Receptors In Rat Brain Research Articles

Differential effects of amyloid-beta 1–40 and 1–42 fibrils on 5-HT1A serotonin receptors in rat brain

Evidence accumulates suggesting a complex interplay between neurodegenerative processes and serotonergic neurotransmission. We have previously reported an overexpression of serotonin 5-HT1A receptors (5-HT1AR) after intrahippocampal injections of amyloid-beta 1–40 (Aβ40) fibrils in rats. This serotonergic reactivity paralleled results from clinical positron emission tomography studies with [18F]MPPF revealing an overexpression of 5-HT1AR in the hippocampus of patients with mild cognitive impairment. Because Aβ40 and Aβ42 isoforms are found in amyloid plaques, we tested in this study the hypothesis of a peptide- and region-specific 5-HT1AR reactivity by injecting them, separately, into the hippocampus or striatum of rats. [18F]MPPF in vitro autoradiography revealed that Aβ40 fibrils, but not Aβ42, were triggering an overexpression of 5-HT1AR in the hippocampus and striatum of rat brains after 7 days. Immunohistochemical approaches targeting neuronal precursor cells, mature neurons, and astrocytes showed that Aβ42 fibrils caused more pathophysiological damages than Aβ40 fibrils. The mechanisms of Aβ40 fibrils–induced 5-HT1AR expression remains unknown, but hypotheses including neurogenesis, glial expression, and axonal sprouting are discussed.

Effects of Subchronic Finasteride Treatment and Withdrawal on Neuroactive Steroid Levels and Their Receptors in the Male Rat Brain

The enzymatic conversion of progesterone and testosterone by the enzyme 5alpha-reductase exerts a crucial role in the control of nervous function. The effects of finasteride in the brain, an inhibitor of this enzyme used for the treatment of human benign prostatic hyperplasia and androgenic alopecia, have been poorly explored. Therefore, the effects of a subchronic treatment with finasteride at low doses (3 mg/kg/day) and the consequences of its withdrawal on neuroactive steroid levels in plasma, cerebrospinal fluid and some brain regions as well as on the expression of classical and non-classical steroid receptors have been evaluated in male rats. After subchronic treatment (i.e., for 20 days) the following effects were detected: (i) depending on the compartment considered, alteration in the levels of neuroactive steroids, not only in 5alpha-reduced metabolites but also in its precursors and in neuroactive steroids from other steroidogenic pathways and (ii) an upregulation of the androgen receptor in the cerebral cortex and beta3 subunit of the GABA-A receptor in the cerebellum. One month after the last treatment (i.e., withdrawal period), some of these effects persisted (i.e., the upregulation of the androgen receptor in the cerebral cortex, an increase of dihydroprogesterone in the cerebellum, a decrease of dihydrotestosterone in plasma). Moreover, other changes in neuroactive steroid levels, steroid receptors (i.e., an upregulation of the estrogen receptor alpha and a downregulation of the estrogen receptor beta in the cerebral cortex) and GABA-A receptor subunits (i.e., a decrease of alpha 4 and beta 3 mRNA levels in the cerebral cortex) were detected. These findings suggest that finasteride treatment may have broad consequences for brain function.

Purinergic signalling during development and ageing.

Extracellular purines and pyrimidines play major roles during embryogenesis, organogenesis, postnatal development and ageing in vertebrates, including humans. Pluripotent stem cells can differentiate into three primary germ layers of the embryo but may also be involved in plasticity and repair of the adult brain. These cells express the molecular components necessary for purinergic signalling, and their developmental fates can be manipulated via this signalling pathway. Functional P1, P2Y and P2X receptor subtypes and ectonucleotidases are involved in the development of different organ systems, including heart, blood vessels, skeletal muscle, urinary bladder, central and peripheral neurons, retina, inner ear, gut, lung and vas deferens. The importance of purinergic signalling in the ageing process is suggested by changes in expression of A1 and A2 receptors in old rat brains and reduction of P2X receptor expression in ageing mouse brain. By contrast, in the periphery, increases in expression of P2X3 and P2X4 receptors are seen in bladder and pancreas.

Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain.

Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1 -LPA6 . LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1 -null mice (a variant of LPA1 -null) lack [(35) S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCR), LPA1 to LPA6 . LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 -binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and LPA-evoked activities are abolished in MaLPA1 -null mice. The phospholipid precursors of LPA are localized by MALDI-IMS. The anatomical distribution of LPA precursors in rodent and human brain suggests a relationship with functional LPA1 receptors.

Hepatic encephalopathy induces site-specific changes in gene expression of GluN1 subunit of NMDA receptor in rat brain.

We investigate changes in gene expression of GluN1 subunit of N-Methyl-D-Aspartate (NMDA) receptor in the prefrontal cortex (PFC), hippocampus and striatum in a rat model of hepatic encephalopathy (HE). We used male Wistar rats in which HE was induced after a common bile duct ligation (BDL). The animals were divided into three sets, and each set included three groups of control, sham operated and BDL. In the first set of animals, blood samples collected for biochemical analysis on day 21 of BDL. In the second set, changes in nociception threshold was assessed on day 21 of BDL using a hotplate test. In the third set, whole brain extracted, and the PFC, the hippocampus and the striatum in each rat were immediately dissected. We used a semi-quantitative RT-PCR method for evaluating the GluN1 gene expression. The biochemical analyses showed that plasma levels of ammonia and bilirubin in BDL rats were significantly increased compared to the sham control group on day 21 of BDL (P < 0.01). Nociception threshold was also increased in rats with BDL compared to sham group (P < 0.001). The results revealed that the GluN1 gene expression at mRNA levels in BDL group was decreased by 19% in the PFC (P < 0.05) but increased by 82% in the hippocampus (P < 0.01) compared to the sham control group; however, no significant change was observed in the striatum. It can be concluded that HE affects the GluN1 gene expression in rat brain with a site-specific pattern, and the PFC and hippocampus are more sensitive areas than striatum.

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain.

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G-protein-coupled receptors, MT1 and MT2 , remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT1 and MT2 localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT1 and MT2 receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT1 and MT2 proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT1 were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT2 receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.

Retraction: Radenovic L, Selakovic V. Mitochondrial superoxide production and MnSOD activity following exposure to an agonist and antagonists of ionotropic receptors in rat brain, Arch Biol Sci. 2005; 57(1):1-10, DOI:10.2298/ABS0501001R

This is a notice of retraction of the article: Mitochondrial superoxide production and MnSOD activity following exposure to an agonist and antagonists of ionotropic glutamate receptors in rat brain, published in the Archives of Biological Sciences in 2005, Vol. 57, Issue 1. The Editor-in-Chief has been informed that this paper plagiarizes an earlier paper: Radenovic L, Selakovic V, Kartelija G, Todorovic N, Nedeljkovic M. Differential effects of NMDA and AMPA/kainate receptor antagonists on superoxide production and MnSOD activity in rat brain following intrahippocampal injection. Brain Res Bull, 2004, 64(1):85-93. This claim is correct and almost the entire paper is a verbatim copy of the earlier one. After confirmation of this fact, the Editor-in-Chief of the Archives of Biological Sciences has decided to retract the paper immediately. We apologize to the readers of the journal that it took so many years to notice this error and to retract the paper. We request readers of the journal to directly get in touch with the editorial office and the editors of the journal for similar cases in the future, so that they can be handled promptly. &lt;br&gt;&lt;br&gt;&lt;font color="red"&gt;&lt;b&gt; Link to the retracted article &lt;u&gt;&lt;a href="http://dx.doi.org/10.2298/ABS0501001R"&gt;10.2298/ABS0501001R&lt;/a&gt;&lt;/b&gt;&lt;/u&gt;

P.5.a.001 Novel cholinesterase inhibitors 1-PC-37 and 1-PC-48 in the treatment of 3-quinuclidinyl benzilate-induced behavioural deficit in rats

Injection of a potent, cholinergic muscarinic antagonist, [3H]3-quinuclidinyl benzilate ([3H]QNB), results in a localization of the drug to muscarinic receptors in rat brain. The distribution of these drug receptors was examined in various brain regions previously thought to contain cholinergic neurons. They were localized to dendritic regions in the hippocampus, corpus striatum, nucleus accumbens and cerebral cortex. Particularly in the hippocampys, the receptor distribution may correspond to that for cholinergic nerve terminals.

Galanin activated Gi/o-proteins in human and rat central nervous systems

The neuropeptide galanin (GAL) is involved in the control of hormone secretion, nociception, feeding behavior, attention, learning and memory. The anatomical localization of galanin receptors in the brain has been described using autoradiography and immunohistochemistry, but both techniques are limited by the availability of specific radioligands or antibodies. Functional autoradiography provides an alternative method by combining anatomical resolution and information of the activity mediated by G-protein coupled receptors. The present study analyzes the functional GAL receptors coupled to Gi/o-proteins in human and rat brain nuclei using [35S]GTPγS autoradiography.The results show the anatomical distribution of Gi/o-proteins activated by GAL receptors that trigger intracellular signaling mechanisms. The activity mediated by GAL receptors in human and rat brain showed a good correlation of the net stimulation in areas such as spinal cord, periaqueductal gray, putamen, CA3 layers of hippocampus, substantia nigra and diverse thalamic nuclei.The functional GAL receptors coupled to Gi/o-proteins showed a similar pattern for both species in most of the areas analyzed, but some discrete nuclei showed differences in the activity mediated by GAL, such as the ventroposteromedial thalamic nucleus, or areas that regulate learning and memory processes in the hippocampus. Taken into consideration the present results, the rat could be used as an experimental model for the study of the physiological role of GAL-mediated neurotransmission and the modulation of GAL receptors activity in the human CNS.

SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C

SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson's disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.

Early-life stress induces persistent alterations in 5-HT1A receptor and serotonin transporter mRNA expression in the adult rat brain

Early-life experience plays a major role in the stress response throughout life. Neonatal maternal separation (MS) is an animal model of depression with an altered serotonergic response. We hypothesize that this alteration may be caused by differences in 5-HT1A receptor and serotonin transporter (SERT) mRNA expression in brain areas involved in the control of emotions, memory, and fear as well as in regions controlling the central serotonergic tone. To test this, Sprague–Dawley rats were subjected to MS for 3 h daily during postnatal days 2–12. As control, age matched rats were non-separated (NS) from their dams. When animals reached adulthood (11–13 weeks) brain was extracted and mRNA expression of 5-HT1A receptor in amygdala, hippocampus and dorsal raphé nucleus (DRN) and SERT in the DRN was analyzed through in situ hybridisation. Densitometric analysis revealed that MS increased 5-HT1A receptor mRNA expression in the amygdala, and reduced its expression in the DRN, but no changes were observed in the hippocampus in comparison to NS controls. Also, MS reduced SERT mRNA expression in the DRN when compared to NS rats. These results suggest that early-life stress induces persistent changes in 5-HT1A receptor and SERT mRNA expression in key brain regions involved in the development of stress-related psychiatric disorders. The reduction in SERT mRNA indicates an alteration that is in line with clinical findings such as polymorphic variants in individuals with higher risk of depression. These data may help to understand how early-life stress contributes to the development of mood disorders in adulthood.

Simvastatin reverses the downregulation of M1/4 receptor binding in 6-hydroxydopamine-induced parkinsonian rats: The association with improvements in long-term memory

BackgroundIt is believed that muscarinic M1/4 receptors are closely correlated to the dopaminergic system and are strongly involved in the pathogenesis of Parkinson’s disease (PD). In addition to regulating lipid metabolism and protection from stroke, statins have been used to regulate the declined cognition. We aimed to explore the regional changes in M1/4 receptors in the 6-hydroxydopamine (6-OHDA)-lesioned rat brain. MethodsPD rat model was set up by injecting 6-OHDA into the unilateral medial forebrain bundle; while simvastatin (10mg/kg/day) or saline was orally administrated for 3weeks, respectively. Long-term memory was measured using the Morris water maze. [3H]pirenzepine binding autoradiography was applied to investigate the alterations of M1/4 receptors in the PD rat brains. Results6-OHDA induced long-term memory deficits along with downregulation of M1/4 receptors in the hippocampus, the medial amygdala, the posteromedial cortical and the piriform cortex; simvastatin administration significantly ameliorated the impaired memory and reversed the downregulation of M1/4 receptors in the examined brain regions. Profound positive correlations were verified between the decline in long-term memory activity and the restoration of M1/4 receptors in different brain regions after simvastatin treatment. Conclusions/significanceOur results provide strong evidence that M1/4 receptor modulation after simvastatin administration did, at least partially, contribute to the improvement in the long-term memory in 6-OHDA-induced PD rats. These results provide a possible mechanism for simvastatin treatment in psycho-neurological diseases such as PD via M1/4 receptors.

Administration of growth hormone and nandrolone decanoate alters mRNA expression of the GABAB receptor subunits as well as of the GH receptor, IGF-1, and IGF-2 in rat brain

ObjectiveThe illicit use of anabolic androgenic steroids (AAS), especially among young adults, is of major concern. Among AAS users it is common to combine the AAS nandrolone decanoate (ND), with intake of growth hormone (GH) and a connection between gonadal steroids and the GH system has been suggested. Both AAS and GH affect functions in the brain, for example those associated with the hypothalamus and pituitary, and several GH actions are mediated by growth factors such as insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2). The GABAergic system is implicated in actions induced by AAS and previous studies have provided evidence for a link between GH and GABAB receptors in the brain. Our aim was to examine the impact of AAS administration and a subsequent administration of GH, on the expression of GABAB receptors and important GH mediators in rat brain. DesignThe aim was to investigate the CNS effects of a high-dose ND, and to study if a low, but physiological relevant, dose of GH could reverse the ND-induced effects. In the present study, male rats were administered a high dose of ND every third day during three weeks, and subsequently the rats were given recombinant human GH (rhGH) during ten days. Quantitative PCR (qPCR) was used to analyze gene expression in hypothalamus, anterior pituitary, caudate putamen, nucleus accumbens, and amygdala. ResultsIn the pituitary gland, the expression of GABAB receptor subunits was affected differently by the steroid treatment; the GABAB1 mRNA expression was decreased whereas a distinct elevation of the GABAB2 expression was found. Administration of ND also caused a decrease of GHR, IGF-1, and IGF-2 mRNA expression in the pituitary while the corresponding expression in the hypothalamus, caudate putamen, nucleus accumbens, and amygdala was unaffected. The rhGH administration did not alter the GABAB2 expression but increased the GABAB1 gene expression in the hypothalamus as compared to the AAS treated group. ConclusionsThese results provide new insights on the impact of ND and GH on the brain and highlight the interaction of these hormones with systems influencing GABAB receptor expression. The physiological significance of the observed effects of these hormones is discussed.

Distribution of Serotonin 4(a) Receptors in the juvenile Rat Brain and Spinal Cord

In the last 20 years there have been many studies investigating the distribution of 5-HT4-receptors in the brain of different species. Most studies are methodically based on RT-PCR or in situ hybridization and have analysed the receptors at the mRNA level.Furthermore there have been some autoradiographic studies using specific 5-HT4-receptor antagonists like [3H]GR113808, [3H]BIMU-1 or [125I]SB207710.This study investigates the topographical distribution of the 5-HT4(a)-receptor in the juvenile rat brain and spinal cord, which is important for neuromodulation of cellular excitability and could be involved in various developmental processes of the central nervous system. We analysed the 5-HT4(a)-receptor at protein level with a monospecific polyclonal antibody by using an immunohistochemical staining.We saw an intensive staining in some areas of the cortex, in the olfactory bulb, in most areas of the cerebellum, in hippocampal areas like the dentate gyrus and in several different areas of the brainstem, especially in the motor nuclei. Overall we have shown comparable results in accordance with the results of other studies investigating the distribution of 5-HT4(a)-receptors.Some areas like the islands of Calleja, the preoptic nucleus or the medial habenular nucleus showed a lower intensity of 5-HT4(a)-receptors in comparison with the results of other studies. As a novel result we found a higher intensity of 5-HT4(a)-receptor in several brain areas associated with motor function than was shown by other studies, especially in the motor cortex, in different areas of the cerebellum, in the red nucleus, in the motor nuclei of the brainstem or in the ventral horn cells of the spinal cord.We conclude that the 5-HT4(a)-receptor may play a more prominent role in the modulation of motor cortico-ponto-cerebellar, cortico-spinal, rubro-spinal, vestibulo-spinal and cortico-nuclear tracts during juvenile development.

Design, Optimization, and Biological Evaluation of Novel Keto-Benzimidazoles as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A)

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

Effect of prenatal stress on density of NMDA receptors in rat brain

N-methyl-d-aspartate (NMDA) receptors are important excitatory receptors which contribute to many brain functions. Altered NMDA receptor levels cause maldevelopment of corticostriatal and corticolimbic pathways, which is a neurobiological predisposing factor for development of epilepsy, schizophrenia and other idiopathic psychotic disorders. It was hypothesized that prenatal stress could play a role in pathophysiology of these disorders by affecting expression of the receptors through releasing corticosterone. Sixty-eight virgin female Wistar rats were selected and mated with male rats with the same genotype. Then, the pregnant rats were subjected to restraint or predator stress on 15th, 16th and 17th gestation days. Prenatal stress consisted of restraint or predator stresses of the dams under normal room conditions. After parturition, the pups were studied in terms of density of NMDA receptors in brain at different time points. Meanwhile, blood sample was obtained and corticosterone blood level (CBL) was measured. The pups were then compared with the pups born to unstressed dams. Stress induced significant rise in CBL and NMDA receptors in brain of the offspring. CBL was significantly higher among the stressed rats compared to the control ones; there was significant difference between the two stresses and between the two sexes. The male pups were affected more severely. Stressful events during gestation had important effects on NMDA receptors of the offspring. It can be concluded that stress-induced elevation of NMDA receptors and corticosterone might mediate altered susceptibility to epilepsy and decrease ability of learning and memory and other stress-induced neurologic disorders.

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca2+ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca2+ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca2+ and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.

Multiple conformations of 5-HT2A and 5-HT2C receptors in rat brain: an autoradiographic study with [125I](±)DOI

Earlier autoradiographic studies with the 5-HT2 receptor agonist [(125)I](±)DOI in human brain showed unexpected biphasic competition curves for various 5-HT2A antagonists. We have performed similar studies in rat brain regions with selective 5-HT2A (M100907) and 5-HT2C (SB242084) antagonists together with ketanserin and mesulergine. The effect of GTP analogues on antagonist competition was also studied. Increasing concentrations of Gpp(NH)p or GTPγS resulted in a maximal inhibition of [(125)I](±)DOI-specific binding of approximately 50%. M100907 competed biphasically in all regions. In the presence of 100μM Gpp(NH)p, M100907 still displaced biphasically the remaining [(125)I](±)DOI binding. Ketanserin showed biphasic curves in some regions and monophasic curves in others. In the latter, Gpp(NH)p evidenced an additional high-affinity site. SB242084 competed biphasically in brainstem nuclei and monophasically in the other regions. In most areas, SB242084 affinities were not notably altered by Gpp(NH)p. Mesulergine competed monophasically in all regions without alteration by Gpp(NH)p. These results conform with the extended ternary complex model of receptor action: receptor exists as an equilibrium of multiple conformations, i.e. ground (R), partly activated (R*) and activated G-protein-coupled (R*G) conformation/s. Thus, [(125)I](±)DOI would label multiple conformations of both 5-HT2A and 5-HT2C receptors in rat brain, and M100907 and ketanserin would recognise these conformations with different affinities.

Pharmacological Characterization of JNJ-40068782, a New Potent, Selective, and Systemically Active Positive Allosteric Modulator of the mGlu2 Receptor and Its Radioligand [3H]JNJ-40068782

Modulation of the metabotropic glutamate type 2 (mGlu2) receptor is considered a promising target for the treatment of central nervous system diseases such as schizophrenia. Here, we describe the pharmacological properties of the novel mGlu2 receptor positive allosteric modulator (PAM) 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-40068782) and its radioligand [(3)H]JNJ-40068782. In guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, JNJ-40068782 produced a leftward and upward shift in the glutamate concentration-effect curve at human recombinant mGlu2 receptors. The EC50 of JNJ-40068782 for potentiation of an EC20-equivalent concentration of glutamate was 143 nM. Although JNJ-40068782 did not affect binding of the orthosteric antagonist [(3)H]2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY-341495), it did potentiate the binding of the agonist [(3)H](2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV), demonstrating that it can allosterically affect binding at the agonist recognition site. The binding of [(3)H]JNJ-40068782 to human recombinant mGlu2 receptors in Chinese hamster ovary cells and rat brain receptors was saturable with a KD of ∼10 nM. In rat brain, the anatomic distribution of [(3)H]JNJ-40068782 was consistent with mGlu2 expression previously described and was most abundant in cortex and hippocampus. The ability of structurally unrelated PAMs to displace [(3)H]JNJ-40068782 suggests that PAMs may bind to common determinants within the same site. It is noteworthy that agonists also increased the binding affinity of [(3)H]JNJ-40068782. JNJ-40068782 influenced rat sleep-wake organization by decreasing rapid eye movement sleep with a lowest active dose of 3 mg/kg PO. In mice, JNJ-40068782 reversed phencyclidine-induced hyperlocomotion with an ED50 of 5.7 mg/kg s.c. Collectively, the present data demonstrate that JNJ-40068782 has utility in investigating the potential of mGlu2 modulation for the treatment of diseases characterized by disturbed glutamatergic signaling and highlight the value of [(3)H]JNJ-40068782 in exploring allosteric binding.

The combined effects of midazolam and propofol sedation on muscle power

We performed a randomised, crossover study to investigate the effects of intravenous sedation on grip strength and bite force. Twenty male volunteers received a bolus intravenous injection of midazolam (0.02 mg.kg(-1)) together with a 30-min propofol infusion designed to achieve an effect-site concentration of 1.0 μg.ml(-1). Observed variables included bispectral index, observer's assessment of alertness/sedation, correct answer rate of Stroop colour-word test, grip strength and bite force. Grip strength decreased from a median (IQR [range]) of 483 (443-517 [380-586]) N to 358 (280-405 [108-580]) N (p < 0.001) during sedation and recovered following flumazenil administration, while bite force increased from 818 (593-1026 [405-1406]) N to 1377 (1243-1585 [836-2357]) N (p < 0.001) during sedation. Although bite force gradually returned to baseline following flumazenil administration, it remained increased throughout the experimental period. We conclude that bite force increased during intravenous sedation and that this may have clinical implications.