Ontogeny of calcitonin receptor mRNA and protein in the developing central nervous system of the rat

In this study, the expression of receptors for calcitonin (CTR), the CTR C1a and C1b isoforms, was investigated during development of the fetal rat central nervous system (CNS) by using in situ hybridization and immunohistochemistry. Coincident expression with both techniques was evident. Immunohistochemical evidence for the expression of the C1a isoform alone was found. Expression was first observed at embryonic day 12/13 (E12/E13) within and adjacent to the ventricular zones known to include primary matrices of proliferation, in regions of the preoptic area, anterior and posterior hypothalamus, anterior and posterior pons, medulla, and spinal cord. At later times, with the decline in the density of immunoreactivity at these loci (E15), expression in primary matrices was found later at distinct loci within the ventricular zones of cerebellum (E17), and at E19, the tectum, lateral ventricle, and cortical subplate. By E19, the density of staining had increased and was widespread throughout the expanding CNS. In the rostral domains, moderate to high density was found in the external plexiform layer; the medial preoptic area and nucleus; the ventromedial, dorsomedial, and arcuate hypothalamic nuclei; and the lateral and posterior hypothalamic areas. In the midbrain, similar levels of expression were noted in the central nucleus of raphe; the deep mesencephalic, dorsal raphe, and laterodorsal tegmental nuclei; and the ventral periaqueductal gray. In the pons, positive loci included the locus coeruleus and the gigantocellular and pontine reticular nuclei. In the medulla, high expression was evident in the gigantocellular, intermediate, magnocellular, and medullary reticular, spinal trigeminal and cuneate nuclei; and the nucleus tractus solitarius. In the spinal cord, moderate to high density of staining was found in the ventral, dorsal, and lateral horns, and in the ventral, dorsal, and cuneate funiculi. On the other hand, transitory expression was found in the diagonal band, bed nucleus of the stria terminalis, amygdala, and the lateral mamillary and anterobasal nuclei of the hypothalamus. These studies indicate a role for CTR in the activation of some premigratory neuroblasts in the CNS as well as a possible role later in an undefined function associated with mature neurons of particular nuclei.     

Chondroitin sulfate proteoglycans in the developing central nervous system. I. Cellular sites of synthesis of neurocan and phosphacan

We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13–16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5′-leader of neurocan may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing central nervous system. © 1996 Wiley-Liss, Inc.     

Expression of a phosphorylated isoform of MAP1B is maintained in adult central nervous system areas that retain capacity for structural plasticity

Microtubule-associated protein IB (MAP1B) is the first MAP to be detected in the developing nervous system, and it becomes markedly down-regulated postnatally. Its expression, particularly that of its phosphorylated isoform, is associated with axonal growth. To determine whether adult central nervous system (CNS) areas that retain immunoreactivity for MAP1B are associated with morphological plasticity, we compared the distribution of a phosphorylated MAP1B isoform (MAP1B-P) to the distribution of total MAP1B protein and MAP1B-mRNA. Although they were present only at very low levels, both protein and message were found ubiquitously in almost all adult CNS neurons. The intensity of staining, however, varied markedly among different regions, with only a few nuclei retaining relatively high levels. MAP1B-P was restricted to axons, whereas total MAP1B was present in cell bodies and processes. Relatively to total MAP1B protein and its mRNA, MAP1B-P levels decreased more dramatically with maturation, and they were detectable in only a few specific areas that underwent structural modifications. These included primary afferents and motor neurons, olfactory tubercles, habenular and raphe projections to interpeduncular nuclei, septum, and the hypothalamus. The distribution pattern of MAP1B-P was compared to that of the embryonic N-CAM rich in polysialic acid (PSA-NCAM). We found that the PSA-NCAM immunostaining was largely overlapped with that of MAP1B-P in the adult CNS. These results suggest that, like PSA-NCAM, MAP1B may be one of the molecules expressed during brain development that also plays a role in structural remodeling in the adult. © 1996 Wiley-Liss, Inc.     

Neuronal expression of the proteolipid protein gene in the medulla of the mouse

The proteolipid protein (PLP) gene (Plp) encodes the major myelin proteins, PLP and DM20. Expression of Plp occurs predominantly in oligodendrocytes, but evidence is accumulating that this gene is also expressed in neurons. In earlier studies, we demonstrated that myelin‐deficient (MD) rats, which carry a mutation in the Plp gene, exhibit lethal hypoxic ventilatory depression. Furthermore, we found that, in the MD rat, PLP accumulated in neuronal cell bodies in the medulla oblongata. In the current study, we sought to determine which neurons expressed the Plp gene in the medulla oblongata and whether Plp gene expression changed in neurons with maturation. A transgenic mouse expressing the Plp promoter driving expression of enhanced green fluorescent protein (Plp‐EGFP) was used to identify neurons expressing this gene. Plp expression in neurons was confirmed by immunostaining EGFP‐positive cells for NeuN and by in situ hybridization for PLP mRNA. The numbers of neurons expressing Plp‐EGFP and their distribution increased between P5 and P10 in the medulla. Immunostaining for surface receptors and classes of neurons expressing Plp‐EGFP revealed that Plp gene expression in brainstem neurons was restricted to neurons expressing specific ligand‐gated channels and biosynthetic enzymes, including glutamatergic NMDA receptors, GABA_A receptors, and ChAT in defined areas of the medulla. Plp gene expression was rarely found in interneurons expressing GABA and was never found in AMPA receptor‐ or tyrosine hydroxylase‐expressing neurons. Thus, Plp expression in the mouse caudal medulla was found to be developmentally regulated and restricted to specific groups of neurons. © 2009 Wiley‐Liss, Inc.     

Expression of the PC4 gene in the developing rat nervous system

PC4 is an early NGF-inducible gene, transiently expressed during the in vitro differentiation of PC12 cells toward a neuronal phenotype. By in situ hibridization analysis, we found that PC4 is expressed at high levels along the whole neural tube of early rat embryos. PC4 mRNA expression is not uniform across the wall of the neural tube, the autoradiographic signal being most intense on the ventricular layer. At later stages, when the rate of proliferation and production of postmitotic neurons decreases, PC4 gene expression also decreases and becomes restricted to the telencephalon, that is the last region to complete neurogenesis. Thus the expression of PC4 gene, although not exclusive of proliferating cells, appears to be correlated to the time span of proliferation of neuronal and glial precursors.     

Expression of the Manduca sexta allatotropin gene in cells of the central and enteric nervous systems

Manduca sexta allatotropin (Mas-AT) was isolated and first characterized as a peptide that stimulated juvenile hormone biosynthesis in adult lepidopteran corpora allata and was subsequently shown to have cardioacceleratory activity in the pharate adult. In this study, we identified the cells in the nervous system of the insect that contain mRNA encoding Mas-AT and immunoreactivity against a polyclonal antiserum to Mas-AT. In larvae, Mas-AT mRNA and immunoreactivity was most abundant in two cells in the frontal ganglion, which project their axons down the recurrent nerve toward the gut, and in cells in the terminal abdominal ganglion. Lower levels of Mas-AT mRNA were detected in the brain and subesophageal ganglion. In the pupal and pharate adult stages, we detected Mas-AT mRNA and immunoreactivity in cells of the abdominal ganglia and in additional cells in the terminal abdominal ganglion. These additional cells in the ventral nerve cord that express Mas-AT during the pupal and pharate adult stages include cells that differentiate during metamorphosis as well as cells that exist in larvae but do not begin to express Mas-AT until these later developmental stages. Some of the cells that exhibit Mas-AT immunoreactivity lack Mas-AT mRNA, suggesting that the antisera used in this and previous studies recognizes other peptides in addition to Mas-AT. This pattern of expression suggests that Mas-AT may mediate multiple physiological functions during the life cycle of the insect, including the larval stage in which no function has yet been described for the peptide. J. Comp. Neurol. 403:407–420, 1999. © 1999 Wiley-Liss, Inc.     

Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo.

Expression of myelin protein genes by myelinating Schwann cells in vivo is dependent on axonal influences. This report investigated the effect of axons on myelin protein mRNA levels in the central nervous system (CNS). In situ hybridization studies of rat spinal cord sections localized mRNAs encoding proteolipid protein (PLP) and myelin basic protein (MBP) 20 and 40 days after unilateral rhizotomy. Compared with control tissue, hybridization intensity was reduced in transected tissue, but there was little change in the number of oligodendrocytes labeled. Cellular RNA was extracted from transected and age-matched control optic nerves 5, 10, 20, and 40 days after surgery, and levels of the following mRNAs were determined by slot blot procedures: PLP, MBP, myelin-associated glycoprotein (MAG), and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP). In transected nerves, PLP and MBP mRNA levels were approximately 85%, 45%, and 25% of control values at 5, 20 and 40 days posttransection, respectively. Axonal transection had a lesser effect on CNP and MAG mRNA levels, which declined to approximately 60% of control levels at 40 days. Immunocytochemical studies indicated that the number of oligodendrocytes was not decreased 40 days after optic nerve transection. These data demonstrate that axons modulate myelin protein mRNA levels in oligodendrocytes. In contrast to Schwann cells, however, oligodendrocytes continue to express significant levels of myelin protein mRNA in vivo following loss of axonal contact.     

Plexin-B family members demonstrate non-redundant expression patterns in the developing mouse nervous system: an anatomical basis for morphogenetic effects of Sema4D during development

Semaphorins and their receptors play important roles in patterning the connectivity of the developing nervous system and recent data suggest that members of the plexin-B family of semaphorin receptors may be involved in axonal guidance. Here we show that the mRNAs of the three plexin-B genes, plxnb1, plxnb2 and plxnb3 (plexin-B1, plexin-B2 and plexin-B3), respectively, are expressed in highly specific and non-redundant patterns in peripheral and central components of the nervous system over defined periods during murine development. Whereas plexin-B1 and plexin-B2 are strongly expressed in the neuroepithelium and developing neurons, plexin-B3 mRNA is selectively localized to the white matter. Moreover, plexin-B1 and its ligand Sema4D are expressed in complementary patterns in several regions such as the developing neopallial cortex, the dorsal root ganglia and the spinal cord over embryonic stages. The Sema4d gene demonstrates a dramatic switch from prenatal expression in neuronal populations to a postnatal expression in oligodendrocytes. In contrast to its collapsing activity on growth cones of embryonic retinal ganglion cells and hippocampal neurons, soluble Sema4D enhances axonal outgrowth in embryonic cortical explants cultured in collagen matrices. Thus, plexin-B family members and Sema4D are likely to play complex and non-redundant roles during the development of the nervous system.     

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.     

Organization of the corticotropin-releasing hormone and corticotropin-releasing hormone-binding protein systems in the central nervous system of the sea lamprey Petromyzon marinus

The expression of the corticotropin-releasing hormone (PmCRH) and the CRH-binding protein (PmCRHBP) mRNAs was studied by in situ hybridization in the brain of prolarvae, larvae, and adults of the sea lamprey Petromyzon marinus. We also generated an antibody against the PmCRH mature peptide to study the distribution of PmCRH-immunoreactive cells and fibers. PmCRH immunohistochemistry was combined with antityrosine hydroxylase immunohistochemistry, PmCRHBP in situ hybridization, or neurobiotin transport from the spinal cord. The most numerous PmCRH-expressing cells were observed in the magnocellular preoptic nucleus-paraventricular nucleus and in the superior and medial rhombencephalic reticular formation. PmCRH expression was more extended in adults than in larvae, and some cell populations were mainly (olfactory bulb) or only (striatum, ventral hypothalamus, prethalamus) observed in adults. The preopto-paraventricular fibers form conspicuous tracts coursing toward the neurohypophysis, but many immunoreactive fibers were also observed coursing in many other brain regions. Brain descending fibers in the spinal cord mainly come from cells located in the isthmus and in the medial rhombencephalic reticular nucleus. The distribution of PmCRHBP-expressing neurons was different from that of PmCRH cells, with cells mainly present in the septum, striatum, preoptic region, tuberal hypothalamus, pretectum, pineal complex, isthmus, reticular formation, and spinal cord. Again, expression in adults was more extended than in larvae. PmCRH- and PmCRHBP-expressing cells are different, excluding colocalization of these substances in the same neuron. Present findings reveal a complex CRH/CRHBP system in the brain of the oldest extant vertebrate group, the agnathans, which shows similarities but important divergences with that of mammals.     

Comparative localization of two serotonin receptors and sensorin in the central nervous system of Aplysia californica

Aplysia californica is a powerful model for understanding the cellular and molecular mechanisms underlying modulation of neuronal plasticity and learning. In the central nervous system of Aplysia, serotonin is associated with various behaviors. For example, it induces short-, intermediate-, and long-term synaptic changes in sensory neurons during learning and inhibits the afterdischarge of the bag cells that initiate egg-laying behavior. Little is known about the nature and contribution of serotonin receptors involved in the numerous serotonin-mediated physiological responses in Aplysia. Recently, two G(i)-coupled serotonin receptors (5-HT(ap1) and 5-HT(ap2)) were cloned. We now report that, by using in situ hybridization to express the profile of these receptors, we are able to gain critical insight into their roles in the behavior of Aplysia. We compared their distribution to that of sensorin-A, a peptide specifically found in sensory neurons. We wished to determine their involvement in some simple forms of behavioral modifications. 5-HT(ap1) and 5-HT(ap2) mRNAs are expressed in all ganglia of the Aplysia central nervous system. Stronger signal was observed with the 5-HT(ap2) antisense probe than with the 5-HT(ap1) antisense probe. Notably, mRNA coding for the receptors was found in several identified neurons, in the bag cells, in characterized serotonergic neurons, and in neurons of the mechanosensory clusters that expressed sensorin. We also observed heterogeneity of receptor expression between R2 and LPl1 and among neurons of a single cluster of sensory neurons. These results suggest that 5-HT(ap1) and 5-HT(ap2) receptors may regulate the response to serotonin and/or its release in several neurons.     

Localization of the neuromedin K receptor (NK3) in the central nervous system of the rat

The distribution of the neuromedin K receptor (NK₃; NKR) in the central nervous system was investigated in the adult rat by using in situ hybridization and immunohistochemical techniques. The rabbit anti-NKR antibody was raised against a bacterial fusion protein containing a C-terminal portion of NKR and affinity purified with a Sepharose 4B column conjugated to the fusion protein. Immunoblot analysis was performed to test the reactivity and specificity of the antibody. Crude membrane was prepared from cDNA-transfected Chinese hamster ovary (CHO) cells expressing each of the rat NKR, substance P receptor (NK₁; SPR), and substance K receptor (NK₂; SKR) and from the hypothalamus, cerebral cortex, and cerebellum. Immunoreactive bands were observed specifically in the NKR-CHO cells, hypothalamus, and cerebral cortex but not in the SPR- or SKR-CHO cells, nor in the cerebellum. Molecular weights of the immunoreactive bands ranged from 73 to 89 kDa and from 59 to 83 kDa in the NKR-CHO cells and tissues, respectively. The distribution of NKR-like immunoreactivity coincided with that of NKR mRNA. The expression of NKR was indicated on neuronal cell bodies and dendrites. NKR was found to be expressed intensely or moderately in neurons in the glomerular and granule cell layers of the main olfactory bulb; glomerular and mitral cell layers of the accessory olfactory bulb; layers IV and V of the cerebral neocortex; medial septal nucleus; nucleus of the diagonal band; bed nucleus of the stria terminalis; globus pallidus; ventral pallidum; paraventricular nucleus; supraoptic nucleus; zona incerta; dorsal, lateral, and posterior hypothalamic areas; amygdaloid nuclei; medial habenular nucleus; ventral tegmental area; midbrain periaqueductal gray; interpeduncular nuclei; substantia nigra pars compacta; linear, median, dorsal, and pontine raphe nuclei; posteromedial tegmental nucleus; sphenoid nucleus; nucleus of the solitary tract; intermediate and rostroventrolateral reticular nuclei; and lamina II of the caudal spinal trigeminal nucleus and spinal dorsal horn. These findings are discussed in relation to the physiological functions associated with neuromedin K. © 1996 Wiley-Liss, Inc.