Phosphohippolin expression in the rat central nervous system

The FXYD family is a small single-span membrane protein family; recently, we have identified a novel member of this family from the cDNA library of the rat hippocampus and named phosphohippolin (Php) (Mol. Br. Res. vol. 86, 2001). The deduced amino acid sequence of this novel Php comprises 93 residues with a core motif of FXYD and a single transmembrane domain. This indicates that Php belongs to FXYD6 subfamily of the seven FXYD subfamilies (FXYD1-7). Php shows a 48.1% homology with rat phospholemman (FXYD1), a transmembrane family protein. In this study, polyclonal antibodies against the carboxyl-terminal sequence of rat Php were raised and purified. The spatial expression of the Php protein was in the neuronal fibers of the medial part of lateral habenula nucleus, thalamus, hypothalamus, stria terminalis, zona incerta, amygdaloid body and cingulum, olfactory bulb, hippocampus, cerebral cortex and cerebellum. A unique Php distribution was identified in the cerebellum, with a predominant expression pattern in the granule layer of lobules VI-IX of the posterior lobe. Developmental studies demonstrated that the highest level of Php expression was seen in the postnatal (PN) 3-week-old rat brain, and a significant amount of Php still existed in the adult brain. These findings suggest that Php may play an important role in the excitability of neurons in the central nervous system during postnatal development, as well as those in the adult brain.     

Ethanol-induced vacuolation in rat peripheral nervous system

The effect of chronic (3–4 weeks), heavy ethanol exposure on neuronal vacuolation in rat peripheral nervous system was studied in male Wistar rats. The rats were force-fed with 25% ethanol 3 times a day, which resulted in blood ethanol levels of 53.1 ± 18.8 mmol/l, i.e., marked intoxication. In the superior cervical ganglia (SCG), the ethanol exposure increased the proportion of vacuolated neurons c. 13-fold (0.2 ± 0.0% in the control ganglia, 2.7 ± 0.6% in the EtOH-ganglia, P < 0.001). A considerable population of vacuolated neurons (VN) was seen in the sensory inferior vagal (nodose) ganglia, and occasional neurons with large cytoplasmic vacuoles in the sensory dorsal root ganglia (DRG) of the EtOH-rats. In the hypogastric ganglia, where VN are regularly found in the adult rat, ethanol exposure did not affect the amount or the appearance of the vacuolated neurons. The number of VN in the SCG decreased significantly between 2 days and 1 week after cessation of the exposure, but did not return to control level by 1 month after ethanol withdrawal. In electron microscopy, most of the vacuolated SCG neurons showed normal ultrastructure, apart from the large cytoplasmic vacuoles. Some vacuolated neurons, however, showed neuropathologic changes, e.g., dilated endoplasmic reticulum, mitochondrial alterations and increased numbers of myelin figures. These degenerative changes were more frequent in the vacuolated DRG neurons than in the sympathetic ones. The occurrence of VN in rat peripheral ganglia may represent a reaction to increased stimulation during prolonged ethanol exposure and, especially, during repeated phases of ethanol withdrawal.     

Extensive aspartoacylase expression in the rat central nervous system

Aspartoacylase (ASPA) catalyzes deacetylation of N-acetylaspartate (NAA) to generate acetate and aspartate. Mutations in the gene for ASPA lead to reduced acetate availability in the CNS during development resulting in the fatal leukodystrophy Canavan disease. Highly specific polyclonal antibodies to ASPA were used to examine CNS expression in adult rats. In white matter, ASPA expression was associated with oligodendrocyte cell bodies, nuclei, and some processes, but showed a dissimilar distribution pattern to myelin basic protein and oligodendrocyte specific protein. Microglia expressed ASPA in all CNS regions examined, as did epiplexus cells of the choroid plexus. Pial and ependymal cells and some endothelial cells were ASPA positive, as were unidentified cellular nuclei throughout the CNS. Astrocytes did not express ASPA in their cytoplasm. In some fiber pathways and nerves, particularly in the brainstem and spinal cord, the axoplasm of many neuronal fibers expressed ASPA, as did some neurons. Acetyl coenzyme A synthase immunoreactivity was also observed in the axoplasm of many of the same fiber pathways and nerves. All ASPA-immunoreactive elements were unstained in brain sections from tremor rats, an ASPA-null mutant. The strong expression of ASPA in oligodendrocyte cell bodies is consistent with a lipogenic role in myelination. Strong ASPA expression in cell nuclei is consistent with a role for NAA-derived acetate in nuclear acetylation reactions, including histone acetylation. Expression of ASPA in microglia may indicate a role in lipid synthesis in these cells, whereas expression in axons suggests that some neurons can both synthesize and catabolize NAA.     

Localization of Bcl-xbeta in the developing and adult rat central nervous system

bcl-xbeta is a novel apoptosis-regulating member of the bcl-x family that has recently been isolated from rats and mice. To explore the functional role of Bcl-xbeta, we raised a monoclonal antibody against rat Bcl-xbeta protein and investigated the cellular localization of the molecule in the rat CNS. Immunohistochemistry revealed that, in the fetal and neonatal stages, Bcl-xbeta was intensively and widely expressed in the CNS. Many neurons in the diencephalon and brain stem showed intense cytoplasmic labeling. The immunoreactivity decreased during the postnatal development and reached to the level of adulthood by P14. In the adult brain and spinal cord, labeling was restricted to specific types of neurons and distributed throughout their somata and dendrites. Weak immunoreactivity was present in many CNS regions such as the cerebral cortex, hippocampal dentate gyrus, caudate-putamen, globus pallidus, thalamus, locus ceruleus, pontine nuclei, inferior olive, reticular formation, cerebellar cortex and spinal anterior horn. Amygdaloid nuclei and hippocampal CA1 to CA3 sectors showed restricted expression of Bcl-xbeta in a subset of neurons. Neuronal labeling was almost undetectable in several regions, including the piriform cortex, hypothalamus, posterior column nuclei and spinal posterior horn. These results suggest that Bcl-xbeta plays an important role throughout the CNS in developing stage and may regulate the apoptosis of postnatal CNS neurons.     

Developmental expression of Neuregulin-3 in the rat central nervous system

Neuregulin-3 (Nrg3) is a member of the Nrg family of growth factors identified as risk factors for schizophrenia. There are three Nrgs expressed in the nervous system (Nrg1-3) and of these Nrg1 has been the best characterized. To set the groundwork for elucidating neural roles for Nrg3, we studied its expression in the rat brain at both the RNA and protein levels. Using an antibody developed against Nrg3, we observed a developmental increase of Nrg3 protein expression from embryonic stages to adulthood and determined that it carries O-linked carbohydrates. In cortical neuronal cultures, transfected Neuro2a cells, and brain tissue sections Nrg3 protein was localized to the soma, neurites, and to the Golgi apparatus, where it is prominently expressed. Nrg3 was detected in excitatory, GABAergic and parvalbumin-expressing inhibitory neurons while expression in glia was limited. Nrg3 mRNA and protein were widely expressed during both embryonic and postnatal ages. At E17, Nrg3 was detected within the cortical plate and ventricular zone suggesting possible roles in cell proliferation or migration. At postnatal ages, Nrg3 was abundantly expressed throughout the cerebral cortex and hippocampus. Multiple thalamic nuclei expressed Nrg3, while detection in the striatum was limited. In the cerebellum, Nrg3 was found in both Purkinje cells and granule neurons. In the rodent brain, Nrg3 is the most abundantly expressed of the Nrgs and its patterns of expression differ both temporally and spatially from that of Nrg1 and Nrg2. These findings suggest that Nrg3 plays roles that are distinct from the other Nrg family members.     

Expression of s-laminin and laminin in the developing rat central nervous system.

The extracellular matrix component, s-laminin, is a homologue of the B1 subunit of laminin. S-laminin is concentrated in the synaptic cleft at the neuromuscular junction and contains a site that is adhesive for motor neurons, suggesting that it may influence neuromuscular development. To ascertain whether s-laminin may also play roles in the genesis of the central nervous system, we have examined its expression in the brain and spinal cord of embryonic and postnatal rats. S-laminin was not detectable in synapse-rich areas of adults. However, s-laminin was present in discrete subsets of three laminin-containing structures: (1) In the developing cerebral cortex, laminin and s-laminin were expressed in the subplate, a transient layer through which neuroblasts migrate and cortical afferents grow. Both laminin and s-laminin disappeared as embryogenesis proceeded; however, laminin was more widely distributed and present longer than s-laminin. (2) In the developing spinal cord, laminin was present throughout the pia. In contrast, s-laminin was concentrated in the pia that overlies the floor plate, a region in which extracellular cues have been postulated to guide growing axons. (3) In central capillaries, s-laminin appeared perinatally, an interval during which the blood-brain barrier matures. In contrast, laminin was present in capillary walls of both embryos and adults. To extend our immunohistochemical results, we used biochemical methods to characterize s-laminin in brain. We found that authentic s-laminin mRNA is present in the embryonic brain, but that brain-derived s-laminin differs (perhaps by a posttranslational modification) from that derived from nonneural tissues. We also used tissue culture methods to show that glia are capable of synthesizing "brain-like" s-laminin, and of assembling it into an extracellular matrix. Thus, glia may be one cellular source of s-laminin in brain. Together, these results demonstrate that s-laminin is present in the developing central nervous system, and raise the possibility that this molecule may influence developmental processes.     

Erythropoietin gene expression in different areas of the developing human central nervous system

Evidence from cell culture and animal experiments suggests a neuroprotective and neurotrophic function of erythropoietin (EPO). We have quantitated the distribution of EPO mRNA expression in the developing human central nervous system (CNS). PATIENTS AND METHODS: Up to seven biopsies from different areas of the CNS of four preterm fetuses (gestational age 23-37 weeks) were obtained at routine postmortem examinations. EPO mRNA was quantitated by competitive PCR in samples from the CNS, the kidneys, and the liver where the EPO gene is predominantly expressed at this gestational age. RESULTS: EPO mRNA was most abundant in one sample from the cerebellum (0.29 amol/microg total RNA [amol=10(-18)mol]) and two from the pituitary gland (0.23 amol/microg total RNA), but levels varied considerably. EPO mRNA in the cortex cerebri (median 0.12 amol/microg total RNA; n=4) dominated over the expression in the corpora amygdala (median 0.05 amol/microg total RNA; n=4), the hippocampus (median 0.03 amol/microg total RNA; n=4), or the basal ganglia (median 0.01 amol/microg total RNA; n=3). Only little EPO mRNA (<0.01 and 0.06 amol/microg total RNA) was found in the spinal cord. EPO mRNA levels in the cerebellum, pituitary gland, or the cerebral cortex were within the same range as in the liver (0.03-1.67 amol/microg total RNA; n=4), or the kidneys (0.06-0.79 amol/microg total RNA; n=4). CONCLUSION: We found the EPO gene expressed throughout the fetal human CNS. Our data provide the basis to discuss a function for EPO in the brain of humans as well.     

Expression of receptor tyrosine kinase RYK in developing rat central nervous system

Receptor tyrosine kinase RYK is a mammalian homologue of Drosophila Lio, which is involved in learning and memory and in axon guidance. We cloned a rat ryk gene and characterized its expression pattern in the central nervous system. Northern blot analysis of the whole brain revealed that the RYK mRNA was abundant during the period from 13 to 18 embryonic days (E13-18) and it decreased by E20. In the postnatal brain, the RYK signal was higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization revealed that RYK was expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA was detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlapped the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis showed that the same cells expressed both RYK and nestin in the ventricular zone. In the postnatal brain, RYK was predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system.     

D-serine in the developing human central nervous system

To elucidate the role of D-serine in human central nervous system, we analyzed D-serine, L-serine, and glycine concentrations in cerebrospinal fluid of healthy children and children with a defective L-serine biosynthesis (3-phosphoglycerate dehydrogenase deficiency). Healthy children showed high D-serine concentrations immediately after birth, both absolutely and relative to glycine and L-serine, declining to low values at infancy. D-Serine concentrations were almost undetectable in untreated 3-phosphoglycerate dehydrogenase-deficient patients. In one patient treated prenatally, D-serine concentration was nearly normal at birth and the clinical phenotype was normal. These observations suggest a pivotal role for D-serine in normal and aberrant human brain development.     

Selective vulnerability in the developing central nervous system

Selective patterns of cerebral injury are observed after a variety of insults at different ages during development. Distinct populations of cells demonstrate selective vulnerability during these specific developmental stages, which may account for the observed patterns of injury. We review the evidence that injury to preoligodendrocytes and subplate neurons contributes to periventricular white matter injury in preterm infants, whereas thalamic neuronal cell vulnerability and neuronal nitric oxide synthase-expressing striatal interneurons resistance result in deep gray nuclei damage in the term infant. The unique roles of particular mechanisms including oxidative stress, glutamatergic neurotransmission, and programmed cell death are discussed in the context of this selective vulnerability.     

Structure and expression of the glycine cleavage system in rat central nervous system

This study investigated the expression of corticotropin releasing hormone (CRH) and its receptor CRHR-1, and arginine vasopressin (AVP) mRNAs during the stress hyporesponsive periods of late pregnancy and lactation (day-3) and in virgin stress-responsive females. In situ hybridization histochemistry showed that basal CRH mRNA in the paraventricular nucleus (PVN) decreased in pregnant and increased in lactating rats (compared with virgin controls), whereas it increased after restraint stress only in virgin rats. Basal PVN CRHR-1 mRNA increased markedly in all groups but reached lower levels in pregnant rats. Basal AVP mRNA in the parvocellular PVN was higher in lactating rats, and in contrast to CRH mRNA, it increased after stress in all groups. In medial preoptic area (MPOA) CRH mRNA levels were higher in lactating females compared with virgin and pregnant rats, and unexpectedly they decreased markedly after stress only in virgin rats. CRH mRNA levels in the central and medial nuclei of the amygdala were higher in lactating rats than in virgin or pregnant ones, and stress had no effect in either group. These data suggest that these stress hyporesponsive periods: (1) do not depend on basal CRH mRNA expression in the PVN; (2) appear to have intact stress-activated afferent pathways to the PVN, as shown by preservation of CRHR-1 and AVP responses to stress, but the information may be differently processed; (3) are associated with an alteration in a CRH mediated pathway from the MPOA.     

Differential expression of mRNAs for the NGF family of neurotrophic factors in the adult rat central olfactory system.

The cellular localization of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), in the rat central olfactory system was evaluated with in situ hybridization of 35S-labeled cRNA probes. In the main olfactory bulb, low levels of NGF and BDNF mRNA expression were detected. NGF mRNA was restricted to the glomerular region while BDNF mRNA was predominantly localized to the granule cell layer. No cellular hybridization to NT3 cRNA was seen. The accessory olfactory bulb did not express detectable levels of mRNA for any of the three related neurotrophic factors. Areas which receive olfactory bulb afferents expressed comparatively high levels of both NGF and BDNF mRNA. Cell labeling with cRNAs for NGF and BDNF occurred throughout the cellular layers of the anterior olfactory nucleus and in layers 2 and 3 of rostral piriform cortex. BDNF mRNA expression in these areas appeared more robust than that of NGF mRNA, while NT3 mRNA was not detectable. In contrast, tenia tecta exhibited dense labeling with the cRNAs for all three neurotrophic factors. The localization of NGF mRNA to primary target neurons of the olfactory nerve in the periglomerular region of the main olfactory bulb suggests that bulb cells may influence the ingrowth and continual turnover of olfactory sensory afferents. However, as there is a strong correlation between the distribution of neurotrophic factor mRNAs within rostral olfactory structures and the distribution of centrifugal cholinergic afferents, it is more likely that bulb-derived NGF, and possibly BDNF, act on the cholinergic neurons of the basal forebrain.     

The effects of protein malnutrition on the developing central nervous system in the rat

We have carried out a multi-disciplinary study of the effects of prenatal protein malnutrition on the developing rat brain. These experiments, involving anatomical, physiological, biochemical, and behavioral approaches, have revealed that malnutrition induced prenatally can affect various parameters of brain growth and development. Some of these effects can be reversed depending on when dietary restitutions are carried out. However, if protein malnutrition is maintained during the brain growth spurt or critical growth periods there are many permanent sequelae that cannot be reversed by subsequent restitution of high protein diets. We have reviewed the concept of critical periods of brain growth relative to the various aspects of neural morphogenesis in the rat, that is, the birth of neurons, migration of neurons, differentiation of neurons, and synapse formation. We have also discussed the rapid phases of brain growth in the rat as compared to similar phases in other species as a basis for determining whether the rat model can provide time-tables for brain growth in other species, including man. Different components of the brain, both morphological and chemical, have their own cycles of rapid development so that insults to the brain at particular periods affect particular aspects of brain chemistry and neuronal systems. Development of chemical circuits in the brain, such as the aminergic neurons, and their eventual adequate functioning, depends on development of the neurotransmitters themselves. These latter are markedly affected by protein malnutrition. Major physiological-behavioral states, such as the sleep-waking continuum, are markedly affected by protein malnutrition as are many behaviors. Some of these latter are merely late or retarded in development but others remain permanently altered. By approaching the problem of protein malnutrition from multiple points of view we have been able to pinpoint several brain areas showing the most drastic residua of early protein malnutrition and are beginning, by use of morphometric, electro-ontogenetic, biochemical development and behavioral studies, to define brain locales and basic mechanisms by which these insults produce their effects.     

Neuronal expression of caspase-1 immunoreactivity in the rat central nervous system

Caspase-1/interleukin-1beta (IL-1beta)-converting enzyme (ICE) cleaves IL-1beta and IL-18 precursor proteins to the active forms of these proinflammatory cytokines. Since both cytokines are constitutively expressed in the brain, we investigated whether this is also the case for caspase-1. Using an antibody raised against the p10-subunit of the active enzyme, constitutive expression of caspase-1 immunoreactivity was found in nerve cells in the arcuate nucleus and in nerve fibres throughout the brain. Co-localisation with alpha-melanocyte stimulating hormone was demonstrated. The distribution pattern of caspase-1 immunoreactive structures is consistent with a role to produce mature IL-1beta in regions where IL-1beta mediates fever and sleep.     

Topographical distribution in the adult rat brain of neurotrophic activities directed to central nervous system targets

Cortex, hippocampus, septum and striatum of day 18 rat embryos were grafted to several brain regions of young adult rats which had been lesioned in the chosen area 4 days earlier. Thirty days after transplantation, the grafts were fixed and morphometrically analysed under light microscope. The volumes, neuronal densities and total number of neurons of the transplants were compared. Each graft survived best when transplanted to its original region. Good survival was also achieved by heterotopic grafts between regions that are anatomically related. Striatal grafts showed reasonable survival only when transplanted to their original site. In a second series of experiences, the neurons from the same embryonic brain regions were cultured in a defined medium, to which was added tissue extracts from the lesioned regions of the adult brain. The neuronal survival was estimated. The in vitro results are closely related to those obtained in vivo. This experimental evidence agrees with the theory of the existence of a retrograde transport of NGF from the hippocampus to the septum, sustaining the survival of the latter. On the other hand, our results demonstrate the existence of other unidentified neurotrophic factors in the central nervous system which differ from one region to another.     

Deposition of the myelin-associated glycoprotein in specific regions of the developing rat central nervous system

In developing optic nerve and medulla of the rat, the concentration of the myelin-associated glycoprotein (MAG) increases rapidly between 10 and 30 days of age to reach adult levels of 4.4 and 4.1 ng per micrograms total protein, respectively. The general shapes of the developmental curves for MAG and basic protein (BP) are similar, but the rapid accumulation of MAG progresses earlier than that of BP by about 3-6 days.