VIP-like and GFAP-like immunoreactivities in the chicken brain stem. I. The medulla

An immunocytochemical analysis was performed on the chicken medulla according to the PAP-DAB procedure, to study the distribution here of the neurons reacting to anti-VIP antibodies and the gliocytes reacting to anti-GFAP antibodies. Positive and negative controls were carried out in both the immunoreactions. The findings seem to indicate that more numerous VIP-immunoreactive neurons are located in the chicken than in the rat or mouse medulla, mainly in its intermediate 1/3, that VIP-immunoreactive fibres connect the medullar group of immunoreactive neurons with at least the first three cervical neuromeres, and confirm also for the chicken medulla, as already reported in other species by various researchers, the close relation of some VIP-immunoreactive neuronal processes with the small blood vessel walls, and the distribution of the GFAP-immunoreactive gliocytes.     

Double-labelling data on somatostatin-like and tyrosine-hydroxylase-like immunoreactivities in chick embryo brain stem. I. Pons and mesencephalon.

With the aim of investigating some factors and mechanisms of the chicken brain development, the same thick sections of brain stems from twelve E13-to-E21-aged chick embryos were sequentially tested with a rabbit anti-Somatostatin antiserum, using a PAP-DAB technique, and with anti-tyrosine hydroxylase (-TH) monoclonal antibodies, using an indirect immuno-fluorescence technique. As regards the pons and mesencephalon, the following main results were obtained. At E21 almost the same distribution of the TH-like immunoreactivity (ir) as at E13 was observed. Neuroblasts in a central, relatively wide region of mesencephalic tegmentum and in the central portion of the pons showed TH-like ir. A co-localization of the 2 immunoreactivities was detected only at E18, within some neuroblasts of the mesencephalic and pontine regions with TH-like ir. It is possible that this transitory co-localization plays a role in the development of the pons and mesencephalon of this species.     

Vasoactive intestinal peptide-immunoreactive cerebrospinal fluid-contacting neurons in the reptilian lateral septum nucleus accumbens

By means of immunocytochemical demonstration of vasoactive intestinal peptide (VIP) an accumulation of cerebrospinal fluid (CSF)-contacting neurons was found in a circumscribed region of the nucleus accumbens/lateral septum of eleven reptilian (chelonian, lacertilian, ophidian, crocodilian) species. Basal processes of these cells contribute to a subependymal plexus whose density displays considerable interspecific variation. VIP-immunoreactive nerve fibers occur also in the lateral septum and the nucleus accumbens where they encompass immunonegative cells in a basket-like pattern. The CSF-contacting neurons are surrounded by columnar ependymocytes frequently arranged in a pseudostratified manner. These specialized arrays of ependymal cells, however, occupy a more extended area than the VIP-immunoreactive CSF-contacting neurons and can be traced from the rostro-ventral pole of the lateral ventricle to the interventricular foramen. These observations suggest the existence of a telencephalic site of CSF-contacting neurons which may be more widespread than hitherto thought and which may participate in a circumventricular system of the lateral ventricle. Previous studies mainly performed with birds indicate that the VIP-immunoreactive CSF-contacting neurons of the nucleus accumbens might form a part of the encephalic (extraretinal and extrapineal) photoreceptor. However, further experiments are required to test this supposition since the VIP-immunoreactive neurons of the nucleus accumbens remained unlabeled by antibodies against bovine rodopsin and chicken cone-opsin in all eleven species analysed in this investigation.     

Local and transient expression of E-cadherin involved in mouse embryonic brain morphogenesis.

We found that E-cadherin (uvomorulin) is transiently expressed in restricted regions of the metencephalon, mesencephalon and diencephalon of mouse embryonic brain. This expression first occurred in parts of the mesencephalon and diencephalon at around E9.5, and subsequently extended to the primordia of cerebellum, the dorsal midline of mesencephalon and some other regions of the embryonic brain. These E-cadherin expressions ceased by E15 except at the dorsal midline. Immunohistological analyses showed that E-cadherin-positive cells are radially arranged in the neural tube and the E-cadherin-positive regions are sharply demarcated from E-cadherin-negative regions. Axons extending from some of the E-cadherin-positive regions also expressed this molecule. When embryonic brains were dissociated into single cells and cultured as monolayers, E-cadherin-positive cells formed clusters that were segregated from E-cadherin-negative cells. E9.5 brain fragments containing metencephalon and mesencephalon were isolated, explanted on Nucleopore filters and cultured in the absence or presence of antibodies to E-cadherin. This antibody treatment removed most of the E-cadherin molecules from the explants and consequently affected their growth pattern. To analyze cellular events induced by the antibody treatment, we stained these explants with an antiserum to En whose distribution was found to overlap in part with that of E-cadherin and found that the pattern of En staining was altered by the anti-E-cadherin antibody treatment. These results suggest that the local and transient expression of E-cadherin in embryonic brain is involved in regional pattern formation in this organ.     

Neonatal development of ACTH-immunoreactive neurons in the rat

Using immunohistochemical techniques, the distribution of ACTH 25-39 immunoreactive perikarya and fibers was studied in the newborn rat. Perikarya and fibers were demonstrated on day 1 after birth in the diencephalon, mesencephalon, pons and medulla. However, the amount of material increased rapidly from the first to third day of postnatal development.     

Immunocytochemical localization of methionine enkephalin: preliminary observations.

Antiserum directed against methionine enkephalin (metenkephalin) was used to determine its anatomical distribution in rat brain. Cross reactivity of that antiserum was not detectable against leucine enkephalin (leu-enkephalin), β-lipotropin (β-LPH), β-endorphin or assorted peptide fragments of met-enkephalin; alpha-endorphin was 370 times less active than met-enkephalin. The localization of met-enkephalin was carried out in the presence of excess leu-enkephalin and yet could be blocked with equal amounts of met-enkephalin. Met-enkephalin was detected in several structures in the spinal cord, medulla, pons, mesencephalon, diencephalon and telencephalon. No met-enkephalin was detected in cerebellum or cerebral cortex.     

Distribution of reticulospinal neurons in the chicken by retrograde transport of WGA-HRP

To determine the distribution of reticulospinal (RS) neurons in the chicken, WGA-HRP was injected into the cervical or lumbosacral enlargement either unilaterally or bilaterally. The brainstem reticular nuclei sent largely descending fibers to both the spinal enlargements. The mesencephalon (medial and lateral mesencephalic reticular formation) and the rostral pons (nucleus reticularis [n.r.] pontis oralis) project mainly to the cervical enlargement. RS neurons were mainly distributed from the pontomedullary junction to the rostral medulla including n. r. pontis caudalis and pars gigantocellularis, n. r. gigantocellularis, n. r. parvocellularis, n. r. paragigantocellularis, and n. r. subtrigeminalis. It is suggested that the majority of these neurons send axons at least as far as the lumbosacral enlargement. In the lower medulla, RS neurons were distributed in the dorsal and ventral parts of the central nucleus of the medulla.     

Immunohistochemical analysis of human neuronectin expression in normal, reactive, and neoplastic tissues

Neuronectin (NEC1) is a human extracellular matrix protein of central nervous system (CNS) parenchyma found throughout the white matter of rostral brain segments (telencephalon, diencephalon, some areas of mesencephalon), but not in rostral CNS gray matter, most areas of mesencephalon, pons, cerebellum, medulla, spinal cord, or peripheral nerves. The present immunohistochemical study, using two monoclonal antibodies to distinct epitopes on the NEC1 molecule, examined NEC1 expression in normal non-neural tissues, malignant tumors of diverse histological types, non-malignant skin lesions, and dermal incision wounds. We show that (a) NEC1 is expressed in normal fetal precartilage blastemas and fetal and adult vascular and visceral smooth muscle, but not in most loose connective tissues and skeletal or cardiac muscle; (b) NEC1 is found along epithelial-mesenchymal junctions, with marked differences in prevalence and histological patterns in different organs; and (c) mesenchymal activation associated with wound healing, actinic keratosis, psoriasis, and neoplasia leads to strong induction of NEC1 expression. Parallel studies with cultured human cells suggest that region-specific NEC1 expression in normal developing tissues and localized induction in wound healing and disparate diseases is under the control of extrinsic signals provided by regulatory polypeptides.     

Development of serotonergic neurons in the brain of the mackerel, Scomber scombrus. An immunohistochemical study

The ontogenetic development of serotonin immunoreactivity (5-HTir) neurons was investigated by immunocytochemistry in the brain of the mackerel, from fertilization to the complete resorption of the yolk sac (201 h). 5-HTir appears first in cell bodies in the diencephalon, on both sides of the third ventricle. At hatching time 5-HTir perikarya are also present in the mesencephalon and the inferior raphe but in small numbers. During larval stages, immunoreactivity appears consecutively in the area thalamo piaetectalis, the nuclei raphe pallidus and obscurus, the nuclei tegmenti dorsalis lateralis, recessus posterioris, lateralis hypothalami and paragigantocellularis lateralis. These results, together with previous findings, suggest a common pattern of distribution of the different 5-HTir neuronal populations in the brain of a teleost during ontogeny but a different sequence of appearance of initial 5-HTir in these populations between species.     

Brain morphology and immunohistochemical localization of the gonadotropin-releasing hormone in the bluefin tuna, Thunnus thynnus

The present study was focused on the morphology of the diencephalic nuclei (likely involved in reproductive functions) as well as on the distribution of GnRH (gonadotropin-releasing hormone) in the rhinencephalon, telencephalon and the diencephalon of the brain of bluefin tuna (Thunnus thynnus) by means of immunohistochemistry. Bluefin tuna has an encephalization quotient (QE) similar to that of other large pelagic fish. Its brain exhibits well-developed optic tecta and corpus cerebelli. The diencephalic neuron cell bodies involved in reproductive functions are grouped in two main nuclei: the nucleus preopticus-periventricularis and the nucleus lateralis tuberis. The nucleus preopticus-periventricularis consists of the nucleus periventricularis and the nucleus preopticus consisting of a few sparse multipolar neurons in the rostral part and numerous cells closely packed and arranged in several layers in its aboral part. The nucleus lateralis tuberis is located in the ventral-lateral area of the diencephalon and is made up of a number of large multipolar neurones. Four different polyclonal primary antibodies against salmon (s)GnRH, chicken (c)GnRH-II (cGnRH-II 675, cGnRH-II 6) and sea bream (sb)GnRH were employed in the immunohistochemical experiments. No immunoreactive structures were found with anti sbGnRH serum. sGnRH and cGnRH-II antisera revealed immunoreactivity in the perikarya of the olfactory bulbs, preopticus-periventricular nucleus, oculomotor nucleus and midbrain tegmentum. The nucleus lateralis tuberis showed immunostaining only with anti-sGnRH serum. Nerve fibres immunoreactive to cGnRH and sGnRH sera were found in the olfactory bulbs, olfactory nerve and neurohypophysis. The significance of the distribution of the GnRH-immunoreactive neuronal structures is discussed.     

Pluripotentiality of the 2-day-old avian germinative neuroepithelium

In a previous study using chick/quail chimeric embryos with homotopic transplants (Martinez & Alvarado-Mallart, 1989b), we have delimited in the 2-day-old avian embryo the areas of the neural tube giving rise to optic tectum and mesencephalic grissea as well as to isthmic grissea and cerebellum: respectively, "mesencephalic" and "metencephalic" alar plates. To investigate the determination or the competence of these areas, portions of these germinative neuroepithelia from a quail embryo were transplanted in substitution for other areas of the chick neural tube. The analysis of the chimeric brains was done by comparing alternating transverse sections stained for cytoarchitecture and with two different techniques to recognize transplanted versus host cells: either the Feulgen and Rossenbeck DNA histochemical reaction and/or immunohistochemical methods with a monoclonal antibody recognizing quail but not chick cells. The eventual visual innervation of the quail graft was analyzed in many cases by injecting anterograde axonal tracers in the eye contralateral to the graft. The results are as follows: (1) caudal metencephalon transferred to mesencephalon maintained in all cases its presumptive cerebellar phenotype, whereas (2) rostral metencephalon transferred to mesencephalon changed its fate to a tectal phenotype but maintained its cerebellar fate when transferred to diencephalon; (3) caudal mesencephalon maintained its tectal fate in 65% of the cases when transferred to diencephalon, whereas (4) rostral mesencephalon transferred to a cerebellar domain changed its fate and became influenced by the surrounding structures in all cases, but only in 85% of the cases when it was transplanted to diencephalon; (5) the in situ host diencephalon, isolated from its normal environment by a mesencephalic graft, is competent to change its fate and express a mesencephalic phenotype. These results demonstrate that at least some regions of the germinative neuroepithelium from either metencephalon, mesencephalon, and diencephalon are still pluripotent in the 2-day-old avian embryo and that their fate seems to be under the influence of the surrounding structures. Rostral mesencephalon and rostral metencephalon have been more easily influenced by environmental factors than their caudal counterparts, suggesting that regions providing instructive positional factors exist within the 2-day-old germinative neuroepithelium. These regions might play an important role in the determination of the various segments of the neural tube.     

Influence of pinealectomy on levels of PACAP and cAMP in the chicken brain

One of the recently found functions of pituitary adenylate cyclase activating polypeptide (PACAP) is the modulation of circadian rhythms. Widespread distribution of PACAP-containing neurons and receptors has been shown in the chicken. Recently, we have demonstrated that PACAP levels oscillate in a circadian manner in the chicken brain. Daily variation in PACAP levels might be influenced by several regulatory mechanisms. Among the structures that may regulate PACAP levels, one candidate is the pineal gland. Therefore, in the present study, we investigated the effect of pinealectomy on the levels of PACAP in the chicken brain. Animals were kept under 12:12-h light-dark schedule. Pinealectomy was performed at 3 weeks of age; sham-operated animals were used as controls. The animals were sacrificed at 15 and 24 h 1 week after pinealectomy. The brainstem and diencephalon were removed, and tissue samples were processed for PACAP and cAMP radioimmunoassay (RIA).PACAP and cAMP levels showed nighttime elevations in both the sham-operated and pinealectomized animals, except for the PACAP content in the diencephalon of pinealectomized chicken. PACAP levels of pinealectomized animals were significantly higher in the diencephalon and brainstem as compared to the control animals at both time-points. Levels of cAMP correlated well with levels of PACAP. The present results provide evidence that the pineal gland has an inhibitory impact on PACAP-neurons in the chicken brainstem and diencephalon.     

Ependymal and neuronal specializations in the lateral ventricle of the Pekin duck,Anas platyrhynchos

Summary The lateral ventricles of the Pekin duck, Anas platyrhynchos, display characteristic ependymal and hypendymal specializations. Adjacent to the nucleus accumbens and the basal pole of the lateral septum the ventricular surface shows a highly folded pattern either with protrusions into the ventricular lumen or deep invaginations into the brain tissue. These medial and basal ependymal folds are found exclusively in a circumscribed region extending over a range of 600 m in the rostrocaudal direction. Ependymal folds occurring in the lateral wall of the ventricles were traced up to the level of the interventricular foramen. Numerous capillaries are observed in the subependymal layer of these folds.By means of immunocytochemistry with antibodies against chicken vasoactive intestinal polypeptide (VIP) an aggregation of classical cerebrospinal fluid-contacting neurons is shown in the region of the nucleus accumbens and the lateral septum. These neurons are closely related to the ependymal folds. Additional VIP-immunoreactive neurons are scattered in deeper layers of the lateral septum and the nucleus accumbens. The latter are richly innervated by VIP-immunoreactive nerve fibers.The results of the present study are discussed with particular reference to the hypothesis of Kuenzel and van Tienhoven (1982) that ependymal specializations demonstrated in the lateral ventricles of the domestic fowl might represent a new circumventricular organ (lateral septal organ).The authors are greatly indebted to Professor A. Oksche for stimulating discussionsSupported by the Deutsche Forschungsgemeinschaft (Ko 758/2-2; 2-3) and the P. Carl Petersen Foundation     

Localization of the peptide Hydra morphogen in the pontine neurons of the human brain

Localization of neurons with peptide "Hydra morphogen" in the human pons has been investigated by means of immunocytochemical methods. Single neurons are found in the cranial part of the pons, in its grey substance, that immediately adjoins the lateral walls of the IV ventricles and in the nucleus tegmenti dorsalis. These neurons are round and polygonal with body diameter 22-25 mcm, they contain light brown precipitate of various density. The data obtained corresponds to general evolutionary regularities on distribution of biologically active peptides in the nervous system of vertebrate and invertebrate animals.     

Natriuretic peptide binding sites in the brain of the Atlantic hagfish, Myxine glutinosa.

We have previously used immunohistochemistry to show that the brain of the hagfish, Myxine glutinosa, contains a rich distribution of natriuretic peptide-immunoreactive elements with the densest distribution occurring in the telencephalon and the diencephalon. In this study, the distribution of (125)I-rat ANP and (125)I-porcine CNP binding sites was determined in the brain of M. glutinosa. The binding pattern of (125)I-rat ANP and (125)I-porcine CNP showed similarities; however, some differences were observed in the olfactory bulb and the caudal brain regions. Specific (125)I-rat ANP and (125)I-porcine CNP binding was observed in the olfactory bulb, outer layers of the pallium, and in regions of the diencephalon. Very little specific binding was observed in the habenula and the primordium hippocampi. In the diencephalon, a distinct zone of specific (125)I-rANP binding separated a region of moderate binding in the lateral regions of the diencephalon from the thalamic and hypothalamic nuclei. Moderate levels of specific (125)I-rANP binding were observed in the mesencephalon and medulla oblongata; little or no (125)I-porcine CNP binding was observed in these regions. The data, in combination with previous immunohistochemical studies, show that the natriuretic peptide system of the hagfish brain is well-developed and suggest that natriuretic peptides have a long evolutionary history as neurotransmitters and/or neuromodulators in the vertebrate brain. J. Exp. Zool. 284:407-413, 1999.     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

The effect of unilateral medial forebrain bundle lesion on 5-hydroxytryptamine in the pigeon brain

Summary The amount of 5-HT was spectrofluorimetrically determined in the brains of normal pigeons and pigeons that had been operated in the left diencephalon. The highest concentration of 5-HT was encountered in a small medial and anterior part of mesencephalon, where both tryptaminergic nerve cell bodies and preterminals occurred. Considerably lower concentrations were found in the diencephalon after removal of the organon vasculosum, which is very rich in both 5-HT and catecholamines. The lowest concentration was recorded in corpora striata, where 5-HT occurs in the form of mostly sub-light-microscopical nerve terminals. After a transversal section of the medial forebrain bundle it could be concluded, on both histochemical and biochemical grounds, that 5-HT is formed and transported in special neurons with their cell bodies in the mesencephalon and nerve terminals in both diencephalon and corpora striata. The most apparent evidence for this was the clear reduction of 5-HT level in the corpora striata of the operated side and the apparent accumulation of fluorescent material proximal to the lesion. An interesting change was also the increase in 5-HT concentration in mesencephalon, diencephalon and corpora striata of the unoperated half of the brain. The opinion was put forward that such an increase could imply some kind of compensation for the reduced transmitter content in the contralateral hemi-brain.