Genetic alteration of the alpha2-adrenoceptor subtype c in mice affects the development of behavioral despair and stress-induced increases in plasma corticosterone levels.

alpha2-Adrenoceptors (alpha2-AR) modulate many central nervous system functions, such as regulation of sympathetic tone, vigilance, attention, and reactivity to environmental stressors. Three alpha2-AR subtypes (alpha2A, alpha2B, and alpha2C) with distinct tissue-distribution patterns are known to exist, but the functional significance of each subtype is not clear. Since specific, alpha2-AR subtype-selective pharmacological probes are not available, mice with genetically altered alpha2C-AR expression were studied in order to investigate the possible involvement of the alpha2C-AR in physiological and behavioral responses to acute and repeated stress. A modified version of Porsolt's forced swimming test was used to assess the possible effects of altered alpha2C-AR expression on the development of behavioral despair. alpha2C-Overexpression increased and the lack of alpha2C-AR (alpha2C-KO) decreased the immobility of mice in the forced swimming test, ie alpha2C-AR expression appeared to promote the development of behavioral despair. In addition, alpha2C-KO was associated with attenuated elevation of plasma corticosterone after different stressors, and overexpression of alpha2C-ARs was linked with increased corticosterone levels after repeated stress. Moreover, the brain dopamine and serotonin balance, but not norepinephrine turnover, was dependent on alpha2C-AR expression, and the expression of c-fos and junB mRNA was increased in alpha2C-KO mice. Since alpha2C-KO produced stress-protective effects, and alpha2C-AR overexpression seemed to promote the development of changes related to depression, it is suggested that a yet-to-be developed subtype-selective alpha2C-AR antagonist might have therapeutic value in the treatment of stress-related neuropsychiatric disorders.     

Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression

Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans.     

Cytokines regulate alpha(1)-adrenergic receptor mRNA expression in human monocytic cells and endothelial cells

The family of adrenergic receptors (AR) plays a central role in regulation of the activity of many organ systems. Consequently, regulated expression of the various subtypes of AR is an important mechanism in maintaining homeostasis. Previously, we have shown that alpha(1)-AR triggering of peripheral blood mononuclear cells from patients with juvenile chronic arthritis results in increased IL-6 production. In contrast, alpha(1)-AR agonists do not alter cytokine production by cells of healthy individuals.The aim of the present study was to investigate whether pro-inflammatory cytokines can regulate the expression of mRNA encoding AR of the alpha(1)-family. We show that human THP-1 monocytic cells express mRNA encoding of two of the three cloned subtypes of alpha(1)-AR: alpha(1b)-AR and alpha(1d)-AR mRNA. The cytokines TNF-alpha and IL-1beta decrease level of mRNA for alpha(1d)-AR in THP-1 monocytic cells. In contrast, alpha(1b)-AR mRNA levels are not affected by these two cytokines. Interestingly, IL-1beta and TNF-alpha induce the expression of alpha(1a)-AR mRNA in THP-1 monocytic cells.In human umbilical vein endothelial cells (HUVEC), IL-1beta and TNF-alpha decrease both alpha(1b)-AR and alpha(1d)-AR mRNA levels in HUVEC. alpha(1a)-AR mRNA is not detectable in HUVEC.IL-6 and IL-8, two other pro-inflammatory cytokines tested in this study, do not change alpha(1)-AR subtype levels in HUVEC or monocytic cells. Our data demonstrate that TNF-alpha and IL-1beta can regulate expression of alpha(1)-AR mRNA and that cytokine regulation of alpha(1)-AR expression is subtype- and tissue-specific.     

Changes in the expression of alpha(2)-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia.

The expression of presynaptic alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion (SCG). Cells were incubated with chicken antibodies against alpha(2)A-, alpha(2)B- or alpha(2)C-AR subtypes either alone or together with antibodies against dopamine-beta-hydroxylase (DbetaH, a marker for adrenergic neurons) or against choline acetyl transferase (ChAT, a marker for cholinergic neurons). We found immunoreactivity for all three alpha(2)-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the alpha(2)A-subtype was approximately 1/3 of that of alpha(2)B- and alpha(2)C-AR. Co-localisation of all three alpha(2)-AR subtypes was observed in cells expressing DbetaH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbetaH and decreased the expression of the alpha(2)A- and alpha(2)C-subtype without altering the expression of the alpha(2)B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the alpha(2)B-subtype without altering the expression of alpha(2)A- and alpha(2)C-subtypes. Our results indicate that the three alpha(2)-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the alpha(2)B-subtype over that of the alpha(2)A- and alpha(2)C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the alpha(2)A- and alpha(2)C-subtypes over that of the alpha(2)B-subtype. Our results suggest that the alpha(2)B-receptor is preferentially associated with noradrenergic nerve endings.     

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.     

Distribution of alpha2-adrenoceptor mRNAs in the rat lumbar spinal cord in normal and axotomized rats.

Using riboprobe in situ hybridization we have studied the distribution of alpha2A-, alpha2B- and alpha2c-adrenoceptor (AR) mRNAs in the lumbar spinal cord in normal rats and after peripheral axotomy. A strong alpha2A- and alpha2C AR mRNA labelling was found in motoneurons and other cells in the ventral horns. In the dorsal horns strong alpha2A-AR mRNA labelling was found in all layers and in the lateral spinal nucleus, whereas alpha2C-AR mRNA was found in lower numbers of cells in various layers. The alpha2B-AR mRNA signal was only detected in some small cells superficially in the dorsal horn. With regard to axotomy only a marginal effect was observed for alpha2C-AR mRNA in the ventral horn. The results suggest that alpha2-ARs are involved both in sensory and motor processing.     

Neuroendocrine mediators up-regulate alpha1b- and alpha1d-adrenergic receptor subtypes in human monocytes

Beta2- and alpha2-adrenergic receptors (AR) are thought to be the main AR subtypes to exert the effects of catecholamines on the immune system. However, in the present study, we demonstrate that another subtype of AR can be induced in human monocytes. Expression of alpha1b- and alpha1d-AR mRNA can be obtained by culturing freshly isolated human peripheral blood monocytes with the neuroendocrine mediators dexamethasone or the beta2-AR agonist terbutaline. Using the human monocytic cell line THP-1, we demonstrate that increased levels of alpha1b- and alpha1d-mRNA are accompanied by increased levels of receptor protein as determined by Western blot analysis and radioligand binding assays. This study describes for the first time regulated expression of alpha1-AR subtypes in human monocytes.     

Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat

Previous studies have revealed the existence of a gene family that encodes a group of neuronal nicotinic acetylcholine receptor (nAChR) subunits. Four members of this family have been characterized thus far; three of these subunits (alpha 2, alpha 3, and alpha 4) are structurally related to the ligand binding subunit expressed in muscle and form functional nAChRs when combined with the beta 2 gene product in Xenopus oocytes. In addition, the alpha 4 gene appears to encode two different products (alpha 4-1 and alpha 4-2) that have been proposed to arise by alternative mRNA splicing. Nine different [35S]-complementary ribonucleic acid (cRNA) probes were used in the present study to map the distribution of these nAChR subunit mRNAs throughout the central nervous system (CNS) of the rat. It was found that the beta 2 gene is expressed in most regions of the CNS, as are the alpha subunit genes as a group. However, each alpha gene is expressed in a unique, although partly overlapping, set of neuronal structures. Alpha 4 is the most widely expressed alpha gene, and the evidence suggests that mRNAs for the alpha 4-1 and alpha 4-2 products are virtually always found in the same regions, in approximately the same ratios (alpha 4-2 greater than alpha 4-1). In addition, there are several examples of cell groups that express beta 2 but none of the alpha subunit mRNAs examined here (particularly in the hypothalamus), as well as all groups that express the converse, thus suggesting that additional neuronal nAChR subunits remain to be characterized. Finally, the extensive expression of multiple alpha subunits in certain regions, particularly for alpha 3 and alpha 4 in the thalamus, suggests that there is microheterogeneity in a small population of cells or that some neurons may express more than one alpha subunit. This problem needs to be examined directly with double labeling methods but raises the possibility that some neuronal nAChRs may be composed of more than one kind of alpha subunit. The wide expression of these receptor genes suggests that nAChRs constitute major excitatory systems in the CNS.     

Stress induces increases in IL-6 production by leucocytes of patients with the chronic inflammatory disease juvenile rheumatoid arthritis: a putative role for alpha(1)-adrenergic receptors

Juvenile rheumatoid arthritis (JRA) is characterized by chronic inflammation of the joints. In the present study we demonstrate that exposure of JRA patients to a noradrenergic stressor (cold pressor test) results in enhanced LPS-induced IL-6 production by peripheral blood cells of these patients. Healthy, age-matched controls had the same rise in norepinephrine, but do not respond with changes in IL-6 production after exposure to the cold pressor test. Moreover, PBMC of patients with JRA express mRNA encoding alpha(1)-adrenergic receptors (AR), predominantly of the alpha(1d)-AR subtype. In contrast, we could not detect mRNA encoding for alpha(1)-AR in PBMC of healthy controls. The results of this study suggest that expression of alpha(1)-AR mRNA in PBMC during chronic inflammation is associated with altered responses of the immune system to stress.     

Comparative localization of serotonin1A, 1C,and 2 receptor subtype mRNAs in rat brain

Serotonin (5-HT) mediates its effects on neurons in the central nervous system through a number of different receptor types. To gain better insight as to the localization of 5-HT responsive cells, the distribution of cells expressing mRNAs encoding the three 5-HT receptor subtypes 1A,1C, and 2 was examined in rat brain with in situ hybridization using cRNA probes. 5-HT1A receptor mRNA labeling was most pronounced in the olfactory bulb, anterior hippocampal rudiment, septum, hippocampus (dentate gyrus and layers CAI-3), entorhinal cortex, interpeduncular nucleus, and medullary raphe nuclei. 5-HT 1C receptor mRNA labeling was the most abundant and widespread of the three 5-HT receptor subtypes examined. Hybridization signal was denset in the choroid plexus, anterior olfactory nucleus, olfactory tubercle piriform cortex, septum, subiculum, entorhinal cortex, claustrum, accumbens nuclues, striatum, lateral amygdala, paratenial and paracentral thalamic nuclei, subthalamic nucleus, substantia nigra, and reticular cell groups. 5-HT2 receptor mRNA was localized to the olfactory bulb, anteriorhippocampal rudiment, frontal cortex, piriform cortex, entorhinal cortex, claustrum, pontine nuclei, and cranial nerve motor nuclei including the oculomotor, trigeminal motor, facial, dorsal motor nucleus of the vagus, and hypoglossal nuclei. The distributions of mRNAs for the three different 5-HT receptor subtypes overlap with regions that bind various 5-HT receptor-selective ligands and are present in nearly all areas known to receive serotonergic innervation. The results of this study demonstrate that nervous which express these 5-HT receptor subtypes are very widespread in the central nervous system, yet possess unique distributions with in the rat brain. Moreover, previously unreported regions of 5-HT receptor subtype expression were observed, particulary with the 5-HT receptor riboprobe in the brainstem. Finally, several brain areas contain multiple 5-HT receptor subtype mRNAs, which leads to the possibility that individual cells may express more than one 5-HT receptor subtype. © 1995 Willy-Liss, Inc.     

Synchronous postnatal increase in alpha1 and gamma2L GABA(A) receptor mRNAs and high affinity zolpidem binding across three regions of rat brain

The objective of this study was to correlate postnatal changes in levels of mRNAs encoding predominant GABA(A) receptor subunits with a functional index of receptor development. This study is the first to quantify the temporal relationship between postnatal changes in predominant GABA(A) receptor mRNAs and zolpidem-sensitive GABA(A) receptor subtypes. In Experiment 1, we measured zolpidem displacement of 3H-flunitrazepam from rat cerebral cortex, hippocampus, and cerebellum at 0, 6, 14, 21, 29, and 90 postnatal days. Three independent 3H-flunitrazepam sites with high (K(i)=2. 7+/-0.6 nM), low (K(i)=67+/-4.8 nM), and very low (K(i)=4.1+/-0.9 mM) affinities for zolpidem varied in regional and developmental expression. In Experiment 2, we used RNAse protection assays to quantify levels of alpha1, alpha2, beta1, beta2, gamma2S and gamma2L mRNAs in the above regions at the same postnatal ages. Although there was a high degree of regional variation in the developmental expression of zolpidem-sensitive GABA(A) receptors and subunit mRNAs, a dramatic increase in high affinity zolpidem binding sites and alpha1 mRNA levels occurred within all three regions during the second postnatal week. Furthermore, a temporal overlap was observed between the rise in alpha1 mRNA and high affinity zolpidem binding and a more prolonged increase in gamma2L in each region. These results point to the inclusion of the alpha1 and gamma2L subunits in a GABA(A) receptor subtype with a high zolpidem affinity and suggest that a global signal may influence the emergence of this subtype in early postnatal life.     

Differential distribution of voltage-gated calcium channel alpha-2 delta (alpha2delta) subunit mRNA-containing cells in the rat central nervous system and the dorsal root ganglia

Voltage-gated calcium channels (VGCCs) play an essential role in controlling neurotransmitter release, neuronal excitability, and gene expression in the nervous system. The distribution of cells that contain mRNAs encoding the auxiliary alpha2delta-1, alpha2delta-2, and alpha2delta-3 subunits of the VGCCs in the central nervous system (CNS) and the dorsal root ganglia (DRG) was examined in rats by using in situ hybridization. Specific labeling of alpha2delta-1, alpha2delta-2, and alpha2delta-3 mRNAs appeared to be largely confined to neurons and was widely, although differentially, distributed in the brain, the spinal cord, and the DRG. Importantly, alpha2delta-2 mRNA was found to be expressed in interneurons in the cortex, the hippocampus, the striatum, and in regions that contain dense cholinergic neurons. Our results suggest that different alpha2delta subunits may exert distinctive functions in the CNS. The alpha2delta-1 subunit mRNA is localized in brain regions known to be involved in cortical processing, learning and memory, defensive behavior, neuroendocrine secretion, autonomic activation, primary sensory transmission, and general arousal. The alpha2delta-2 subunit mRNA is present in brain regions known to modulate the overall activities of the cortex, the hippocampus, and the thalamus. The alpha2delta-2 subunit is also found in brain regions known to be involved in olfaction, somatic motor control, fluid homeostasis, ingestive and defensive behaviors, neuroendocrine functions, and circadian rhythm. In addition to being localized in brain regions that express alpha2delta-1 and alpha2delta-2 subunit mRNAs, alpha2delta-3 subunit mRNA is highly expressed in regions involved in auditory information processing and somatic movement.