorsolateral regions. In the cerebellum, staining was noted mainly in the deeper cerebellar nuclei and in the Purkinje cells. Immunostaining in the cerebral cortex was localized to the cingulate cortex and the primary olfactory cortex. Light staining was present in the endopiriform cortex and in scattered neurons adjacent to the external capsule. In the olfactory bulbs light neuronal staining was mainly associated with the mitral cell layer. The widespread distribution of angiotensinogen-like immunoreactivity supports the view that it is synthesized in the central nervous system and forms part of a brain renin-angiotensin system. In addition, its presence at sites other than those normally associated with the control of blood pressure and fluid and electrolyte homeostasis suggests that its involvement may not be limited to these regulatory functions.     

Seasonal variations in the immune response of the tortoise Mauremys caspica.

The primary and secondary immune responses to sheep erythrocytes (SRBC) have been characterized in the tortoise Mauremys caspica in terms of circulating antibodies and PFC response in two different seasons: summer and autumn. Primary immunization is followed by the appearance of both 2ME-sensitive antibodies and splenic PFCs in autumn but not in summer. During the secondary response, 2ME-resistant antibodies were found in both seasons, but the number of PFCs was significantly reduced during summer. The results are discussed from the perspective of the role played by glucocorticosteroids in the seasonal variations affecting reptilian immunoreactivity.     

Immunocytochemical localization of the sodium, potassium activated ATPase in knifefish brain

Summary Results presented in this paper demonstrate the feasibility of using immunocytochemical methods to localize the (Na⁺ + K⁺)-ATPase, and its subunits, in the C.N.S. We have shown that in the Black Ghost knifefish,Sternarchus albifrons, the enzyme is located on the plasma membrane of the somata and dendrites of neurons and on the somata and cellular processes of glia. In myelinated axons the enzyme is restricted in localization to those portions of the axolemma not covered by the myelin sheath. The capacity of cell plasma membranes to restrict mobility of functionally important proteins should be considered in models of membrane structure in which lateral mobility of membrane components is considered a major characteristic.     

Immunocytochemical localization of serotonin in intracellularly analyzed and dye-injected ganglion cells of the turtle retina

Epileptiform bursts of population spikes were evoked in the CA1 region of slices of the hippocampus in which the CA3 region had been previously lesioned with kainic acid. D-2-amino-5-phosphonovalerate (D-APV), a specific N-methyl-D-aspartate (NMDA) antagonist, would markedly reduce the number of spikes in the burst but had no effects on the primary population spike or the amplitude of the field excitatory postsynaptic potential (EPSP). In unlesioned control slices only a single population spike was evoked and D-APV had no effect on this response or the field EPSP. Multiple population spike bursts evoked following application of bicuculline to control slices were much less attenuated by D-APV. The results suggest that activation of NMDA receptors contributes to the production of epileptiform activity in the kainic acid-lesioned hippocampus.     

Immunocytochemical localization of D-amino acid oxidase in rat brain

d-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.     

Immunocytochemical localization of dopamine-beta-hydroxylase in neurons of the human brain stem

The distribution of catecholaminergic cells in the human pons and medulla was illustrated by immunocytochemistry using a polyclonal antibody directed against the catecholamine synthetic enzyme, dopamine-beta-hydroxylase. The antibody specifically recognizes dopamine-beta-hydroxylase in putative adrenergic and noradrenergic neurons. The adrenergic and noradrenergic neurons are found in the brain stem from caudal levels of the medulla through the caudalmost levels of the midbrain. Large numbers of dopamine-beta-hydroxylase-positive cells were observed in cell groups of the medulla. Additionally, dopamine-beta-hydroxylase-positive cells were concentrated in the nucleus locus coeruleus and the nucleus subcoeruleus in the pons. These studies confirm that immunocytochemical localization of dopamine-beta-hydroxylase can be used to identify noradrenergic and adrenergic neurons and their terminal varicosities in the pons and medulla in routine autopsy material. These studies have also illustrated that the distribution of dopamine-beta-hydroxylase in the adult human brain is comparable to the distribution in other species.     

Immunocytochemical ultrastructural study of hypothalamic neurons containing corticotropin-releasing factor in normal and adrenalectomized rats

The neurons of the rat hypothalamus which secrete corticotropin-releasing factor were studied by using a pre-embedding immunocytochemical staining technique that improves both the penetration of immunoreagents within the tissue and the preservation of the ultrastructural morphology of labeled structures. Comparison was made between the subcellular location of corticotropin-releasing factor-41 in perikarya of the paraventricular nucleus and axons of the median eminence, both in intact and adrenalectomized animals either untreated or 24 h after the intracerebral injection of colchicine. Morphometric analysis of the numerical density and of the diameter of corticotropin-releasing factor immunoreactive neurosecretory granules in axons of the median eminence of rats not treated with colchicine, indicated that the main modifications induced by adrenalectomy concerned (1) the differential repartition of labeled granules within the preterminal and terminal axonal portions of the median eminence, and (2) the enlargement of the diameter of labeled granules contained in these axons (from 98 nm to 165 nm). In the hypothalamus of intact and adrenalectomized rats, colchicine treatment increased the number of corticotropin-releasing factor-immunoreactive granules in the neuronal perikarya and reduced their number in the axons, but both these variations were much more marked in adrenalectomized rats. Although the corticotropin-releasing factor immunoreactive granules that accumulated in the perikarya after colchicine treatment were slightly smaller than those in the corresponding axons, the diameter of perikaryal-labeled granules was larger in adrenalectomized than in intact animals (129 nm vs 93 nm). These findings fit the idea that adrenalectomy markedly stimulates both the synthesis and axonal excretion of secretory granules in the hypothalamic neurons secreting corticotropin-releasing factor. They also indicate that suppression of circulating corticosteroids induces qualitative modifications in these neurons leading to the visualization of larger neurosecretory granules, which may reflect differential synthesis and granular packing of synergistic peptides other than corticotropin-releasing factor and/or changes in the process of intragranular maturation of hormonal material.     

Immunocytochemical localization of endopeptidase-24.11 in the nucleus tractus solitarius of the rat brain

Recently, it has been hypothesized that the N-terminal portion of substance P (SP), SP(1-7), which results from the action of endopeptidase 24.11 (EC3.4.24.11), could be involved in mediating the depressor effects of baroreceptor afferent activation via its action on cells in the nucleus tractus solitarius (NTS). In this study, the binding of a monoclonal antibody to endopeptidase 24.11 was examined immunohistochemically at the level of the caudal medulla of the rat brain. By light microscopy, intense immunoreactivity was seen in the NTS, in fibers bordering the area postrema, and in the area postrema itself. After electron microscopy, endopeptidase 24.11-like immunoreactivity was seen to be associated with the cytoskeleton and plasma membrane in axons, dendrites and glial processes. Antigen was also associated with synaptic vesicles and plasma membranes in presynaptic terminals forming mainly axo-dendritic synapses typical of vagal afferent terminals involved in the baroreceptor reflex. Thus, endopeptidase 24.11 appears to be localized at sites where it could effectively process SP prior to its binding to postsynaptic receptors.     

Immunocytochemical detection of corticotropin-releasing factor: multiple cross-reactions of a widely used carboxy-terminally directed corticotropin-releasing factor antiserum (code rC70) in rat hypothalamus

Forty-one-residue corticotropin-releasing factor is a physiologically significant mediator of the hypothalamic control of corticotropin secretion by the anterior pituitary gland. This releasing hormone is produced by parvicellular neurons in the hypothalamic paraventricular nucleus that project to the external zone of the median eminence. Recent immunocytochemical evidence based on work with a rabbit antiserum against rat corticotropin-releasing factor (code rC70) suggests that about half of the parvicellular corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus synthesize vasopressin, another potent corticotropin secretagogue, while the rest of the cells do not. If this is indeed the case, the neurohumoral control of corticotropin release may be mediated via distinct hypothalamic effector pathways utilizing releasing hormone cocktails of varying composition. In the present study we have examined the specificity of various antisera against rat corticotropin-releasing factor in immunocytochemical staining. Male Wistar rats pretreated with colchicine were used throughout. The brain was fixed by perfusion with a Zamboni type fixative solution. Vibratome sections of the hypothalamus were immunostained with three different primary antisera (codes rC70, rCRF-3, oCRF-N) using the peroxidase-antiperoxidase or avidin-biotin complex methods. All three antisera stained cell groups previously described to be immunopositive for corticotropin-releasing factor. Most notably, however, rC70 labelled a significant number of additional cells, most readily identified in the arcuate and suprachiasmatic nuclei, as well as in the dorsolateral hypothalamic area caudal to the paraventricular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of a granuliberin-like peptide in Rana pipiens brain

The histamine-releasing peptide “granuliberin”, originally isolated from the skin of Rana rugosa, was localized by immunofluorescence within nerve cell bodies and fibers in the brain of Rana pipiens. The granuliberin-positive neurons were characterized ultrastructurally by electron microscopic observation of ultrathin sections stained either with immunoperoxidase or with conventional stains. Granuliberin-positive nerve cell bodies were seen throughout the hypothalamus, from the suprachiasmatic area rostrally to the full length of the periinfundibular grey matter caudally. Similarly positive nerve fibers were localized in the hypothalamus radiating upwards to the optic vesicles in the midbrain, extending through the preoptic area into the subpallium in the forebrain, and throughout the white matter surrounding the floor and lateral walls of the fourth ventricle in the brainstem. The granuliberin-positive nerve cells showed the presence of variable numbers of small cytoplasmic neurosecretory granules. Possessing an electron dense elongated core, and measuring 250–350 nm in their largest diameter. The functional significance of a granuliberin-like peptide in neurons of the frog brain is not known.     

Immunocytochemical localization of acetylcholine receptors in locust brain using auto-anti-idiotypic acetylcholine antibodies

Auto-anti-idiotypic antibodies have been detected in antisera of rabbits immunized with an acetylcholine (ACh) conjugate. These antibodies were found to bind to ACh receptor (ACh-R) purified from different species membranes. They competed with the ACh-R antagonist alpha-bungarotoxin and some agonists such as ACh conjugate and ACh itself. They did not recognize acetylcholinesterase. Their characterization 'in vitro' suggested their employment as an immunohistological marker for ACh-R. In the locust brain, specific immunoreactivity was found in neuropils of the protocerebrum, the optic lobes, the deutocerebrum and the tritocerebrum.     

Immunohistochemical localization of corticotropin-releasing factor (CRF)-like immunoreactivity in the hypothalamus of the newt, Triturus cristatus

The distribution of corticotropin-releasing factor (CRF) immunoreactivity was investigated in the hypothalamus and preoptic area of the newt by single and double immunocytochemical procedures. CRF immunopositive cell bodies were seen in the preoptic area (from the anterior wall of the preoptic recess to the dorsal parts of the preoptic nucleus) and in the tuberal portions of the posterior hypothalamus. Abundant nerve fibres are seen in the outer zone of the median eminence, while the pars nervosa lacks CRF-immunoreactivity. CRF immunoreactive material is seemingly separated from neurophysins- and somatostatin immunoreactive cell bodies and fibres.