Typology,distribution and development of the catecholamine-containing neurons in the chicken brain

Summary Fluorescence-histochemical investigations by use of the FAGLU method show the presence of several groups of catecholamine (CA)-containing neurons in the chicken brain. The distribution, shape and orientation of the fluorescent perikarya as well as the number and orientation of primary dendrites have been systematically examined. In the chick embryo, the first neurons displaying specific catecholamine fluorescence are identifiable on the 9th day of incubation. The onset of this type of specific fluorescence is consistent with biochemical data reported in the literature for the chick embryo. The main complexes of CA-containing cell bodies, shown at medullary, pontine and mesencephalic levels, display a pattern of distribution that is quite similar in both the chicken and the rat. In the hypothalamus of chick embryos and newly hatched chicks, CA-containing neurons have been localized within the paraventricular organ, and the periventricular and mammillary regions. By the fourth week after hatching, within the hypothalamus the paraventricular organ alone continues to display fluorescent neurons.This investigation was supported by grants from the Italian National Research Council (CNR) No 82.02079.04 (R.G.), No 83.00492.04 (G.C.P.) and MPI (40%).Part of this study was previously presented at the 7th ENA meeting, Hamburg, FRG, September 1983 (Guglielmone and Panzica 1983).     

Interaction of neuronal NOS and catecholamines in regulation of expression of proteins of apoptosis by vasopressinergic hypothalamic neurons

The work studied vasopressinergic neurons of hypothalamic supraoptic and paravenricular nuclei of the wild type mice and the neuronal nitric oxide synthase (nNOS) gene knockouted mice at a decrease of the brain catecholamine (CA) level caused by administration of the blocker of activity of tyrosine hydroxylase alpha-methyl-paratyrosine (alpha-MPT) and at the CA level decrease on the background of functional activity of the vasopressinergic neurons caused by dehydration of animals. There were analyzed changes in the number of neurons in both magnocellular hypothalamic nuclei expressing proapoptotic proteins caspase-8 and caspase-9, p53, and antiapoptotic protein Bcl-2. The disturbance of the CA-ergic innervation was shown to be a strong damaging factor leading to apoptosis of neurons regardless of the presence of nNOS in the cells. However, at disturbance of the CA-ergic innervation due to the 5-day mouse dehydration, no death of neurons by apoptosis was revealed. Thus, it is possible that functional activation prevents the hypothalamic vasopressinergic neurons from death at a decrease of the CA level in brain. The main difference of the nNOS gene knockouts is the absence of activation of the Bcl-2 expression under all used actions. This confirms our suggestion about interaction of CA and NO in triggering of expression of the antiapoptotic protein Bcl-2.     

Interaction of neuronal NOS and catecholamines in regulation of expression of proteins of apoptosis by vasopressinergic hypothalamic neurons

The work deals with studies on vasopressinergic neurons of hypothalamic supraoptic and paravenricular nuclei in the wild type mice and the neuronal nitric oxide synthase (nNOS) in the gene knockouted mice at a decrease of the brain catecholamine (CA) level caused by administration of the blocker of activity of tyrosine hydroxylase α-methyl-paratyrosine (α-MPT) and at the CA level decrease on the background of functional activity of the vasopressinergic neurons caused by dehydration of animals. There were analyzed changes in the number of neurons in the magnocellular hypothalamic nuclei expressing proapoptotic proteins caspase-8 and caspase-9, p53, and antiapoptotic protein Bcl-2. Disturbance of the CAergic innervation was shown to be a strong damaging factor leading to apoptosis of neurons regardless of the presence of nNOS in the cells. However, at disturbance of the CAergic innervation due to the 5-day mouse dehydration, no death of neurons by apoptosis was revealed. Thus, it is possible that functional activation prevents the hypothalamic vasopressinergic neurons from death at a decrease of the CA level in brain. The main difference of the nNOS gene knockouts is the absence of activation of the Bcl-2 expression under all used actions. This confirms our suggestion about interaction of CA and NO in triggering of expression of the antiapoptotic protein Bcl-2.     

Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus

The lentiviral Gag polyprotein (Pr55(Gag)) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55(Gag) is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55(Gag) and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55(Gag), several CA-containing cleavage intermediates of Pr55(Gag), and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55(Gag) can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55(Gag) and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55(Gag) and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.     

A fluorescence microscopic study of the distribution of monoamines in the hypothalamus of the cat

Three distinct groups of monoamine (MA)-containing nerve cell bodies have been visualized in the hypothalamus and preoptic area of the cat by means of the Falck-Hillarp fluorescence histochemical technique. First, numerous small-sized catecholamine (CA) type neurons were disclosed within the ventral half of the periventricular area in the supraoptic and middle hypothalamic regions. The round to oval neurons of this medio-ventral group were more especially abundant around the base of the third ventricle, within the arcuate and supraopticus diffusus nuclei. Numerous medium-sized CA perikarya identified as the dorsal group, were also mapped out in the dorsal and posterior hypothalamic areas. Finally, a small population of both CA and serotonin (5-hydroxytryptamine, 5-HT)-containing neurons was disclosed within the lateral area of the middle and mammillary hypothalamic regions. These multipolar or elongated neurons which compose the lateral group were lying either along the ventrolateral surface of the hypothalamus or around the ventrolateral aspect of the fornix. In addition to these three MA cell groups, a few cells displaying a fluorescence of the CA type were also visualized in the so-called “dorsal chiasmatic nucleus” after α-methyl-dopa treatment. High density of CA axon terminals were found, on the other hand, in the external layer of the median eminence, in the dorsomedial, paraventricular, supraoptic and suprachiasmatic nuclei, and also within nucleus interstitialis of stria terminalis. In the present study, however, it was not possible to identify with certainty any concentration of 5-HT axon terminals in the cat hypothalamus. Therefore, except for the lateral cell group which could be peculiar to the cat, the topographical distribution of MA nerve cell bodies and axon terminals in the hypothalamus of the cat appears similar to the morphological organization of the MA neuronal elements in the hypothalamus of the rat.     

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.     

Participation of catecholamines and NO in regulation of apoptosis of nonapeptidergic neurons of neonatal rat pups

Effects of catecholamines (CA) and the character of interaction of CA and NO in regulation of apoptosis were studied in vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei of rat pups in early postnatal ontogenesis. To study role of CA in regulation of programmed cell death in SON and PVN in the course of embryonal development, pregnant female rats were intraperitoneally injected daily from the 13th to the 20th day with αMPT—a blocker of CA synthesis. The second group of pregnant rats was injected from the 13th to the 20th day with the same volume of saline. The third group was composed of intact animals. The born rat pups were sacrificed at the 3rd and 15th days of life. Caspase 9, Bcl-2, tyrosine hydroxylase, and neuronal NO-synthase (nNOS) in SON and PVN neurons were revealed immunohistochemically, and the amount of immunoreactive substance in neuronal bodies was estimated using the computerized digital analyzer of TV image and Video Test software. Caspase-9 was shown to play an important role in postnatal formation of cellular composition of hypothalamic nonapeptidergic centers by leading to initiation of apoptosis and rejection of “useless” postmitotic SON and PVN neurons. Survival of “useless” nonapeptidergic neurons in early postnatal ontogenesis seems to be connected with antiapoptotic action of Bcl-2. Death of postmitotic neurons, and therefore formation of cellular composition begins earlier and, accordingly, is completed earlier in SON, in which neurons were noted to have a considerable decrease of the caspase-9 expression and, therefore, also a decrease of intensity of neuronal death via caspase-9-dependent pathway. In PVN, neurons continue to die also at the 15th day of rat life, i.e., almost two weeks later than in SON. The observed high correlation between the content of nNOS, caspase-9, and Bcl-2 in the SON and PVN neurons of intact rats of both age groups allows suggesting participation of NO in realization of apoptosis in the course of early postnatal development. The increase of nNOS expression in hypothalamic neurons as a result of disturbances in CA-ergic innervation in embryogenesis might be a possible cause of the long preserved enhancement of expression of apoptosis signal proteins. It can be suggested that CA participate in morphogenesis of hypothalamic neurons by increasing expression of nNOS in neurons and thereby affecting expression of apoptotic proteins.     

The relationship of cell division to the acquisition of adrenergic characteristics by developing sympathetic ganglion cell precursors

The relationship of the acquisition of defining characteristics by precursor cells of sympathetic ganglia to the withdrawal of these cells from the cell cycle was investigated in the developing chick. The characteristics studied included the ability to synthesize catecholamines (CA), the development of characteristic subcellular storage granules, and the specific uptake of norepinephrine (NE). All were present in presumptive sympathicoblasts and adrenal medullary precursors, which also became labeled after the injection of tritiated thymidine and so retained the ability to divide. These dividing CA-containing cells were found in both primary ganglia and secondary preand paravertebral ganglia. The developing sympathetic neuronal population was found to be a heterogeneous one. Some sympathetic precursor cells appeared to become postmitotic (or to enter a pause in division) early in ontogeny, while others continued to divide throughout the time of hatching. As embryogenesis proceeded, the proportion of CA-containing cells or their precursors which were dividing decreased. However, those cells which did divide probably divided repeatedly. It is concluded that some of the definitive characteristics of mature neurons are expressed by dividing precursor cells. The specific characteristic that marks the transition from immature dividing cells to mature postmitotic neurons has not yet been determined.     

Participation of neuronal NO-synthase in regulation of hypothalamus vasopressinergic neurons of rat pups at early stages of postnatal ontogeny

To reveal character of interaction of catecholamines (CA) and NO in regulation of development and of the functional state of vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei, the female rats were injected intraperitoneally with the inhibitor of CA synthesis α-methyl-p-tyrosine, daily, from the 13th to the 20th days of pregnancy. Rat pups born by the females administered with saline at the same period of pregnancy as well as intact pups and adult rats were used as control. Expression of neuronal NO-synthase (nNOS) in neurons of SON and PVN of rat pups at early stages of postnatal development was found to be significantly higher than the definitive level, which allows suggesting participation of NO in development of hypothalamic VP-ergic neurons. The revealed differences of periods of the maximal nNOS expression in the SON and PVN neurons have permitted suggesting development of SON to be completed earlier than that of PVN. The pups exposed to stress at the last third of embryonic development had a long-lasting effect on the state of VP-ergic neurons of the pups after birth. The nNOS expression in neurons does not change, which suggests that NO is not involved in regulation of VP-ergic neurons after exposure to stress at early stages of ontogenesis. A decrease of CA level in the brain at the last third of embryogenesis led to a long preserved decrease of the functional activity of VP-ergic neurons. The nNOS expression in VP-ergic neurons of SON and PVN rose substantially under effect of a compensatory enhancement of tyrosine hydroxylase (TH) expression in neurons of SON and of an increase of the level of CA-ergic innervation of PVN. Thus, we have shown that a decrease of CA level in the embryonic brain leads to an increase of nNOS expression of hypothalamic VP-ergic neurons of rat pups after birth and that the character of NO action on function of VP-ergic neurons does not differ from that of adult animals as soon as at early stages of ontogenesis.     

Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons

The mechanism by which pluripotent progenitors give rise to distinct classes of mature neurons in vertebrates is not well understood. To address this issue we undertook a genetic screen for mutations which affect the commitment and differentiation of catecholaminergic (CA) [dopaminergic (DA), noradrenergic (NA), and adrenergic] neurons in the zebrafish, Danio rerio. The identified mutations constitute five complementation groups. motionless and foggy affect the number and differentiation state of hypothalamic DA, telencephalic DA, retinal DA, locus coeruleus (LC) NA, and sympathetic NA neurons. The too few mutation leads to a specific reduction in the number of hypothalamic DA neurons. no soul lacks arch-associated NA cells and has defects in pharyngeal arches, and soulless lacks both arch-associated and LC cell groups. Our analyses suggest that the genes defined by these mutations regulate different steps in the differentiation of multipotent CA progenitors. They further reveal an underlying universal mechanism for the control of CA cell fates, which involve combinatorial usage of regulatory genes.     

Immunocytochemical Localization of TASK-3 Protein (K2P9.1) in the Rat Brain

Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.     

Apparent effect of rabbit endogenous lentivirus type K acquisition on retrovirus restriction by lagomorph Trim5αs

To test the hypothesis that rabbit endogenous lentivirus type K (RELIK) could play a role in shaping the evolution of TRIM5α, the susceptibility of viruses containing the RELIK capsid (CA) to TRIM5 restriction was evaluated. RELIK CA-containing viruses were susceptible to the TRIM5αs from Old World monkeys but were unaffected by most ape or New World monkey factors. TRIM5αs from various lagomorph species were also isolated and tested for anti-retroviral activity. The TRIM5αs from both cottontail rabbit and pika restrict a range of retroviruses, including HIV-1, HIV-2, FIV, EIAV and N-MLV. TRIM5αs from the European and cottontail rabbit, which have previously been found to contain RELIK, also restricted RELIK CA-containing viruses, whereas a weaker restriction was observed with chimeric TRIM5α containing the B30.2 domain from the pika, which lacks RELIK. Taken together, these results could suggest that the pika had not been exposed to exogenous RELIK and that endogenized RELIK might exert a selective pressure on lagomorph TRIM5α.     

Primary cultures of dispersed hypothalamic cells from fetal rats: morphology, electrical activity, and peptide content

Cultures prepared from dispersed fetal hypothalamic tissue have cells which can be identified as neurons by their morphology and electrical activity. The elongation of neuritic processes in these cultures is increased by treatment with 1-beta-D arabinofuranosylcytosine (ara-C). Hypothalamic cultures have measurable quantities of immunoreactive substance P and neurotensin, and the neurons can accumulate (3H)GABA.     

Interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone in rat and guinea-pig neurons: a comparison of Ca2+ - and GABA(A)-CI- -channel current modulation.

A comparison of the interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone (TDOC) on voltage-gated Ca2+ -and the gamma-aminobutyric receptor (GABA(A)) gated-Cl- -channels was examined in freshly dissociated guinea-pig (GP) and rat hippocampal CA1 neurons and rat hypothalamic ventromedial nucleus (VMN) neurons. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarized steps from -80 to -10 mV by TDOC increased in concentration-dependent manner with IC50 values of 1 and 6 pM for rat and GP CA1 neurons, respectively and 3 nM for rat VMN neurons. TDOC rapidly and reversibly inhibited a fraction (up to 26%) of the total Ca2+ channel current in all neurons. Intracellular dialysis with GDP-beta-S (500 microM) significantly diminished the TDOC inhibition of the Ca2+ channel current, suggesting a G-protein involvement. In neurons isolated from pertussis-toxin-treated animals by chronic intracerebroventricular (1000 ng/24/48 h) infusion, the TDOC inhibition was also significantly diminished, suggesting modulation by the Galphai and/or Galphao G-protein subunits. The peak GABA-gated inward Cl- current was enhanced in both species from 0.1 to 10 microM with the greatest increase (48% at 10 microM) seen in the VMN. There was no difference in the enhancement of the GABA current in the CA1 region of both species. The results show that in contrast to the 3a-series, the 3beta-series weakly enhance the GABA-evoked Cl- current but potently inhibit the Ca2+ channel current. In addition, these results also suggest a common mode of action and a lack of interspecies difference for this steroid.     

Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release

In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.     

Identification of two cardioacceleratory neurons in the isopod crustacean, Ligia exotica and their effects on cardiac ganglion cells

We identified two pairs of cardioacceleratory (CA1, CA2) neurons in the central nervous system of the isopod Ligiaexotica and examined their effects on the cardiac ganglion (CG). CA1 neurons had cell bodies in the 2nd thoracic ganglion and had arborizations in the subesophageal ganglion and the 1st and 2nd thoracic ganglia. CA2 neurons had cell bodies in the 3rd thoracic ganglion and had arborizations in the 2nd, 3rd and 4th thoracic ganglia. They sent axons to the heart through the ipsilateral 3rd roots of the ganglia where their cell bodies were located. Repetitive stimulation of the CA1 axon rapidly increased the burst frequency of the CG, and that of CA2 rather slowly. The increased burst rate caused by the CA1 stimulation was significantly higher than that caused by CA2. Overall depolarization of a quiescent CG cell produced by the CA1 stimulation was significantly larger in amplitude than that produced by CA2. Facilitation was obviously seen in the excitatory post-synaptic potentials evoked by the CA1 stimulation. These results show that the synaptic properties of CA1 and CA2 neurons are different, suggesting that they have different functional roles in heart regulation. Accepted: 19 July 1997     

Neuropeptide FF2 receptor distribution in the human brain. An immunohistochemical study

Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.