A semi-quantitative atlas of 5-hydroxytryptamine-1 receptors in the primate brain

The regional distribution of 5-hydroxytryptamine-1 receptors in the primate brain was studied by semi-quantitative autoradiographic analysis of tritiated ligand binding. Areas showing the highest density of 5-hydroxytryptamine-1 receptors (greater than 200 fmol [3H]5-hydroxytryptamine bound per mg tissue), included the cerebral cortex (laminae I-II), claustrum, posterior cell group of the basal nucleus of Meynert, the infracommissural part of the globus pallidus, cortical amygdaloid nucleus, hippocampal formation (CA1-subiculum region, the anterior CA2, CA3 and CA4 regions and the molecular layer of the dentate gyrus), thalamic nuclei (parafascicular, parataenial, paraventricular and superior central lateral nuclei), substantia nigra pars reticulata, dorsal raphe nucleus and choroid plexus. The distribution of 5-hydroxytryptamine-1 receptors is compared to the distribution of both 5-hydroxytryptamine receptors and terminal fields of serotonergic projections as previously described in subprimates.     

Differential effects of stress on presynaptic and postsynaptic 5-hydroxytryptamine-1A receptors in the rat brain: an in vitro electrophysiological study.

Extracellular and intracellular recording techniques were used to assess possible changes in the functional properties of 5-hydroxytryptamine-1A receptors in brain slices prepared from rats subjected to different stress paradigms. Whereas a 30-min restraint stress did not alter the inhibitory influence of ipsapirone on the firing of serotoninergic neurons in the dorsal raphe nucleus, the same session followed by a 24-h isolation produced a significant decrease in the potency of the 5-hydroxytryptamine-1A agonist to inhibit the electrical activity of these cells. Similarly, exposure of the animals to novel uncontrolled environmental conditions for 16 h significantly reduced the potency of ipsapirone to decrease the firing rate of serotoninergic neurons in brain stem slices. The effects of the latter two stressful paradigms were observed in slices from intact rats, but not in those from adrenalectomized animals. Intracellular recording showed that exposure of the animals to novel uncontrolled environmental conditions markedly reduced the potency of 5-carboxamidotryptamine to hyperpolarize serotoninergic neurons in the dorsal raphe nucleus and to decrease the input resistance of their plasma membrane. In contrast, the same stressful paradigm exerted no significant influence on the membrane effects of this 5-hydroxytryptamine-1A agonist on pyramidal cells in the CA1 hippocampal area. These data show that, like the direct application of corticosterone on to brain slices [Laaris N. et al. (1995) Neuropharmacology 34, 1201-1210], the stress-induced in vivo elevation of serum levels of endogenous corticosterone is associated with desensitization of somatodendritic 5-hydroxytryptamine-1A receptors in the dorsal raphe nucleus. The differential changes in 5-hydroxytryptamine-1A receptor sensitivity due to stress in the latter area versus the hippocampus further support the idea that somatodendritic and postsynaptic 5-hydroxytryptamine-1A receptors are regulated differently in the rat brain.     

Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo

Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR₂. We have extended these findings herein by showing the cooperation between PAR₂ and TLR2, TLR3, or TLR4 for activation of nuclear factor-κB-dependent signaling in mucosal EC lines. In contrast, activation of PAR₂ negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR₂^−/− and TLR4^−/− mice, which were refractory to footpad edema induced by PAR₂ agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR₂–TLR4 “receptor cooperativity” and highlight the complexity of signaling integration between heterologous innate immune biosensors.     

In vitro and in vivo modulation of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and calcitonin-gene related peptide-like immunoreactivities in adult rat sensory neurons.

In a previous work we have shown that culturing adult rat dorsal root ganglia neurons modifies their neurotransmitter phenotype in such a way that cultured neurons synthesize transmitters that are not found in situ, while several other transmitters are expressed in a much higher percentage of neurons in culture than in situ [Schoenen J. et al. (1989) J. Neurosci. Res. 22, 473-487]. The aim of the present study was to investigate the origin and the nature of the relevant environmental signals that allow this plasticity to be expressed, focusing on three neurotransmitters: 5-hydroxytryptamine, thyrotropin-releasing hormone and calcitonin-gene related peptide. The main results can be summarized as follows: (1) culturing cells in fetal calf serum or on feeder layers of astrocytes, Schwann cells or fibroblasts partially inhibits the serotoninergic phenotype of dorsal root ganglia neurons; (2) in vivo disconnection of dorsal root ganglia from their spinal targets but not from their peripheral or supraspinal targets induces a significant increase of the percentage of 5-hydroxytryptamine- and thyrotropin-releasing hormone-positive neurons in disconnected ganglia; (3) growth factors such as ciliary neuronotrophic factor or basic fibroblast growth factor but not nerve growth factor repress 5-hydroxytryptamine and calcitonin gene-related peptide immunoreactivity in cultured sensory neurons. In conclusion, neurotransmitter gene expression of adult dorsal root ganglia neurons is controlled by complex influences. Our data suggest that thyrotropin-releasing hormone and 5-hydroxytryptamine gene expression are tonically repressed in vivo by factors originating from the spinal segmental level and that growth factors such as ciliary neurotrophic factor or basic fibroblast growth factor could be potential vectors of this repressing effect.     

Selective 5HT2A and 5HT6 receptor antagonists promote sleep in rats

STUDY OBJECTIVES: Serotonin (5-HT) has long been implicated in the control of sleep and wakefulness. This study evaluated the hypnotic efficacy of the 5-HT6 antagonist RO4368554 (RO) and the 5-HT2A receptor antagonist MDL100907 (MDL) relative to zolpidem. DESIGN: A randomized, repeated-measures design was utilized in which Wistar rats received intraperitoneal injections of RO (1.0, 3.0, and 10 mg/kg), MDL (0.1, 1.0 and 3.0 mg/kg), zolpidem (10 mg/kg), or vehicle in the middle of the dark (active) period. Electroencephalogram, electromyogram, body temperature (Tb) and locomotor activity were analyzed for 6 hours after injection. MEASUREMENTS AND RESULTS: RO, MDL, and zolpidem all produced significant increases in sleep and decreases in waking, compared with vehicle control. All 3 doses of MDL produced more consolidated sleep, increased non-rapid eye movement sleep (NREM) sleep, and increased electroencephalographic delta power during NREM sleep. The highest dose of RO (10.0 mg/kg) produced significant increases in sleep and decreases in waking during hour 2 following dosing. These increases in sleep duration were associated with greater delta power during NREM sleep. ZO Zolpidem induced sleep with the shortest latency and significantly increased NREM sleep and delta power but also suppressed rapid eye movement sleep sleep; in contrast, neither RO nor MDL affected rapid eye movement sleep. Whereas RO did not affect Tb, both zolpidem and MDL reduced Tb relative to vehicle-injected controls. CONCLUSIONS: These results support a role for 5-HT2A receptor modulation in NREM sleep and suggest a previously unrecognized role for 5-HT6 receptors in sleep-wake regulation.     

Serotonin transporters (SERTs) within the rat nucleus of the solitary tract: subcellular distribution and relation to 5HT2A receptors

Serotonergic transmission is terminated by serotonin transporter (SERT)-mediated uptake following activation of serotonin receptors, several subtypes of which are present in the medial nucleus of the solitary tract (mNTS) at the area postrema level. In this region, serotonin (5HT) is a major modulator of the baroreceptor reflex and also affects gastric motility. This serotonin is derived from multiple sources including local neurons and inputs from raphe and visceral vagal afferents. To determine the relevant functional sites for serotonin uptake in the mNTS, we examined the electron microscopic localization of SERTs using both immunoperoxidase and immunogold labeling in rat brain. In addition, we combined these methods for dual labeling of SERTs and 5HT2A receptors to detect whether the SERT in this region was located near or at a distance from the sites of activation of these G-protein coupled receptors. Intensive SERT immunolabeling was seen on plasma membranes of axons and morphologically heterogeneous axon terminals that formed symmetric or asymmetric synapses on dendrites without detectable 5HT2A immunoreactivity (IR). 5HT2A-IR was, however, located in other nearby neuronal and glial profiles, some of which apposed intensively SERT-labeled terminals or terminals containing lower intensity of SERT immunolabeling. In somatodendritic profiles, co-expression of SERT and 5HT2A receptor immunolabeling was seen near synapses and Golgi lamellae. Our results suggest that in the mNTS 5HT activates 5HT2A receptors at a distance from SERT-mediated uptake sites in diverse cell types including some that express both 5HT2A receptors and SERTs.     

Relationships of 5-hydroxytryptamine immunoreactive terminal-like varicosities to 5-hydroxytryptamine-2A receptor-immunoreactive neuronal processes in the rat forebrain

The distributions of 5-hydroxytryptamine (5-HT)-immunoreactive (IR) varicosities and 5-hydroxytryptamine-2A receptor (5-HT2A)-IR neuronal structures in the rat brain have previously been described individually. Using double labeling immunocytochemistry, the relationships between 5-HT2A-IR and 5-HT-IR elements in the forebrain of male rats has been studied at the light microscopic level. In neocortical regions (frontal, parietal and retrosplenial cortex), the strongest 5-HT2A-IR was found in the apical dendrites of pyramidal cells in layers III-V, while 5-HT-IR terminal-like varicosities were present in all layers but most prominently in the outer layers. In other forebrain regions, the olfactory bulb, the hippocampal formation, and the islands of Calleja and Calleja magna, localized discrepancies were present between the 5-HT2A-IR neuronal profiles and the 5-HT-IR terminal-like varicosities. Hardly any additional juxtapositions between the 5-HT2A-IR neuronal profiles and 5-HT-IR terminal-like varicosities were revealed when the intraneuronal level of 5-HT was increased by monoamine oxidase inhibitor pretreatment (nialamide, 250 mg/kg, 3 h). Thus, in most forebrain regions, there were overall few juxtapositions between 5-HT terminal-like varicosities and 5-HT2A-IR neuronal structures. This observation suggests that 5-HT2A receptor mediated 5-HT transmission in the rat forebrain is mainly a volume transmission process mediated via short distance diffusion in the extra-cellular space.     

A comparison of in vivo and in vitro neuroimaging of 5-HT 1A receptor binding sites in the cat brain

To validate the cat as a suitable model for positron emission tomography imaging (PET) and to gain further knowledge on the anatomical distribution of the serotonin-1A receptor (5-HT 1A) in the feline brain, we used PET with [18F]MPPF and in vitro autoradiography with [3H]MPPF, [3H]8-OH-DPAT and [3H]paroxetine. PET radioactivity curves with [18F]MPPF were very reproducible in anaesthetized cats, with the highest radioactivity uptakes recorded in the hippocampus, cingulate cortex, septum, infralimbic cortex and raphe nucleus, whereas the lowest were found in the cerebellum. [3H]8-OH-DPAT binding displayed a comparable, albeit lower, regional distribution than with [3H]MPPF. Autoradiography also revealed the presence of 5-HT 1A receptor binding sites in the cortex and in the interpeduncular nucleus, due to its greater sensitivity and spatial resolution compared with PET imaging. The cat constitutes an interesting experimental model for PET imaging, as many physiological concepts have been well established with this animal. Our study also shows the advantages of combining complementary neuroimaging techniques such as in vivo PET imaging and in vitro autoradiography to visualize the distribution of the 5-HT 1A receptors.     

慢性应激抑郁模型大鼠脑内5-HT1A和5-HT2A受体的变化

为了在5-羟色胺受体水平研究抑郁症的机制和三环类抗抑郁药物(TCAs)阿米替林的药理学机理,将24只SD雄性大鼠随机均分为三组,即对照组、抑郁组、阿米替林治疗组.应用[3H]8-OH-DPAT、[3H]Ketanserin作为标记配基,采用放射性配体受体结合法,分别测定大鼠海马5-HT1A受体、大脑皮层5-HT2A受体结合.结果显示抑郁大鼠海马 [3H]8-OH-DPAT 特异性结合(18.78±5.62 fmol/mg prot),较正常对照组(26.12±5.52fmol/mg prot )明显下降(P＜0.05).抑郁大鼠大脑皮层[3H]Ketanserin特异性结合(112.58±4.21fmol/mg prot),较正常对照组(86.28±4.24fmol/mg prot)明显增加( P＜0.05).阿米替林治疗3周后,可使抑郁大鼠海马5-HT1A受体与大脑皮层5-HT2A受体结合恢复正常.提示 海马5-HT1A受体结合下降、大脑皮层5-HT2A受体结合增加可能与抑郁症病因有关;海马5-HT1A受体、大脑皮层5-HT2A受体是阿米替林发挥抗抑郁作用的环节.     

Antagonist properties of eliprodil and other NMDA receptor antagonists at rat NR1A/NR2A and NR1A/NR2B receptors expressed in Xenopus oocytes

We have studied the effects of a variety of N-methyl-

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-aspartate (NMDA) antagonists acting at different sites of the NMDA receptor complex on NMDA-induced currents in Xenopus oocytes expressing heteromeric NR1A/NR2A and NR1A/NR2B receptors. The polyamine site antagonists eliprodil (IC₅₀=3.0 μM) and ifenprodil (IC₅₀=0.27 μM) antagonized NMDA responses at NR1A/NR2B receptors but not at NR1A/NR2A receptors (IC₅₀>100 μM). The channel blockers dizocilpine, memantine and phencyclidine (PCP) were equally potent antagonists at both receptor subtypes whereas dextromethorphan was four times more potent at NR1A/NR2A receptors. The glycine site antagonists

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-689,560 and 7-Cl-kynurenate were 10 times more potent at NR1A/NR2A than at NR1A/NR2B receptor subtypes. The selectivity of eliprodil and ifenprodil for the NR1A/NR2B receptor subtype may, at least partially, explain their favorable side effect profile.     

Cellular and subcellular distribution of the amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2 in the rat dorsal vagal complex

Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) type glutamate receptors are ligand gated ion channels made up of various combinations of four subunits termed GluR1-4. The GluR2 subunit controls several key features of the receptor including calcium permeability and inward rectification. In the present study, we analysed by immunocytochemistry the cellular and subcellular distribution of the GluR2 subunit in neurons of the dorsal vagal complex of the rat. GluR2 immunoreactivity was found both in the neuropile and in neuronal cell bodies. Perikaryal staining was strong in the dorsal motor nucleus of the vagus nerve and moderate in the medial part of the nucleus tractus solitarii as well as in the area postrema. The lateral part of the nucleus tractus solitarii was almost devoid of immunoreactivity except for the interstitial subnucleus which was filled with numerous strongly immunoreactive perikarya and large cell processes. Ultrastructural examination was carried out in the interstitial subnucleus. Peroxidase staining indicative of GluR2 immunoreactivity was observed in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Additional experiments performed using pre-embedding immunogold showed that most of the labeling in immunoreactive dendrites was intracytoplasmic.These results indicate that GluR2 immunoreactivity is differentially distributed among neurons in the dorsal vagal complex, thereby suggesting differences in the functional properties of AMPA receptors between neuronal populations. These results also suggest that AMPA receptors, at least those containing the GluR2 subunit, have no major role as presynaptic receptors within this region. Finally, they indicate the existence of large intracellular pools of GluR2 subunits within dendrites of immunoreactive neurons.     

A monoclonal antibody against rat platelets. I. Tissue distribution in vitro and in vivo.

In this study we describe a new monoclonal antibody (MoAb PL.1) against rat platelets. Immunohistology of various rat tissues showed staining of platelets, especially in the spleen, and staining of megakaryocytes in bone marrow and spleen red pulp. In the liver small platelet aggregates and endothelial cells were stained. After in-vivo administration of MoAb PL.1 an acute severe thrombocytopenia was observed. In general the distribution of the antibody and/or antibody-coated platelet aggregates showed the same pattern as after in-vitro incubation, i.e. staining of rat platelets and platelet aggregates in spleen red pulp, and staining of megakaryocytes in spleen and bone marrow. Platelet aggregates were observed in the liver and electron microscopy indicated that they were associated with Kupffer cells. Furthermore, liver endothelial cells were positively stained. Comparison of the molecular weight of the antigens recognized by this MoAb and by human anti-platelet MoAbs, as well as comparison of staining patterns of megakaryocytes indicated that MoAb PL.1 is probably directed to a GPIIb/IIIa complex analogue. Since MoAb PL.1 is of the non-complement-binding mouse IgG1 isotype, it can be used for studying clearance of platelet aggregates by Fc-receptors of the MPS. It also promises to be a useful tool in the study of platelet involvement in rats with experimental nephritis.     

Regulation of Streptococcus pneumoniae distribution by Toll-like receptor 2 in vivo

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.     

In vitro and in vivo action of cyclosporin A on the induction of human interleukin-2 receptor alpha and beta chains.

To compare in vitro and in vivo cyclosporin A (CyA) effects on early events involved in human T cell activation, lymphocytes obtained from healthy donors and from diabetic patients undergoing CyA therapy were studied for their interleukin 2 (IL-2) responsiveness, surface IL-2 receptor (IL-2R) expression and IL-2R mRNA accumulation, following stimulation with mitogen or anti-CD3 monoclonal antibody. T cells recovered from eight in vivo CyA-treated patients and stimulated in vitro for 4 h with mitogen or anti-CD3 (in the absence of CyA) showed significant (50-60%) inhibition of Tac mRNA accumulation, as assessed and quantified by scanning densitometry. Conversely, these cells showed no modification in their expression of membrane alpha (p55, Tac) or beta (p70) chains of IL-2R in binding experiments performed with both iodinated anti-Tac and IL-2 following 18 h stimulation with either mitogen or anti-CD3. Normal lymphocytes treated in vitro with CyA showed significant inhibition of alpha chain IL-2R expression both at the mRNA and the membrane level. At variance, expression of the IL-2R beta chain was unaffected; a significant number of high-affinity IL-2 binding sites was still detectable after in vitro CyA treatment. These results suggest that: (1) a residual immunosuppressive effect of CyA on T cell activation may be evidenced in in vivo treated cells by measuring very early events triggered following short-term stimulation; (2) CyA activity on T cell activation seems similar in vivo and in vitro; and (3) the described approach would be potentially useful to monitor the individual in vivo immunosuppressive capacity of CyA.     

In vitro and in vivo evaluation of staphylococcal superantigen peptide antagonists

Superantigen peptide antagonists failed to block T-cell activation and cytokine production as well as toxic shock induced by staphylococcal enterotoxin B (SEB) in HLA class II transgenic mice. They also failed to inhibit the binding of SEB to HLA class II molecules as well as activation of human T lymphocytes in vitro.     

Clozapine downregulates 5-hydroxytryptamine6 (5-HT6) and upregulates 5-HT7 receptors in HeLa cells

Clozapine is an atypical antipsychotic with high affinity for several serotonin receptors. This drug causes paradoxical downregulation of 5-hydroxytryptamine(2A) (5-HT)(2A) receptors, but its modulation of other serotonin receptors has not been studied. We examined the effects of clozapine and several other drugs on the regulation of rat 5-HT(6) and 5-HT(7) receptors individually expressed in transfected HeLa cells. Both 5-HT(6) and 5-HT(7) receptor densities (B(max)) were reduced by 5-carboxamidotryptamine, an agonist, and methiothepin, an inverse agonist. Clozapine reduced 5-HT(6) B(max). This suggests that 5-HT(6) receptors are also paradoxically downregulated by the antagonist clozapine. 5-Hydroxytryptamine(7) receptor B(max), on the other hand, was increased by clozapine. Clozapine's modulation of the 5-HT(6) and 5-HT(7) receptor levels may be important in the action of this atypical antipsychotic.     

In vivo and in vitro effects of doxycycline on leucocyte membrane receptors.

Tetracyclines, particularly doxycycline, have adverse effects on granulocyte function in vitro. We have examined the effects of doxycycline on membrane receptors for IgG (Fc gamma-R) and C3b (C3b-R) on granulocytes and lymphocytes, as well as on the sheep erythrocyte receptor (E-R) on T lymphocytes. Acne patients given doxycycline orally had a lower percentage of Fc gamma-R positive granulocytes (57%) than before treatment (80%) or compared to healthy controls (81%). Following in vitro doxycycline incubation, normal granulocytes showed decreased levels of Fc gamma-R positive cells. This effect was counteracted by the addition of magnesium during incubation. The deleterious effect of doxycycline on granulocyte functions may be due to decreased levels of Fc gamma-R bearing granulocytes. Doxycycline in vivo or in vitro had no significant effect on the proportion of C3b-R bearing granulocytes or lymphocytes or the T lymphocyte percentage. After in vitro irradiation with light at 340-380 nm, however, both granulocytes and lymphocytes preincubated with doxycycline showed up to 50% decrease in Fc gamma-R bearing cells, while control cells without doxycycline were unaffected.