Adult Brain Cortex Research Articles

Establishing gene regulatory networks from Parkinson's disease risk loci.

The latest meta-analysis of genome-wide association studies identified 90 independent variants across 78 genomic regions associated with Parkinson’s disease, yet the mechanisms by which these variants influence the development of the disease remains largely elusive.To establish the functional gene regulatory networks associated with Parkinson’s disease risk variants, we utilized an approach combining spatial (chromosomal conformation capture) and functional (expression quantitative trait loci) data.We identified 518 genes subject to regulation by 76 Parkinson’s variants across 49 tissues, whicih encompass 36 peripheral and 13 CNS tissues. Notably, one-third of these genes were regulated via trans-acting mechanisms (distal; risk locus-gene separated by >1 Mb, or on different chromosomes). Of particular interest is the identification of a novel trans-expression quantitative trait loci–gene connection between rs10847864 and SYNJ1 in the adult brain cortex, highlighting a convergence between familial studies and Parkinson’s disease genome-wide association studies loci for SYNJ1 (PARK20) for the first time. Furthermore, we identified 16 neurodevelopment-specific expression quantitative trait loci–gene regulatory connections within the foetal cortex, consistent with hypotheses suggesting a neurodevelopmental involvement in the pathogenesis of Parkinson’s disease. Through utilizing Louvain clustering we extracted nine significant and highly intraconnected clusters within the entire gene regulatory network. The nine clusters are enriched for specific biological processes and pathways, some of which have not previously been associated with Parkinson’s disease.Together, our results not only contribute to an overall understanding of the mechanisms and impact of specific combinations of Parkinson’s disease variants, but also highlight the potential impact gene regulatory networks may have when elucidating aetiological subtypes of Parkinson’s disease.

Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

We previously showed that zinc (Zn) deficiency affects the STAT3 signaling pathway in part through redox-regulated mechanisms. Given that STAT3 is central to the process of astrogliogenesis, this study investigated the consequences of maternal marginal Zn deficiency on the developmental timing and key mechanisms of STAT3 activation, and its consequences on astrogliogenesis in the offspring. This work characterized the temporal profile of cortical STAT3 activation from the mid embryonic stage up to young adulthood in the offspring from dams fed a marginal Zn deficient diet (MZD) throughout gestation and until postnatal day (P) 2. All rats were fed a Zn sufficient diet (control) from P2 until P56. Maternal zinc deficiency disrupted cortical STAT3 activation at E19 and P2. This was accompanied by altered activation of JAK2 kinase due to changes in PTP1B phosphatase activity. The underlying mechanisms mediating the adverse impact of a decreased Zn availability on STAT3 activation in the offspring brain include: (i) impaired PTP1B degradation via the ubiquitin/proteasome pathway; (ii) tubulin oxidation, associated decreased interactions with STAT3 and consequent impaired nuclear translocation; and (iii) decreased nuclear STAT3 acetylation. Zn deficiency-associated decreased STAT3 activation adversely impacted astrogliogenesis, leading to a lower astrocyte number in the early postnatal and adult brain cortex. Thus, a decreased availability of Zn during early development can have a major and irreversible adverse effect on astrogliogenesis, in part via multistep alterations in the STAT3 pathway.

Early Developmental Marginal Zinc Deficiency Affects Neurogenesis Decreasing Neuronal Number and Altering Neuronal Specification in the Adult Rat Brain.

During pregnancy, a decreased availability of zinc to the fetus can disrupt the development of the central nervous system leading to defects ranging from severe malformations to subtle neurological and cognitive effects. We previously found that marginal zinc deficiency down-regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and affects neural progenitor cell (NPC) proliferation. This study investigated if marginal zinc deficiency during gestation in rats could disrupt fetal neurogenesis and affect the number and specification of neurons in the adult offspring brain cortex. Rats were fed a marginal zinc deficient or adequate diet throughout gestation and until postnatal day (P) 2, and subsequently the zinc adequate diet until P56. Neurogenesis was evaluated in the offspring at embryonic day (E)14, E19, P2, and P56 measuring parameters of NPC proliferation and differentiation by Western blot and/or immunofluorescence. At E14 and E19, major signals (i.e., ERK1/2, Sox2, and Pax6) that stimulate NPC proliferation and self-renewal were markedly downregulated in the marginal zinc deficient fetal brain. These alterations were associated to a lower number of Ki67 positive cells in the ventricular (VZs) and subventricular zones (SVZs). Following the progression of NPCs into intermediate progenitor cells (IPCs) and into neurons, Pax6, Tbr2 and Tbr1 were affected in the corresponding areas of the brain at E19 and P2. The above signaling alterations led to a lower density of neurons and a selective decrease of glutamatergic neurons in the young adult brain cortex exposed to maternal marginal zinc deficiency from E14 to P2. Current results supports the concept that marginal zinc deficiency during fetal development can disrupt neurogenesis and alter cortical structure potentially leading to irreversible neurobehavioral impairments later in life.

Isolation of vascular endothelial cells from intact and injured murine brain cortex-technical issues and pitfalls in FACS analysis of the nervous tissue.

CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vessel-forming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1(+) , CD45(-) , c-Kit(+) ). Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies.

Abstract W P218: Interleukin 21 receptor: A Major Modulator of Infarct Volume

Using the permanent middle cerebral artery occlusion (MCAO) model of stroke, we have demonstrated that different inbred mouse strains show profound differences in infarct volume, indicating that infarction is under strong genetic control. To identify natural genetic determinants modulating infarction, we employed quantitative trait locus (QTL) linkage analysis and a genome-wide association study of cerebral infarct volume. We identified a locus on distal chromosome 7 that contributes over 50% of the variation in infarct volume, as well as other loci of smaller effect. Using interval-specific ancestral haplotype analysis, we fine-mapped the chromosome 7 locus to only 12 candidate genes. To identify the gene(s) underlying this locus, we determined the strain-specific transcript levels of all 12 genes in relevant tissues that included P1 and adult brain cortex, and embryonic macrophages, the latter due to their importance in the development of the cerebrovascular system. One gene, interleukin 21 receptor (Il21r), showed a 7-fold expression difference between strains and harbors a coding SNP difference that segregates with infarct volume. To determine whether Il21r is a major modulator of infarction, we examined Il21r in mice for their cerebrovascular anatomy as well as the cerebral infarct volume after MCAO. While Il21r-/- mice show a moderate reduction in collateral vessel connections compared to wild-type littermate mice cerebral infarct volume in Il21r-/- mice is increased 3-fold. This suggests that Il21r has effects on both cerebrovascular anatomy and innate neuroprotection. To examine the latter, we performed an ex vivo study of brain slices under in vitro oxygen deprivation. In this system devoid of any potential circulatory effects, but retaining appropriate tissue architecture, Il21r-/- brain slices showed an increase in oxygen-deprivation induced cell death, showing that Il21r is also involved in cerebrovascular-independent neuroprotection. Biochemical studies of the brain slices show that Il21R regulates ischemia-induced apoptosis. The identification of Il21R as a cerebrovascular-independent modulator of infarct volume provides a fundamental advance in the understanding of genetic modulation of ischemic stroke.

Kainate-induced calcium overload of cortical neurons in vitro: Dependence on expression of AMPAR GluA2-subunit and down-regulation by subnanomolar ouabain

Whereas kainate (KA)-induced neurodegeneration has been intensively investigated, the contribution of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in neuronal Ca2+ overload ([Ca2+]i) is still controversial. Using Ca2+ imaging and patch-clamp techniques, we found different types of Ca2+ entry in cultured rat cortical neurons. The presence of Ca2+ in the extracellular solution was required to generate the [Ca2+]i responses to 30μM N-methyl-d-aspartate (NMDA) or KA. The dynamics of NMDA-induced [Ca2+]i responses were fast, while KA-induced responses developed slower reaching high [Ca2+]i. Ifenprodil, a specific inhibitor of the GluN2B subunit of NMDARs, reduced NMDA-induced [Ca2+]i responses suggesting expression of GluN1/GluN2B receptors. Using IEM-1460, a selective blocker of Ca2+-permeable GluA2-subunit lacking AMPARs, we found three neuronal responses to KA: (i) IEM-1460 resistant neurons which are similar to pyramidal neurons expressing Ca2+-impermeable GluA2-rich AMPARs; (ii) Neurons exhibiting nearly complete block of both KA-induced currents and [Ca2+]i signals by IEM-1460 may represent interneurons expressing GluA2-lacking AMPARs and (iii) neurons with moderate sensitivity to IEM-1460. Ouabain at 1nM prevented the neuronal Ca2+ overload induced by KA. The data suggest, that cultured rat cortical neurons maintain functional phenotypes of the adult brain cortex, and demonstrate the key contribution of the Na/K-ATPase in neuroprotection against KA excitotoxicity.

Proteomic analysis of human fetal and adult brain cortex

To study the differences of protein expression levels in the brain cortex of human fetus and adult with proteomics technique, and provide preliminary data on the change of proteins during brain development. Proteins extracted from human temporal lobes in fetal (3 month and 5 month respectively) and adult (30 years old) brain were separated by two-dimensional gel electrophoresis (2DE). The proteins were then stained with colloidal Coomassie blue to produce a high-resolution map of the proteiome. The differential protein spots were analyzed by PDQuest 7.0 software and 8 spots, which were gradually reduced or gradually increased in brain development process and the protein spots of difference over two-fold in the brain, were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF). (1) On average, 642, 511 and 527 protein spots could be obtained in the temporal lobes of adult, 3 month and 5 month fetus. The matching rate of images was 87%. The basic proteins in adult brain were obviously much more than that in the fetus; (2) There were 172, 171 and 152 singular protein spots in temporal lobes of adult, 3 month and 5 month fetus respectively.(3) Compared with adult, there were 131 and 115 different protein spots in the 3 month and 5 month fetus respectively. There were 60 and 40 protein spots with more than 2 fold difference, among which 24 and 17 were down-regulated, and 36 and 23 were up-regulated respectively. (4) There was different expression in proteins such as serum albumin, triosephosphate isomerase, etc. in the 3 groups. Fatty acid binding protein 7 and unnamed proteins were only highly expressed in the 3 month brain; ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit and transducin beta-1 subunit were up-regulated in adult brain. Serum albumin decreases gradually with brain development. However, ATP synthase, mitochondrial F0 complex, and triosephosphate isomerase increase gradually with brain development. The proteins of human brain cortex were obviously changed from embryonic stage to adult. The differentially displayed proteins may provide further insight into the understanding of development of human brain.

Spatial and temporal expression of vasodilator-stimulated phosphoprotein (VASP) in fetal and adult human cerebral cortex

Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP protein family and was for the first time identified in human platelets. VASP plays a role for cell adhesion and cell migration because of E/DFPPPPXD/E amino acid sequence in its structure which gives reaction with focal adhesion proteins. In this study we suggest that VASP expression may have important role for neural cell migration, differentiation, axonal growth and angiogenesis during prenatal cerebral cortical development. Our aim is to detect VASP expression by means of immunohistochemistry and Western blot analysis in developing human neocortex and adult brain cortex (n=12 samples from first, second, and third trimesters and n=3 adult normal cerebral cortex). Our results suggest that VASP showed different immunostaining patterns between cerebral cortical plates in prenatal and adult human brain samples. We observed that the expression patterns of the VASP protein are clearly identified in fibers, cytoplasm of neural cells and endothelial cells of vessels. We detected that VASP indicates progressive expression from the adult brain to second trimester neocortexes. Therefore, we suggest that VASP may play a crucial role in the regulation of human neonatal cerebral cortical development.

Met158 variant of the catechol-O-methyltransferase genotype is associated with thicker cortex in adult brain

Cortical thickness has been proposed as a new promising brain imaging endophenotype in elucidating the nature of gene–brain relationships. Here, we define the morphological impact of the Val 158Met polymorphism in the catechol-O-methyltransferase (COMT) gene on human brain anatomy. One hundred and forty-nine adult healthy subjects (mean age: 40.7±16.1; ranging from 19 to 76 years) were genotyped (38 in the homozygous Val 158 group; 80 in the Val 158 Met group; 31 in the homozygous Met 158 group) for the COMT polymorphism and underwent morphological examination. Surface-based analysis of the cortical mantle showed that the COMT genotype was associated with structural differences in the right superior temporal sulcus and inferior prefrontal sulcus, where the individuals carrying the Met 158 allele had a thicker cortex with respect to their Val 158 counterparts. Our study extends the previous evidence found on pediatric population to the adult population, demonstrating that the higher synaptic dopamine levels associated with the presence of the Met 158 allele may influence neuronal architecture in brain structures important for executive and emotional processing.

Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3).

CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about the expression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRalpha beta T cells in the spleen and thymus of naive mice express surface mCD223 (<3 %), approximately 18 % TCR gamma delta T cells and approximately 10 % NK cells are positive. This small population of TCRalpha beta T cells are cycling memory T cells (BrdU(+), CD44(hi), CD62L(lo)). In contrast, all T cells express mCD223 2-3 days post activation. This study and the anti-CD223 mAb should greatly assist in the elucidation of CD223 function.

Varied Tropism of HIV-1 Isolates Derived from Different Regions of Adult Brain Cortex Discriminate between Patients with and without AIDS Dementia Complex (ADC): Evidence for Neurotropic HIV Variants

A number of infected individuals develop neuropathological disorders, such as AIDS dementia complex (ADC), as a consequence of HIV/AIDS. The biological features governing HIV entry and tropism in different brain cell types remain unclear, as do the genetics of the virus regulating these events and the neuropathogenic processes within the brain tissues. HIV-1 was isolated from the right and left parietal, occipital, and frontal lobes of the brain cortex of three HIV-1-infected patients: two with ADC and one without. The viral strains were studied from the innate tissues and various primary cell cultures. The kinetics and tropism of viral strains from different brain regions showed clear differences on various primary cell types (monocytes, monocyte-derived macrophages, and T cells), which could discriminate between biological behavior of HIV-1 strains from patients with and without ADC. The variable effect of different donor cells on tropism was also clearly evident. The majority (with a few exceptions) of isolates from different brain regions of all three patients used CCR5 as coreceptor for entry. The consistent CCR5 use, macrophage tropism, and non-syncytium-inducing phenotype were the main characteristics of the brain-derived HIV-1 strains from all three patients. Importantly, viral strains derived directly from innate brain tissue of the patient without ADC showed some differences from the cultured variants of the same patient, whereas those from brain tissue of the patients with ADC were more similar to the culture-adapted strains. This suggests that the emergence of primary cell type-adapted isolates during ADC may play a crucial role in the development and progression of the neuropathology associated with ADC. The different genotypes residing in different areas of brain combined with their differential tropism and coreceptor use suggest that neurotropic variants exist that may be governing the neurological manifestation of HIV disease in infected patients.

Transport of α-Ketoisocaproate in Rat Cerebral Cortical Neurons

Transport of α-ketoisocaproic acid (KIC), the product of leucine transamination, was studied in the cerebral cortex cells isolated from the adult rat brain. The process of [14C]KIC accumulation was followed in the presence of aminooxyacetate, an inhibitor of transaminases. Accumulation of KIC was not affected by Na+ replacement, its initial velocity was observed to be higher upon lowering of external pH. Addition of KIC promoted acidification of cytoplasmic pH, monitored with 2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. The detected inhibition of KIC accumulation by α-cyano-4(OH)cinnamate pointed to an involvement of one of monocarboxylate transporters (MCT), although 4,4′-diisothiocyano-2,2′-stilbenedisulphonate was without effect. Accumulation of KIC was inhibited by lactate; the effect of pyruvate was detected to be much weaker. Other branched-chain α-ketoacids (ketoisovalerate, keto-methylvalerate), as well as β-hydroxybutyrate and valproate decreased the transport of KIC by 30, 60, and 80%, respectively. The observed characteristics of KIC accumulation in the cortical neurons indicate an involvement of one of the MCT transporters. A crucial role of SH group(s) in the process of KIC accumulation, excluding MCT2, indicates the MCT1, although an involvement of another isoform of MCT in the process of KIC transport in neurons from cerebral cortex of adult brain has not been definitely excluded.

Amyloid beta peptide 25-35 modulates hydrolysis of phosphoinositides by membrane phospholipase(s) C of adult brain cortex.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C. Cholinergic pathways are important in learning and memory, and deficits in cholinergic transmission have been implicated in Alzheimer's disease (AD). AD is also associated with increased beta-amyloid plaques. In the present study, we have investigated the effect of the amyloid beta (A beta) synthetic peptide homologous to residue 25-35 of A beta in nonaggregated and aggregated forms on the degradation of inositol phospholipids. Synaptic plasma membranes (SPM) and the cytosolic fraction from rat brain cortex served as a source of enzymes. The studies were carried out with radioactive inositol phospholipids in the presence of endogenous and 2 mM CaCl2. The enzyme(s) activity was evaluated by determination of the product formation of [3H]inositol-1-phosphate (IP1) or [3H]inositol-1,4,5-trisphosphate (IP3). Results show that the PI-PLC activity was significantly higher in cytosol compared to SPM, and this enzyme was stimulated by 2 mM CaCl2, but not by GTPgammaS or carbachol, a cholinergic receptor agonist. Activity of the SPM-bound PIP2-PLC was similar to that in cytosol and was not activated by 2 mM CaCl2. The SPM PIP2-PLC was significantly stimulated by GTPgammaS together with the cholinergic agonist, carbachol. Fresh-water-soluble A beta 25-35 activated PI-PLC in SPM markedly by two- to threefold, but this effect was absent in the presence of 2 mM CaCl2. Moreover, A beta 25-35 had no effect on basal PIP2-PLC activity and cytosolic PI-PLC and PIP2-PLC. The aggregated form of A beta 25-35 significantly inhibited PIP2-PLC only in the presence of endogenous CaCl2. It also inhibited the carbachol and GTP(gamma)S-stimulated PIP2-PLC. Our findings show that depending on the aggregation state and Ca2+ concentration, A beta modulates phosphoinositide degradation differently and exclusively in brain synaptic plasma membranes. Our data suggested that aggregated A beta peptide may be responsible for the significant impairment of phosphoinositide signaling found in brain membranes during AD.

Human 12(R)-Lipoxygenase and the Mouse Ortholog: MOLECULAR CLONING, EXPRESSION, AND GENE CHROMOSOMAL ASSIGNMENT

Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.

Aging Diminishes Serotonin‐Stimulated Arachdonic Acid Uptake and Cholinergic Receptor‐Activated Arachidonic Acid Release in Rat Brain Cortex Membrane

Synaptoneurosomal and synaptosomal fractions from the brain cortex of adult (4-month-old) and aged (27-month-old) rats were used for studies on the uptake and subsequent release of [14C]arachidonic acid ([14C]AA) from brain lipids. The incorporation of AA and the pattern of its uptake into lipids of the aged brain cortex synaptoneurosomes and synaptosomes were not significantly different when compared with those in the adult brain cortex fractions. Serotonin (5-HT), at 10 microM to 1 mM in the presence of pargyline and the agonist of the 5-HT1A receptor, buspirone, stimulated AA uptake into membrane lipids, mainly into phosphatidylinositol, by about 40% exclusively in adult brain synaptoneurosomes. Aging significantly diminished the effect of 5-HT on AA uptake. Synaptoneurosomal and synaptosomal fractions prelabeled with [14C]AA were used subsequently for investigation of voltage-dependent, muscarinic and 5-HT receptor-mediated AA release. Aging diminished markedly carbachol-stimulated Ca(2+)-dependent AA liberation from membrane lipids of synaptoneurosomes and synaptosomes. Moreover, aging decreased voltage-dependent and 5-HT2 receptor-mediated AA release. These results show that aging affects receptor-dependent AA uptake and pre- and postsynaptic receptor-mediated AA release. These modulations of AA incorporation and release in aged brain may be of pathophysiological significance, in view of the importance of these processes for signal transmission in the brain. The changes of receptor-dependent processes of deacylation and reacylation may be responsible for alteration in the function of neuronal cells and may affect learning and memory ability and brain plasticity during aging.

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.

Examination of autologous and embryonic cortical brain tissue transplantation to adult brain cortex in rats.

Autologous and embryonic cortical brain tissue was transplanted to adult rats in order to reconstruct experimentally degenerated cortical brain tissue. Rats were decapitated within 6 or 12 weeks. Viability of the graft tissues was studied by light and electron microscopy. Embryonic cortical brain tissue grafts became enlarged but adult cortical brain tissue grafts were found to be unaltered. Electron-microscopically observed mitochondria and other cell organellae and the newly vascularized areas clearly showed that the graft tissues were alive.

Development of binding sites for vasoactive intestinal polypeptide in mouse cerebral cortex and cultured cortical neurons

Binding sites for vasoactive intestinal polypeptide (VIP) and the content of immunoreactive VIP were measured in the foetal and neonatal mouse brain cortex and primary cultures of foetal murine brain-cortical neurons and astrocytes. The amount of cortical VIP binding sites and the concentration of immunoreactive VIP were low before birth, but increased postnatally reaching adult level at about 3 weeks of age. In cultures, a similar rise in neuronal binding sites occurred after 10 days, whereas the VIP concentration remained lower than in adult brain cortex. No binding sites or immunoreactive VIP could be detected in cultured astrocytes. The VIP binding sites were heterogeneous both in developing brain cortex and cultured neurons, and consisted of two classes of binding sites. The high affinity constants (2.5–2.9 nM) as well as low affinity constants (50–76 nM) were unchanged during the development, whereas the number of binding sites increased. In cultured neurons the binding constants were similar to those found in the adult mouse brain cortex. The peptide specificity for the VIP binding was similar in brain cortex and neuronal culture. In conclusion, the maturation of murine brain cortical neurons is accompanied by the development of synaptosomal VIP receptors in support of the neurotransmitter function of VIP.

Studies on neurotransmitter-stimulated phospholipid metabolism with cerebral tissue suspensions: A possible biochemical correlate of synaptogenesis in normal and undernourished rats

The phenomenon of neurotransmitter-stimulated incorporation of 32Pi into phosphatidic acid and inositol phosphatides (neurotransmitter effect) in developing brain was studied in vitro as a possible measure of synaptogenesis. While the neurotransmitter effect was not observed with brain homogenates, highly consistent and significant effects were noted with brain tissue suspensions obtained by passing the tissue through nylon bolting cloth. The magnitude of the effect decreased with the increase in mesh number. Maximum stimulations obtained with the 33 mesh adult brain cortex preparations (mean±S.E.M. of6experiments) were203 ± 8%, 316 ± 11 % and150 ± 8% with 10 −3 M acetylcholine (ACh) + 10 −3 M eserine; 10 −2 M norepinephrine (NE) and 10 −2 M serotonin (5-HT), respectively. Experiments with developing rat brain at 7, 14 and 21 days of age showed that the neurotransmitter effects due to ACh, NE and 5-HT increase progressively in different regions of the brain but that there are marked regional differences. It is suggested that the neurotransmitter effect is a valid biochemical correlate of synaptogenesis. In rats undernourished from birth t0 21 days of age, by increasing the litter size, the neurotransmitter effect with ACh, NE or 5-HT was not altered in the cortex but was significantly reduced in the brain stem. In cerebellum the effects due to ACh and NE were significantly altered, while that with 5-HT was unaffected. It is concluded that cholinergic, adrenergic and serotonergic synapses are relatively unaffected in the cortex but are significantly affected in the brain stem by undernutrition. In the cerebellum of undernourished rats the adrenergic and cholinergic, but not serotonergic systems, are altered.

Acetoacetate metabolism in infant and adult rat brain in vitro.

1. Acetoacetate or dl-beta-hydroxybutyrate increases the rate of oxygen consumption to a smaller extent than that brought about by glucose or pyruvate in adult rat brain-cortex slices but to the same extent as that in infant rat brain-cortex slices. 2. The rate of (14)CO(2) evolution from [1-(14)C]glucose considerably exceeds that from [6-(14)C]glucose in respiring infant rat brain-cortex slices, in contrast with adult brain-cortex slices, suggesting that the hexose monophosphate shunt operates at a greater rate in the infant rat brain than in the adult rat brain. 3. The rate of (14)CO(2) evolution from [3-(14)C]acetoacetate or dl-beta-hydroxy[3-(14)C]butyrate, in the absence of glucose, is the same in infant rat brain slices as in adult rat brain slices. It exceeds that from [2-(14)C]glucose in infant rat brain but is less than that from [2-(14)C]glucose in adult rat brain. 4. Acetoacetate is oxidized in the brain through the operation of the citric acid cycle, as shown by the accelerating effect of glucose on acetoacetate oxidation in adult brain slices, by the inhibitory effects of malonate in both infant and adult brain slices and by its conversion into glutamate and related amino acids in both tissues. 5. Acetoacetate does not affect glucose utilization in adult or infant brain slices. It inhibits the rate of (14)CO(2) formation from [2-(14)C]glucose or [U-(14)C]-glucose the effect not being wholly due to isotopic dilution. 6. Acetoacetate inhibits non-competitively the oxidation of [1-(14)C]pyruvate, the effect being attributed to competition between acetyl-CoA and CoA for the pyruvate-oxidation system. 7. Acetoacetate increases the rate of aerobic formation of lactate from glucose with both adult and infant rat brain slices. 8. The presence of 0.1mm-2,4-dinitrophenol diminishes but does not abolish the rate of (14)CO(2) formation from [3-(14)C]acetoacetate in rat brain slices. This points to the participation of ATP in the process of oxidation of acetoacetate in infant or adult rat brain. 9. The presence of 5mm-d-glutamate inhibits the rate of (14)CO(2) formation from [3-(14)C]acetoacetate, in the presence or absence of glucose. 10. Labelled amino acids are formed from [3-(14)C]acetoacetate in both adult and infant rat brain-cortex slices, but the amounts are smaller than those found with [2-(14)C]glucose in adult rat brain and greater than those found with [2-(14)C]glucose in infant rat brain. 11. Acetoacetate is not as effective as glucose as a precursor of acetylcholine in adult rat brain but is as effective as glucose in infant rat brain slices. 12. Acetoacetate or beta-hydroxybutyrate is a more potent source of acetyl-CoA than is glucose in infant rat brain slices but is less so in adult rat brain slices.