Comparative analyses of influenza virus receptor distribution in the human and mouse brains

Accumulating evidence suggests a potential link between influenza A virus infection and the occurrence of influenza-associated neurological disorders. As influenza infection is mediated by specific receptors on the host cell surface, it is important to understand the distribution patterns of influenza receptors in target organs. We carried out comprehensive experiments to localize influenza receptors in the brains of two different mouse strains and the human brain for comparison using lectin histochemistry. We further compared the brain regions in which influenza receptors were expressed and the regions in which experimental influenza infection was observed. Our results show that the expression patterns for influenza receptors in mouse and human brains are different. In the mouse brain, human influenza virus receptors (HuIV-R) were expressed in part of brainstem and cerebellar white matter while avian influenza virus receptors (AIV-R) were expressed in the cerebellar Purkinje neurons. In contrast, in the human brain, many neurons and glia in widespread regions, including the cerebral cortex, hippocampus, brainstem, and cerebellum, express both AIV-R and HuIV-R. Importantly, vascular endothelial cells, choroid plexus epithelial cells and ependymal cells in both mouse and human brains express high levels of HuIV-R and AIV-R. The regional reciprocity was not observed when comparing regions with influenza receptor expression and the regions of influenza infection within the mouse brain. Our results demonstrate a differential influenza receptor expression pattern in mouse and human brains, and a disparity between influenza receptor distribution and regions with actual influenza infection.     

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.     

Expression of P2X purinoceptors during rat brain development and their inhibitory role on motor axon outgrowth in neural tube explant cultures

Extracellular ATP is well known as a neurotransmitter and neuromodulator in the CNS of adults. However, little is known about the involvement of ATP during the development of mammalian brain. In the present study, we have examined the expression pattern of P2X receptor subtype mRNA and protein during perinatal rat brain development (from embryonic day (E) 10 to postnatal day (P) 16 brain). While P2X3 receptors appeared early at E11, they declined in the stages that follow. P2X2 and P2X7 receptors were expressed from E14 onwards, while P2X4, P2X5 and P2X6 receptors were expressed from P1 onwards. P2X1 receptor expression was not observed in any of the developmental ages examined. We investigated the effect of 100 microM ATP and alpha,beta-methylene ATP (alpha,beta-meATP; selective agonist for P2X1, P2X2/3 and P2X3 receptors) on motor axon outgrowth in collagen-embedded neural tube explant cultures. Both ATP- and alpha,beta-meATP-treated neural tubes showed a significant reduction in neurite outgrowth compared with the control explants. This inhibitory effect could not be reproduced by uridine triphosphate. In conclusion, all P2X receptor subtypes, except for P2X1, were strongly represented in the developing rat brain. ATP was shown to inhibit motor axon outgrowth during early embryonic neurogenesis, most likely via the P2X3 receptor. It is speculated that P2X7 receptors may be involved in programmed cell death during embryogenesis and that P2X4, P2X(5) and P2X6 receptors might be involved in postnatal neurogenesis.     

Melanocortin-4 receptor messenger RNA expression is up-regulated in the non-damaged striatum following unilateral hypoxic-ischaemic brain injury

Melanocortin peptides (alpha-melanocyte-stimulating hormone, adrenocorticotropin and fragments thereof) have been shown to have numerous effects on the central nervous system, including recovery from nerve injury and retention of learned behaviour, but the mechanism of action of these peptides is unknown. A family of five melanocortin receptors have recently been discovered, two of which (melanocortin-3 and melanocortin-4 receptors) have been mapped in the rat brain. We have tested the hypothesis that the expression of one or more of the messenger RNAs for three melanocortin receptors (melanocortin-3, melanocortin-4 and melanocortin-5 receptors) would be altered in rat brain following unilateral transient hypoxic-ischaemic brain injury. In this study, using in situ hybridization, we show that melanocortin-4 receptor messenger RNA was up-regulated in the striatum in the non-damaged hemisphere within 24 h after severe hypoxic-ischaemic injury compared with control brains (P<0.05). In a small group of animals, this induction was not blocked by treatment with the anticonvulsant, carbamazepine. Expression of melanocortin-3 receptor messenger RNA in the brain was not altered in this hypoxic-ischaemic injury model and melanocortin-5 receptor messenger RNA was not detected in either control or hypoxic-ischaemic injured rat brains. We hypothesize that the up-regulation of melanocortin-4 receptor messenger RNA expression in the contralateral striatum may be involved in transfer of function to the uninjured hemisphere following unilateral brain injury.     

Spatio-temporal expression of cysteinyl leukotriene receptor-2 mRNA in rat brain after focal cerebral ischemia

Cysteinyl leukotrienes (CysLTs) induce inflammatory responses mediated by activating CysLT(1) and CysLT(2) receptors. We have recently reported that CysLT(1) receptor expression is increased in rat brain after focal cerebral ischemia and the increased expression is spatio-temporally related to acute neuronal injury and late astrocyte proliferation. Here we report spatio-temporal expression of CysLT(2) receptor mRNA in rat brain after focal cerebral ischemia induced by 30min of middle cerebral artery occlusion. We found that the neuron density was gradually decreased or disappeared in the ischemic core and boundary zone during 14 days after reperfusion, and the astrocyte population in the boundary zone was increased 3-14 days after reperfusion. In the ischemic core, the expression of CysLT(2) receptor mRNA was increased at 6, 12 and 24h and then recovered at 3, 7 and 14 days after reperfusion. In the boundary zone, the expression was significantly increased 3, 7 and 14 days after reperfusion. The results suggest that CysLT(2) receptor may be related to the acute neuronal injury and late astrocyte proliferation in the ischemic brain.     

Alpha-2 adrenergic receptor development in rat CNS: an autoradiographic study

During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of adrenergic receptors. alpha-2 Adrenergic receptors, one of three adrenergic receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined alpha-2 receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [(3)H]RX821002 for autoradiography. Binding kinetics and pharmacology for alpha-2 adrenergic receptors were the same in adults and neonates. There was an overall increase in alpha-2 receptor levels during postnatal development with great variability in pattern and timing of receptor density changes among brain regions. Three major patterns were apparent. First, in many regions receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of alpha-2 adrenergic receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for alpha-2 receptors during development.This study investigates the development of alpha-2 adrenergic receptors in the rat CNS. It demonstrates there is region-specific regulation of alpha-2 receptor development and identifies brain regions where these receptors may play a specific and critical role in the regulation normal development.     

Specific luteinizing-hormone-releasing hormone receptor binding sites in hippocampus and pituitary: an autoradiographical study

Using the iodinated luteinizing-hormone-releasing hormone analogue [D-Ala6, N alpha MeLeu7, Pro9 NEt]-luteinizing-hormone-releasing hormone as radioligand, specific binding sites have been visualized in the rat both in the pituitary and the hippocampal formation of the brain. In the hippocampus, the CA1, CA2 and particularly CA3 regions were heavily labelled. These hippocampal sites have a pharmacological specificity resembling that of luteinizing-hormone-releasing hormone receptors in pituitary homogenates and could therefore represent true luteinizing-hormone-releasing hormone receptors. The luteinizing-hormone-releasing hormone superagonist [D-Ala6, Pro9 NEt]-luteinizing-hormone-releasing hormone and the potent antagonist [D-pGlu1, D-Phe2, D-Trp3,6]-luteinizing-hormone-releasing hormone were highly potent in displacing the iodinated luteinizing-hormone-releasing hormone analogue. The weak agonist [Gln8]-luteinizing-hormone-releasing hormone, however, was at least two orders of magnitude less potent. Somatostatin was inactive. Hippocampal luteinizing-hormone-releasing hormone receptors were species-specific, being present in the rat but not in the mouse, guinea-pig, hamster, rabbit and human brains. In order to identify the cellular location of these hippocampal receptors, various lesions were performed. Electrolytic lesions of the septal afferents did not reveal any receptor density change. Colchicine as well as kainic acid injections did, however, reduce considerably the number of hippocampal receptors. Interestingly, in the electrolytically and kainic-acid-lesioned animals, the appearance of non-displaceable luteinizing-hormone-releasing hormone binding sites within a well-defined area corresponding to the lesioned, gliosis-rich area was observed. The present results suggest the presence of pharmacologically specific, species-dependent, luteinizing-hormone-releasing hormone receptors located, at least partly, on intrinsic hippocampal neurons, in particular granule and pyramidal cells.     

Cigarette smoking decreases neurotrophin-3 expression in rat hippocampus after transient forebrain ischemia

Stroke is a common cause of death and severe disability among adults in developed countries. Cigarette smoking adversely affects human health in many ways and is considered to be a risk factor for a stroke. However, the mechanism that determines the relative importance of neurotrophins in this process remains unclear. To study the effect of chronic cigarette smoking on ischemic stroke, in situ hybridization and immunohistochemistry were employed to detect the mRNA and protein expression of neurotrophin-3 (NT-3), respectively, which is thought to play a critical role in protection against neuronal death in brain ischemia. Rats, with or without chronic cigarette smoking, were subjected to 20 min of transient forebrain ischemia. Distribution and quantification of mRNA and protein of NT-3 in the whole hippocampus and the cell death in the hippocampal CA1–CA3 regions were determined in these rats. Experimental results show that chronic cigarette smoking produces a significantly delay and persistent down-regulation of ischemia-induced NT-3 mRNA and protein changes at 6–24 h post-ischemia, and seemly increases neuron death 7 days after reperfusion. These experimental results indicate that by influencing NT-3 expression, directly or indirectly, chronic cigarette smoking has a potentially harmful effect when acute brain ischemia attacks.     

Localization of 5-HT(7) receptors in rat brain by immunocytochemistry, in situ hybridization, and agonist stimulated cFos expression

5-HT(7) receptors are recently identified members of the serotonin receptor family that have moderate to high affinity for several important psychotropic drugs. However, the lack of selective ligands has impeded the study of the brain distribution of these receptors. In this report, we describe the localization of 5-HT(7) receptor in rat forebrain by immunocytochemistry, in situ hybridization of 5-HT(7) mRNA, and functional stimulation of cFOS expression by 5-HT(7) receptor activation. The anatomical localization of 5-HT(7) mRNA in situ hybridization signal. Prominent immunostaining was apparent in numerous sites within the cerebral cortex, hippocampal formation, tenia tecta, thalamus and hypothalamus. 5-HT(7) receptors were detected in suprachiasmatic nucleus by both immunocytochemistry and in situ hybridization. At a microscopic level, both cell bodies and proximal fibers were strongly stained in these regions, suggesting a somatodendritic subcellular distribution. 5-HT(7) receptor-like immunoreactivity was further compared with 5-HT(7) mediated biological function by administering 8-OH-DPAT intracerebroventricular injection (icv)with WAY 100135 (to block 5-HT(1A) receptors) followed by double immunostaining localization of cFos activation and 5-HT(7) receptors. In all regions examined, cFos stimulation and 5-HT(7)-like immunoreactivity colocalized to the same neurons. Furthermore, cFos activation by 8-OH-DPAT was blocked by pimozide--a 5-HT(7) antagonist. Therefore, by using multiple strategies, we were able to localize 5-HT(7) receptors in rat brain unequivocally. The distribution of these receptors is consistent with their involvement in the control of circadian activity and the action of anti-depressants and atypical neuroleptics.     

Bilateral control of brain activity by dopamine D1 receptors: evidence from induction patterns of regulator of G protein signaling 2 and c-fos mRNA in D1-challenged hemiparkinsonian rats

Recent reports show that striatal dopamine D1-type receptors from one side of the normal rat brain can control brain activity (as measured by c-fos induction) on both sides of the brain. However, this phenomenon has not yet been studied in the presence of sensitized dopamine D1-type receptors. Here we address this issue by investigating the extent to which dopamine D1-type receptors control brain activation in rats with unilaterally sensitized dopamine D1-type receptors. Gene induction assays were used to identify activated regions from midbrain to forebrain in unilaterally 6-hydroxydopamine lesioned (hemiparkinsonian) rats challenged with the full dopamine D1-type agonist SKF82958 (3 mg/kg, 0.5 and 2 h). The genes used are c-fos, the proven neuronal activity marker, and Regulator of G protein Signaling 2, a gene we propose as a marker of signaling homeostasis. SKF82958-mediated induction of both genes is greatly enhanced in hemiparkinsonian rats compared with shams, in both the lesioned and the intact hemisphere. For example, in the denervated caudate-putamen at 2 h postinjection, this enhancement is more than 80-fold for c-fos and up to 20-fold for Regulator of G protein Signaling 2; for the intact side this is 35-fold for c-fos and 27-fold for Regulator of G protein Signaling 2. Cortical induction of c-fos and Regulator of G protein Signaling 2 was generalized to most neocortical regions and was essentially equivalent in both the denervated and intact hemispheres. Interestingly, hippocampal structures also showed strong bilateral induction of both genes. This overall pattern of brain activation can be accounted for by the basal-ganglia thalamocortical and hippocampal circuits which both contain hemisphere-crossing connections and which can be initially activated in the lesioned hemisphere. Some regions, such as the intact striatum or the CA1 region, showed relatively low c-fos induction and relatively high Regulator of G protein Signaling 2 induction, possibly indicating that these regions are engaged in unusually strong signaling regulation activities. Our results show that, besides basal ganglia-thalamocortical circuits, dopamine D1-type-mediated brain activation in hemiparkinsonian rats also involves hippocampal circuits.     

Increased expression of cysteinyl leukotriene receptor-1 in the brain mediates neuronal damage and astrogliosis after focal cerebral ischemia in rats

Cysteinyl leukotrienes are potent pro-inflammatory mediators. Cysteinyl leukotriene receptor 1 is one of the two cysteinyl leukotriene receptors cloned. We recently reported that cysteinyl leukotriene receptor 1 antagonists protected against cerebral ischemic injury, and an inducible expression of cysteinyl leukotriene receptor 1 was found in neuron- and glial-appearing cells after traumatic injury in human brain. To determine the role of cysteinyl leukotriene receptor 1 in ischemic brain injury, we investigated the temporal and spatial profile of cysteinyl leukotriene receptor 1 expression in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion, and observed the effect of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, on the ischemic injury. We found that cysteinyl leukotriene receptor 1 mRNA expression was up-regulated in the ischemic core both 3-12 h and 7-14 days, and in the boundary zone 7-14 days after reperfusion. In the ischemic core, cysteinyl leukotriene receptor 1 was primarily localized in neurons 24 h, and in macrophage/microglia 14 days after reperfusion; while in the boundary zone it was localized in proliferated astrocytes 14 days after reperfusion. Pranlukast attenuated neurological deficits, reduced infarct volume and ameliorated neuron loss in the ischemic core 24 h after reperfusion; it reduced infarct volume, ameliorated neuron loss and inhibited astrocyte proliferation in the boundary zone 14 days after reperfusion. Thus, we conclude that cysteinyl leukotriene receptor 1 mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia.     

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.     

Species differences in the localization of 'peripheral' type cholecystokinin receptors in rodent brain

A comparison of cholecystokinin (CCK) receptor binding to sections of rat, mouse and guinea pig brain has been performed using 125I-Bolton Hunter CCK and the selective peripheral CCK receptor antagonist L-365,031. In both rat and mouse, 125I-Bolton Hunter CCK binding in the region of the interpeduncular nucleus (IPN) was inhibited by L-365,031 indicating that these receptors resemble CCK receptors found in peripheral tissues. In the mouse especially, dense regions of peripheral CCK receptors were detected either side of the IPN. By contrast, in the guinea pig IPN no evidence of L-365,031-sensitive binding could be found. The present reports shows that in different species, regional variations in brain CCK receptor binding occur not only in the case of classical 'brain' receptors, but also for the more discretely localised 'peripheral' type CCK receptors.     

Methamphetamine-induced reduction in D1 and D2 dopamine receptors as evidenced by autoradiography: comparison with tyrosine hydroxylase activity

As determined by autoradiographic techniques, multiple high doses of methamphetamine elicited a reduction in dopamine receptor population (both D1 and D2) in several areas of the rat central nervous system. D1 receptors were labeled with the D1-selective antagonist, [3H]SCH 23390, and D2 receptors were labeled with the D2-selective neuroleptic, [3H]sulpiride. Scatchard analysis, obtained from saturation data in caudate-putamen, indicated that the receptor alterations were due to a decrease in the number of receptors (Bmax) without an apparent change in affinity (Kd). A time course demonstrated that five doses of methamphetamine were required to elicit significant changes in receptors in most brain areas examined. The onset of the receptor alterations in various brain regions correlated with the development of methamphetamine-induced depression of striatal tyrosine hydroxylase activity. In most brain areas, the dopamine receptors returned to normal within 7 days following methamphetamine.     

Upregulation of erbB receptors in rat brain after middle cerebral arterial occlusion

We have previously demonstrated that neuregulin-1 (NRG-1) is upregulated and is neuroprotective in ischemic brain injury, however the expression and localization of its receptors during ischemia has not been investigated. Therefore, we used a rat middle cerebral artery occlusion (MCAO) model to examine the distribution of erbB receptors following ischemic stroke. Like neuregulin-1, we observed a dramatic induction of erbB4 in the peri-infarct regions of the ipsilateral cortex 24 h following MCAO. Using Fluoro-Jade B (FJB) staining as a marker of neurodegeneration, erbB4 was upregulated in FJB-positive cells, suggesting that erbB receptors are induced in injured neurons. The increase in erbB receptors was seen in neurons and a subpopulation of macrophages/microglia. There was no erbB co-localization with GFAP-positive astrocytes. These results demonstrate that erbB receptors are upregulated in neurons and macrophages/microglia following ischemic stroke and may be involved in neuroprotection and repair.     

Effect of unilateral perinatal hypoxic-ischemic brain injury on striatal dopamine uptake sites and D1 and D2 receptors in adult rats.

The effect of unilateral perinatal hypoxic-ischemic brain injury on striatal dopamine uptake sites and on D1 and D2 receptors was investigated in rat by using in vitro quantitative receptor binding autoradiography, 9-11 weeks after the insult. Saturation experiments revealed a significant 20% decrease in maximal binding capacity (Bmax) for [3H]spiperone-labeled D2 receptors on the side of the lesion in comparison to the non-lesioned contralateral side or to either side of control animals. There was no significant change in [3H]mazindol-labeled dopamine uptake sites or in [3H]SCH 23390-labeled D1 receptor characteristics (Bmax and Kd) on the lesioned side. We conclude that the decrease in D2 receptor binding previously observed in immature animals is persistent, whereas the decrease in D1 binding is not.