Avian Central Nervous System Research Articles

Biosynthesis of progesterone derived neurosteroids by developing avian CNS : in vitro effects on the gabaa receptor complex

It has been demonstrated in different vertebrate species that the GABAA receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [[14]C]progesterone metabolites as well as the characterization of their in vitro effects on the GABAA receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5β-pregnanedione, 5β-pregan-3β-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5β-pregnanedione was converted to 5β-pregnan-3β-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [[3]H]flunitrazepam binding by 5β-pregnan-3β-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5β-pregnan-3β-ol-20-one EC50 values were 0.195±0.049, 0.101±0.017, 0.147±0.009, and 0.569±0.114 μM, and Emax were 22.37±1.57, 23.67±4.02, 29.01±1.08, and 15.11±2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively.In conclusion, the biosynthesis of 5β-pregnan-3β-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABAA receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.

Neurosteroid modulation of GABA binding sites in developing avian central nervous system

Our aim was to examine the effect of the potent neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-P) on [ 3H]-GABA binding to its receptor sites in the chick optic lobe. Binding was performed on synaptic membranes isolated at different stages of development and two different membrane preparation procedures were applied to expose high and low affinity GABA binding sites. The addition of 3α,5α-P was shown to increase [ 3H]-GABA binding in an age- and concentration-dependent manner. Maximal stimulation for low affinity GABA binding sites was observed at hatching (130% enhancement), in fresh-washed as well as in frozen membranes. Saturation analysis performed on both membrane types disclosed that 3α,5α-P increases the affinity of low affinity GABA binding sites without altering their maximal binding capacity. On the other hand, the augmenting effect at high affinity sites, displayed only in frozen membranes, was roughly 50% for all developmental stages. However, their saturation binding parameters remained unaltered in the presence of the steroid, suggesting that stimulation of such sites seems due to interference exerted by the low affinity site population. Findings indicate that 3α,5α-P acts as an allosteric modulator only for low affinity GABA binding sites, displaying an age-dependent profile probably related to plastic events during visual pathway development.

Chick cerebellar purkinje cells express ω-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels

Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds 7 and later in mammals. 9 A specific calcium channel, the P-type channel, 10 has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. 13, 25 P-type channels play an essential role in synaptic transmission of mammalian cerebellum. 15, 16, 20, 22 It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex 1, 12 and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. 21 We found that ω-conotoxin GVIA ω-CgTx GVIA):, a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

Comparative modulation by 3 alpha,5 alpha and 3 beta,5 beta neurosteroids of GABA binding sites during avian central nervous system development.

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABAA receptor complex. The presence of a 3 alpha-hydroxyl group and a 5 alpha-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [3H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3 alpha-hydroxy,5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 beta-hydroxy,5 beta-pregnan-20-one (3 beta,5 beta-P). Both steroids were found able to enhance [3H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3 alpha,5 alpha-P had greater potency (EC50 0.22 microM) and enhancing effect (Emax: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABAA receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3 alpha,5 alpha-P by its 3 beta,5 beta isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5 alpha-reduced progesterone metabolites present since early development, while 3 alpha,5 alpha-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5 beta-steroids in early avian development, we propose that 3 beta,5 beta-P, instead of the classical potent 3 alpha,5 alpha-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.

Expression and distribution of the dentatorubral-pallidoluysian atrophy gene product (atrophin-1/drplap) in neuronal and non-neuronal tissues

Utilizing an affinity-purified antiserum directed against the carboxyl terminal region of atrophin-1/drplap (residues 1170–1185), we have examined the expression and distribution of the protein in a variety of neuronal and non-neuronal tissues. Immunohistochemical analyses of gelatin-embedded sections of monkey brain demonstrated a wide-spread distribution of the protein throughout the cerebrum and cerebellum. Labeling was primarily cytoplasmic within neuronal cell bodies and dendrites. Prominently staining regions included layers II, III, V, and VI of cerebral cortex, CA1-4 of the hippocampus, caudate nucleus, putamen, globus pallidus, amygdala, thalamus, red nucleus, pons, Purkinje cells, and deep cerebellar nuclei. Immunoblot analysis of extracts of frontal cortex from a wide variety of mammalian species (human, monkey, rabbit, rat, mouse, and bovine) detected a 190 kDa band in each extract. No cross-reactive material of similar molecular weight was detected in an extract of avian (chicken) central nervous system (CNS) tissue. Furthermore, in the rat, expression of the protein was predominantly neuronal in origin as immunoblot analyses of non-neuronal tissue extracts detected little or no 190 kDa protein. Collectively these investigations suggest a ubiquitous expression of atrophin-1/drplap in mammalian CNS tissue and provide initial immunochemical data for the study of the neuroanatomic and perhaps, phylogenetic relationships between mammalian and non-mammalian forms of the protein. © 1997 Elsevier Science B.V. All rights reserved.

Expression of the zinc-finger gene PLZF at rhombomere boundaries in the vertebrate hindbrain.

To investigate the potential biological role(s) of the PLZF gene, discovered as a fusion with the RARA locus in a patient with acute promyelocytic leukemia harboring a t(11;17) chromosomal translocation, we have isolated its murine homologue (mPLZF) and studied its patterns of developmental expression. The levels of mPLZF mRNAs increased perinatally in the liver, heart, and kidney, but with the exception of the heart, they were either absent or very low in the adult tissues. In situ analysis of mPLZF expression in mouse embryos between 7.0 and 10.5 days of development revealed that mPLZF mRNAs and proteins were coexpressed in spatially restricted and temporally dynamic patterns in the central nervous system. In the hindbrain region, a segmental pattern of expression correlated with the development of the rhombomeres. From 9.0 days of development, starting first in rhombomeres 3 and 5, there was an ordered down-regulation of expression in the center of each rhombomere, so that 1 day later elevated levels of mPLZF mRNAs and proteins were restricted to cells surrounding the rhombomeric boundaries. The chicken homologue of the PLZF gene, which we have also cloned, demonstrated a similar segmental pattern of expression in the hindbrain. To date, PLZF represents the only example of a transcription factor with elevated expression at rhombomeric boundaries. The high degree of evolutionary conservation between the patterns of PLZF expression during mammalian and avian central nervous system development suggests that it has an important functional role in the regionalization of the vertebrate hindbrain, potentially regulating boundary cell interactions.

Neurotrophin 3 stimulates the differentiation of motoneurons from avian neural tube progenitor cells.

Neurotrophin 3 (NT-3) promotes differentiation of neural tube progenitors into motoneurons expressing the BEN/SC1 and islet-1 epitopes. A 1.75- to 6.7-fold increase in BEN-positive motoneurons was obtained when quail neural tube cells were cultured with NT-3 at 0.1-10 ng/ml, respectively. In contrast, the overall number of cells, as well as the proportion of motoneurons that developed from cycling precursors, did not change. Addition of NT-3 at 1 ng/ml to cells obtained from ventral half-neural tubes promoted a 2.5-fold stimulation in motoneuron number, confirming the specificity of the effect. Moreover, NT-3 had no significant effect on survival of differentiated avian motoneurons. The distribution of trkC mRNA, which encodes the high-affinity receptor for NT-3, is consistent with these findings. trkC expression is homogeneous in the embryonic day 2 (E2) neural tube, becomes restricted to the mantle layer on E3, where differentiation occurs, and disappears from the ventral third of the E4-E5 spinal cord right before the onset of normal motoneuron death. These results suggest that NT-3 and trkC regulate early neurogenesis in the avian central nervous system.

Neurotrophin-3 affects proliferation and differentiation of distinct neural crest cells and is present in the early neural tube of avian embryos.

Neurotrophin-3 is mitogenic for cultured quail neural crest cells (Kalcheim et al., 1992, Proc. Natl. Acad. Sci. USA 89:1661-1665). We now report that neurotrophin-3 also influences the survival and/or differentiation of a subset of postmitotic neural crest precursors into neurons, provided these progenitors are grown on a cellular substrate. When cultured for 1 day on monolayers of NT-3-producing, chinese hamster ovary cells, 59% of the neural crest clusters growing on the transfected line revealed the presence of intense neuronal outgrowth, compared to 25% of that in controls. Moreover, dissociated neural crest cells grown for 20 h on top of mesodermal cells in the presence of various concentrations of purified recombinant neurotrophin-3 displayed a dose-dependent increase in neuronal number. Localization experiments using specific polyclonal antibodies, revealed that neurotrophin-3 is confined to neuroepithelial cells of quail neural tubes in situ on E2 and E3, and to E2 neural tubes grown in culture for 24 h. At this stage, neural crest cells and somites were negative. At later stages, staining was likewise apparent in peripheral nerves and dorsal root ganglia. We, therefore, propose that NT-3, a factor that is expressed in the early avian central nervous system, has multiple effects both on the proliferation and differentiation of distinct neural crest cells, which depend on the state of commitment of the responsive progenitors.

First appearance, distribution, and origin of macrophages in the early development of the avian central nervous system

A phagocytic cell system of hemopoietic origin exists in the early avian embryo (Cuadros, Coltey, Nieto, and Martin: Development 115:157-168, '92). In this study we investigated the presence of cells belonging to this system in the central nervous system (CNS) of chick and quail embryos by using both histochemical staining for acid phosphatase and immunolabelling with antibodies recognizing cells of quail hemangioblastic lineage. The origin of these cells was traced in interspecific chick-quail yolk sac chimeras. Hemopoietic cells were detected within the CNS from developmental stage HH15 on, and steadily increased in number at subsequent stages. Analysis of yolk sac chimeras revealed that most of these cells were of yolk sac origin, although some hemopoietic cells of intramebryonic origin were also found in the CNS. Immunocytochemical, histochemical, and ultrastructural characterization allowed us to identify hemopoietic cells in the CNS as macrophages. These cells were consistently found in the brain vesicles and spinal cord, appearing (1) between undifferentiated neuroepithelial cells at dorsal levels of the CNS; (2) in areas of cell death; (3) in the marginal layer in close relationship with developing axons; (4) in large extracellular spaces in the subventricular layer; (5) on vascular buds growing through the marginal and subventricular layers; and (6) in the ventricular lumen. Macrophages in different locations varied in morphology and ultrastructure, suggesting that in addition to their involvement in phagocytosis, they play a role in other processes in the developing CNS, such as axonal growth and vascular development. The first macrophages migrate to the CNS independently of its vascularization, apparently traversing the pial basal lamina to reach the nervous parenchyma. Other macrophages may enter the CNS together with vascular buds at subsequent stages during CNS vascularization.

Phosphorylated Tau Epitope of Alzheimer's Disease Is Coupled to Axon Development in the Avian Central Nervous System

The monoclonal antibody PHF-1 recognizes phosphorylated tau isoforms present in paired helical filaments of Alzheimer's disease. We have found that PHF-1 immunoreactivity is present in chick brain, which expresses three major PHF-1-reactive proteins at the same molecular weights seen in humans. The developmental pattern of expression suggests a functional role in differentiation, rather than in programmed nerve cell death. Expression of PHF-1 immunoreactivity in developing retina was highly cell selective, showing robust staining of ganglion cells, the only long-axon neuron of the seven major retina cell types. The majority of ganglion cells were PHF-1 positive. The developmental window of expression extended at least from E6 through P0, well outside the period of embryonic ganglion cell death. Mature cells did not show PHF-1 immunoreactivity. In the embryo, staining was particularly robust in ganglion cell axons (optic fiber layer), and association of PHF-1 reactivity with axonal tracts also was seen in developing forebrain. PHF-1 polarization occurred at ages when staining with polyclonal anti-tau did not show axonal selectivity. Similarly, in cell cultures, PHF-1 immunoreactivity became localized to single neurites, but polyclonal anti-tau did not. These results indicate that, rather than being associated with cell degeneration, PHF-1 immunoreactivity in the developing nervous system is associated with early stages of axon information, both in vivo and in vitro. Therefore, expression of PHF-1 immunoreactive proteins in Alzheimer's disease suggests that paired helical filament formation might be triggered by mechanisms related to axon regeneration.

Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody

The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor cDNAs (GluRl, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluR1, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.

The distribution of tenascin and its transcript in the developing avian central nervous system

AbstractI have studied the temporal and spatial regulation of the expression of the extracellular matrix protein tenascin in the developing chick brain using immunological and in situ hybridization methods. Tenascin is barely detectable by immuno‐slot blot analysis in the E4 telencephalon, but increases rapidly in abundance until E14, when the level of expression tapers off. The late appearance of tenascin was confirmed by immunohistochemistry. Tenascin can not be detected at E4, but at E7 is distributed homogeneously in most parts of the central nervous system. Tenascin is much less abundant in the retina than in other brain areas. As the avian retina is devoid of both vasculature and fibronectin, this supports the notion that tenascin may mask fibronectin from migrating growth cones and cell bodies. I have also used in situ hybridization with a cDNA probe to determine the origins of tenascin in the developing nervous system. Tenascin transcript is found in regions of glial proliferation and the cell bodies of radial glia. In contrast, tenascin appears to be made by neurons in the retina. As a low‐molecular weight splice variant of tenascin is detected on western blots of retina homogenates, different cell types may be responsible for generating the multiple forms of tenascin found in whole brain homogenates.

Identification and localization of ryanodine binding proteins in the avian central nervous system

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of ∼2000 kd and are composed of two high molecular weight ∼500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Autoradiographic localization of progestin‐concentrating cells in the brain of the zebra finch

The production of song in passerine birds is under the control of steroid hormones, and brain regions involved in song production have been shown to contain androgen and/or estrogen receptors. Studies to date, however, have not considered the possible role of progestins in this behavior. As one approach to this question, the autoradiographic method was used to investigate the distribution of progestin-concentrating cells in the brain of the adult male zebra finch (Poephila guttata) after injection of the radiolabeled synthetic progestin [17 alpha-methyl-3H]-promegestone. In the telencephalon, identifiable groups of progestin-accumulating cells were found in the hyperstriatum dorsale, at the medial edge of the lobus parolfactorius, and in the medial septum. In the diencephalon, labeled groups of cells were found in the preoptic area, through much of the medial hypothalamus--including nucleus periventricularis magnocellularis, nucleus medialis hypothalami posterialis, and area infundibularis--and in the medial spiriform nucleus and dorsomedial thalamus. In the myelencephalon, labeled cells are described at the dorsal edge of the medulla and scattered lateral to nXII. These findings offer no support for the hypothesis that progestin acts on any of the known song regions, but do suggest areas of progestin action in the avian central nervous system outside of the known song system. Not surprisingly, these include many areas of the medial hypothalamus and other midline structures.

Cellular localization of carnosine-like and anserine-like immunoreactivities in rodent and avian central nervous system

Aminoacylhistidine dipeptides are present in the nervous tissue of many species. The olfactory mucosa and bulb of many vertebrates are rich in carnosine (β-alanyl- l-histidine). Two related dipeptides homocarnosine (γ-aminobutyryl- l-histidine) and anserine (β-alanyl- N-methyl- l-histidine ) are present in the CNS of mammals and birds, respectively. This manuscript describes the production, characterization and use in immunolocalization studies of antisera directed against carnosine and anserine. The anserine antiserum is highly specific for anserine while the carnosine antiserum cross-reacts with all three dipeptides. The differential specificity of the antisera, coupled with chemical characterization of the dipeptide composition of various brain regions, has permitted assignment of the cellular localization of the various dipeptides. Immunocytochemical localization of anserine has not been previously reported. Carnosine immunoreactivity in the olfactory system is restricted to the mature neurons in the olfactory mucosa, their axons and synaptic terminations in the glomerular layer of the olfactory bulb. Similar reactivity is seen in the accessory olfactory system. Astrocytes and cerebellar Bergmann glia seem to account for all the non-olfactory carnosine-like immunoreactive staining in the rodent brain. In contrast, in the avian CNS where anserine is chemically abundant, anserine-like immunoreactivity is widespread and apparently exclusively associated with glial cells. Thus, the olfactory receptor neurons appear to be the only neuronal population that expresses carnosine. Elsewhere in the CNS the aminoacylhistidine dipeptides are associated with various populations of glia.

Expression of the SMP antigen by oligodendrocytes in the developing avian central nervous system

ABSTRACT Expression of the avian antigen SMP (Schwann cell Myelin Protein, Mr 75–80 000), first characterized in the PNS with a monoclonal antibody as an early and strictly specific Schwann cell marker, was further studied in the CNS. Comparing SMP immunoreactive areas in the different parts of the CNS with those expressing the Myelin Basic Protein (MBP), we showed a strict colocalisation of both phenotypes. In vitro, MBP+ oligodendrocytes express the surface antigen SMP as well. SMP cellular expression was followed in situ and in culture using nervous tissues from embryos at different stages. We were thus able to detect an early expression of this marker by oligodendroblasts before the first appearance of MBP immunoreactivity. We have also identified a subpopulation of SMP+/ MBP– and SMP+/GC– cells, which persists under our culture conditions as precursors remaining in an immature state.

A transient embryonic dopamine receptor inhibits growth cone motility and neurite outgrowth in a subset of avian retina neurons

To investigate the possible developmental significance of a transient dopamine receptor in the avian central nervous system, we examined the effects of dopamine on the morphology and motility of cultured retina neurons. Neurite arborization was significantly reduced by chronic dopamine stimulation. Using continuous video microscope monitoring, we observed that a subset of neurons responded to short-term dopamine with decreased filopodial activity and retracted neurites. The effects of dopamine could be blocked or reversed by haloperidol or SCH23390 and forskolin produced a response similar to dopamine, indicating the morphological changes were mediated by D 1-receptor stimulation of adenylate cyclase.

Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain

A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 +/- 400 and 72,200 +/- 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system.

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl. An immunohistochemical study.

The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.