A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

Hypothyroidism Alters the Expression of Prostaglandin D2 Synthase/β- Trace in Specific Areas of the Developing Rat Brain

Lipocalin-type prostaglandin D₂ synthase is the enzyme responsible for the synthesis of prostaglandin D₂, a major prostaglandin in the central nervous system. We analysed the effects of thyroid hormone deprivation on prostaglandin D₂ synthase gene expression in the developing rat brain. By in situ hybridization, the strongest prostaglandin D₂ synthase mRNA signal was detected in the leptomeninges and choroid plexus. The signal was greatly reduced in the cerebellar interlaminar meninges of hypothyroid rats aged 15 and 25 days. lmmunohistochemical studies defined changes in the location of the prostaglandin D₂ synthase protein. In control but not in hypothyroid animals, Cajal-Retzius neurons of cortical layer 1, and pyramidal cortical plate neurons were intensely stained on postnatal day 5. Conversely, prostaglandin D₂ synthase protein levels were higher in neurons of the CA1 and CA3 regions and the dentate gyrus of the hippocampus of hypothyroid animals on postnatal days 5, 15 and 25, and also in subplate neurons on postnatal days 15 and 25. In agreement with the in situ hybridization and northern blotting data, the major difference was found in the cerebellar interlaminar meninges of hypothyroid animals, where the protein was clearly down-regulated on postnatal days 15 and 25. These results show that hypothyroidism causes both age- and region-specific alterations in the expression and location of the prostaglandin D₂ synthase during postnatal brain development, probably reflecting a cell-specific regulatory effect of thyroid hormone on the prostaglandin D₂ synthase.     

Increased β-Amyloid Precursor Protein Expression in Astrocytes in the Gerbil Hippocampus Following Ischaemia: Association with Proliferation of Astrocytes

Increases in β-amyloid precursor proteins (APP), which include the β-amyloid senile plaque protein present in patients with Alzheimer's disease, have been shown to occur in models of neuronal damage and neurotoxic cell injury. This observation led us to examine the expression of these proteins after transient ischaemic episodes in the gerbil. Animals were killed 2–28 days after ischaemia and APP were detected by immunocytochemistry at the light and electron microscopic levels with an antibody raised against the C-terminal region of these proteins. The gliotic reaction was also examined using glial fibrillary acid protein (GFAP) immunoreactivity. Two days after ischaemia, neuronal cell death was observed in the hippocampal CA1 region accompanied by astrocyte hypertrophy. These hypertrophic astrocytes were found to be GFAP positive but stained weakly for APP. Seven days after ischaemia both astrocyte hypertrophic and hyperplasia, with identified mitotic figures, were observed. These hyperplasic astrocytes were intensely stained by the APP antibody, and were observed up to 28 days after ischaemia. This shows that neuronal cell death produced by transient ischaemia is followed by an increased APP expression which appears to be associated with the hyperplasic astrocytes but not with the initial hypertrophy of this cell population. These results, when taken together with those obtained in other models of neuronal damage or death, clearly suggest that APP expression follows neuronal death and is associated with astrocyte proliferation.     

Interleukin-1β is increased in the cerebrospinal fluid of patients with small infarcts

Background and purpose:  Interleukin-1beta (IL-1β) and interleukin-6 (IL-6) are involved in inflammatory responses during large vessel occlusion in animal models. The aim of this study was to investigate the intrathecal levels of cytokines in patients with acute small infarcts.
Methods:  Forty patients with acute minor stroke and 32 non-stroke patients (including 29 age- and gender-matched subjects) who received operations with spinal anesthesia were studied prospectively and underwent measurements of cerebrospinal fluid (CSF) IL-1β and IL-6 levels.
Results:  After an age- and gender-matched analysis of 58 patients (29 pairs), the mean intrathecal levels of IL-1β were 0.80 pg/ml in patients with small infarcts and 0.59 pg/ml in non-stroke patients ( P  <   0.0001). In addition, the mean CSF levels of IL-6 were 21.54 pg/ml and 7.52 pg/ml in the stroke and control groups, respectively ( P  =   0.38). These results were consistent with the data without matching. The CSF levels of IL-1β in the 40 stroke patients were significantly higher than in the 32 non-stroke controls ( P  <   0.0001).
Conclusions:  The proinflammatory cytokine IL-1β, but not IL-6, remained elevated in the CSF of patients in the acute stage of small infarcts.     

IL-1β and IL-6 excite neurones and suppress cholinergic neurotransmission in the myenteric plexus of the guinea pig

The effects of the inflammatory mediators interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) on myenteric neurones were investigated by intracellular recordings in a conventional myenteric plexus preparation of guinea pig ileum. Micropressure ejection of IL-1beta and IL-6 (10-7 mol L-1) both caused an excitatory effect in, respectively, 19% (13/70) and 7% (5/70) of the myenteric neurones. The IL-1beta-induced depolarizations were inhibited by superfusion of the IL-1beta receptor antagonist. The responses seen were tetrodotoxin-resistant, indicating a direct neuronal effect. Responses to both cytokines were seen in nitric oxide synthase-immunoreactive as well as choline acetyltransferase-immunoreactive neurones. In addition, both IL-1beta and IL-6 reversibly caused a presynaptic inhibition of acetylcholine release from cholinergic nerve terminals. Both cytokines had no effect on the slow excitatory postsynaptic potentials. Therefore, we can conclude that the inflammatory mediators IL-1beta and IL-6 can act as excitatory neuromodulators of gastrointestinal motility through direct excitatory actions on a subset of myenteric neurones and through the presynaptic inhibition of acetylcholine release.     

Hypophysiotropic Role and Hypothalamic Gene Expression of Corticotropin-Releasing Factor and Vasopressin in Rats Injected with Interleukin-1β Systemically or into the Brain Ventricles

Intact adult male rats were injected intravenously (i.v., 400 ng/kg), intraperitoneally (i.p., 400 ng/kg) or intracerebroventricularly (i.c.v., 100 ng/kg) with interleukin-1β (IL-1β) or its vehicle. In comparison with vehicle-treated animals, IL-1β induced significant (P<0.01) increases in plasma ACTH levels measured 30 min later regardless of the route of cytokine administration. These changes were markedly blunted in rats administered specific antibodies directed against corticotropin-releasing factor (CRF). In contrast, vasopressin (VP) antibodies significantly blunted ACTH released by the i.c.v. injection of IL-1β, but only modestly altered the effect of the systemic injection of the cytokine. We then used semi-quantitative in situ hybridization analysis to measure changes in steady-state mRNA levels, as they might occur in response to these same doses of IL-1β. Following administration of the vehicle, measurement of gene expression in the paraventricular (PVN) portion of the hypothalamus indicated a measurable amount of hybridization signals for both CRF and VP. No detectable changes in either CRF or VP gene expression were observed in rats injected with IL-1β i.v. or i.p. 5 h earlier. In contrast, the i.c.v. administration of the cytokine significantly (P<0.01) increased both CRF and VP mRNA levels measured 5 h later. These results suggest that while endogenous CRF modulates the response of the corticotrophs to this cytokine regardless of the route of administration, the role of VP is more important in rats injected centrally than in those injected peripherally. The observation that at the dose tested and over the time-course studied, systemic injection of IL-1β failed to alter CRF or VP gene expression, supports our earlier hypothesis that blood-born IL-1β acts primarily at the level of nerve terminals in the median eminence.     

Anatomical Gradients in Proliferation and Differentiation of Embryonic Rat CNS Accessed by Buoyant Density Fractionation: α3, β3 and γ2 GABAA Receptor Subunit Co-expression by Post-mitotic Neocortical Neurons Correlates Directly with Cell Buoyancy

Development of the CNS occurs as complex cascade of pre-programmed events involving distinct phases of cell proliferation and differentiation. Here we show these phases correlate with cells of specific buoyant densities which can be readily accessed by density gradient fractionation. Sprague-Dawley dams were pulse-labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at El1–22 dissociated with papain into single-cell suspensions. Proliferative cell populations were assessed by anti-BrdU and propidium iodide staining using flow cytometry. Cell differentiation was evaluated using molecular and immunocytochemical probes against mRNAs and antigens differentiating the neuroepithelial, neuronal and glial cell lineages. The results show the emergence of distinctive spatiotemporal changes in BrdU⁺ populations throughout the CNS during embryonic development, which were followed by corresponding changes in the cellular distributions of antigens distinguishing specific cell types. Fractionation of neocortical cells using discontinuous Percoll gradients revealed that an increasing number of cells increase their buoyancy during corticogenesis. Immunocytochemical and molecular characterization showed that the proliferative and progenitor cell populations are for the most part associated with lower buoyancy or higher specific buoyant densities (> 1.056 g/ml) whereas the post-mitotic, differentiated neurons generally separated into fractions of higher buoyancy or lower specific buoyant densities (<1.043 g/ml). Immunostaining with antibodies against several GABA_A receptor subunits (α3, β3, γ2) revealed that the highest percent (70–90%) of immunopositive cells could be identified in the most buoyant, differentiating neurons found in the cortical plate/subplate regions, with the lowest percent of the immunopositive cells found in the least buoyant, proliferative and progenitor cell populations originating from the ventricular/subventricular zones. Taken together, these results indicate that buoyant density is distinguishing characteristic of embryonic CNS cells transforming from primarily proliferative to mainly differentiating, and that fractionation of these cells according to their buoyant densities provides rapid access to the properties of specific cell lineages during the prenatal period of CNS development.     

Regulation of Multidrug Resistance P‐Glycoprotein in the Developing Blood–Brain Barrier: Interplay between Glucocorticoids and Cytokines

P‐glycoprotein (P‐gp) encoded by Abcb1 provides protection to the developing brain from xenobiotics. P‐gp in brain endothelial cells (BECs) derived from the developing brain microvasculature is up‐regulated by glucocorticoids and inhibited by pro‐inflammatory cytokines in vitro. However, little is known about how prenatal maternal glucocorticoid treatment can affect Abcb1/P‐gp function and subsequent cytokine regulation in foetal BECs. We hypothesised that glucocorticoid exposure increases Abcb1/P‐gp in the foetal brain microvasculature and enhances the sensitivity of Abcb1/P‐gp in BECs to the inhibitory effects of cytokines. BECs isolated from dexamethasone‐ or vehicle‐exposed foetal guinea pigs were cultured and treated with interleukin‐1β, interleukin‐6 or tumour necrosis factor‐α, and Abcb1/P‐gp expression and function were assessed. Prenatal dexamethasone exposure significantly increased Abcb1/P‐gp expression/activity and cytokine receptor levels in BECs of the foetal brain microvasculature. Foetal dexamethasone exposure in vivo also increased the subsequent responsiveness of BECs to pro‐inflammatory cytokines in vitro. In conclusion, maternal treatment with synthetic glucocorticoids appears to prematurely mature P‐gp mediated drug resistance at the foetal BBB in vivo and profoundly impact the subsequent responsiveness of P‐gp to pro‐inflammatory cytokines in the foetal BEC. The significance of these findings to foetal brain protection against xenobiotics and other P‐gp substrates in vivo requires further elaboration. However, the results of the present study may have implications for human pregnancy and foetal brain protection, particularly in cases of preterm birth combined with infection.     

Amyloid βprotein precursors with kunitz-type inhibitor domains and acetyl-cholinesterase in cerebrospinal fluid from patients with dementia of the Alzheimer type

We used the ELISA to measure the concentration of amyloid protein precursor with Kunitz type trypsin inhibitor domains (APPI) in CSF of dementia of the Alzheimer type (DAT) and examined the correlation of APPI with acetylcholinesterase (AChE) and somatostatin (SRIF). We found the APPI concentration in CSF of DAT to be significantly elevated compared with that of multi-infarct dementia and controls. We could significantly correlate APPI with AChE, but not correlate APPI with SRIF. The present results suggest that measurement of CSF APPI levels may be useful for diagnosis of DAT and the change of APPI may closely be associated with abnormality of acetylcholine system in DAT that has been reported.     

Co-localization of β/A4 and cystatin C in cortical blood vessels in Dutch, but not in Icelandic hereditary cerebral hemorrhage with amyloidosis

Based on the recent discovery of co-localization of β/A4 and cystatin C in cortical blood vessels of patients with cerebral hemorrhages due to sporadic amyloid angiopathy and patients with Alzheimer's disease we investigated the presence of these two proteins in the cortical blood vessels of patients suffering from hereditary cerebral hemorrhage with amyioidosis of the Dutch (n = 11) and the Icelandic (n = 2) type. The brains of three patients with sporadic cerebral amyloid angiopathy were also investigated. Blood vessels of the Dutch patients clearly showed immunostaining with β/A4 as well as with cystatin C antibodies, whereas the blood vessels of Icelandic patients showed only staining with cystatin C. In one of the three sporadic amyloid angiopathy patients co-localization was shown as well. The co-localization of mutated β/A4 with normal cystatin C in the Dutch patients suggests that cystatin C deposition occurs secondarily to β/A4 deposition. This is probably also the case in sporadic amyloid angiopathy and Alzheimer's disease. Cystatin C deposition may play a role in the development of cerebral hemorrhages and leukoencephalopathy.     

TGF-βs and cAMP Regulate GAP-43 Expression in Schwann Cells and Reveal the Association of this Protein with the Trans-Golgi Network

We have shown previously that growth-associated protein 43 (GAP-43) is expressed by rat Schwann cells and is restricted to non-myelin-forming Schwann cells in vivo. Here we examined the regulation of GAP-43 using agents that are known to control Schwann cell differentiation in vitro. GAP-43 protein and mRNA levels are decreased by forskolin and other agents that elevate intracellular cAMP (and promote expression of the myelinating Schwann cell phenotype). We also found that expression of GAP-43 protein but not mRNA is down-regulated by transforming growth factor betas (TGF-βs). Moreover, TGF-β treatment of Schwann cells results in cell clumping, process retraction and disappearance of GAP-43 from the plasma membrane, revealing that GAP-43 is associated with the Golgi apparatus. This association was confirmed by partial overlap of GAP-43 with the trans-Golgi network marker (23c) and the disruption of the Golgi with brefeldin A or monensin leading to altered GAP-43 distribution. Golgi-associated GAP-43 appeared to have the same molecular weight as the plasma membrane-associated GAP-43. Thus these results show that GAP-43 expression in Schwann cells is subject to regulation by both extracellular and intracellular signalling molecules and that Schwann cell GAP-43 is often associated with the Golgi apparatus.     

Genetic association analysis of tagging SNPs in alpha4 and beta2 subunits of neuronal nicotinic acetylcholine receptor genes (<Emphasis Type="Italic">CHRNA4</Emphasis> and <Emphasis Type="Italic">CHRNB2</Emphasis>) with schizophrenia in the Japanese population

Several lines of evidence suggest that nicotinic cholinergic dysfunction may contribute to the cognitive impairments in schizophrenia. The majority of high affinity nicotine binding sites in the human brain have been implicated in heteropentameric alpha4 and beta2 subunits of neuronal nicotinic acetylcholine receptors; therefore, these two neuronal nicotinic acetylcholine receptors genes (CHRNA4 and CHRNB2) are considered to be attractive candidate genes for the pathophysiology of schizophrenia. To represent these two genes in a gene-wide manner, we first evaluated the linkage disequilibrium structure using our own control samples. Thirteen SNPs (7 SNPs for CHRNA4 and 5 SNPs for CHRNB2) were selected as tagging SNPs. Using these tagging SNPs, we then conducted genetic association analysis of case-control samples (738 schizophrenia and 753 controls) in the Japanese population. No significant association was detected in the allele/genotype-wise or haplotype-wise analysis. Our results suggest that CHRNA4 and CHRNB2 do not play a major role in Japanese schizophrenia.