Effects of 1-naphthyl acetyl spermine on dendrite formation by cultured cerebellar Purkinje cells

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. To date, the contributions of calcium-permeable AMPA receptors (CP-AMPARs) in calcium signaling and dendrite formation of Purkinje cells remain to be elucidated. In the present study, therefore, we examined the effects of 1-naphthyl acetyl spermine (NAS), a blocker of CP-AMPARs, on dendrite formation by cultured Purkinje cells. NAS markedly inhibited elongation and branching of Purkinje cell dendrites. Calcium imaging experiments using caged glutamate demonstrated that NAS inhibits the increase of intracellular calcium concentration in Purkinje cells after glutamate release. These results suggest that calcium signaling mediated through CP-AMPARs plays an important role in Purkinje cell dendrite formation.     

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum

Previous physiological and pharmacological studies have shown that the serotonin_2A (5-HT_2A) receptor is involved in cerebellar functions. However, the expression of 5-HT_2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT_2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT_2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT_2A receptors during the cerebellar development.     

Cholesterol-dependent induction of dendrite formation by ginsenoside Rh2 in cultured melanoma cells

Herbal remedies containing root extracts of Panax ginseng are commonly used for complementary or alternative therapies. Ginsenosides, the major components of root extracts, are responsible for ginseng's pharmacological and biological effects; however, their mechanisms of action are unclear. We examined whether membrane cholesterol was involved in the mechanism of action of ginsenoside Rh2 in cultured cells. In B16 melanoma cells, Rh2 (18.5 μM) induced dendrite formation within 2 h. Depletion of cholesterol by pretreatment with 10 mM methyl-β-cyclodextrin suppressed this effect of Rh2. Rh2 did not change the cellular cholesterol content and the immunofluorescence staining pattern of the lipid-raft-associated molecules, ganglioside GM3, Caveolin-1, Flotillin-1, and Flotillin-2, for up to 3 or 6 h. However, within 2 min of addition, Rh2 changed the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) but not of 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). DPH is more sensitive than TMA-DPH to changes in the physical properties of membrane lipid bilayers regulated by cholesterol. These results suggest that Rh2 affects the physical properties of cholesterol-regulated membrane lipid bilayers and could lead to changes in cellular functions.     

Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells

Aim: The aim of this study was to determine the effect of long‐term serotonin (5‐hydroxytryptamine, 5‐HT) treatment on the human serotonin transporter (hSERT) function and its expression. Methods: This study was carried out in the enterocyte‐like cell line Caco‐2. These cells constitutively express the hSERT and have been shown to be an excellent model for the study of this protein. We measured serotonin transport, levels of mRNA expression and of the SERT protein after treating the cells with serotonin. Results: Serotonin treatment diminished hSERT activity in a concentration and period‐dependent way by increasing the K_t value and reducing V_max. This inhibition was reversible and was not mediated by either the action of 5‐HT₂, 5‐HT₃ or 5‐HT₄ receptors, or by the intracellular second messengers, protein kinase C and cAMP. 5‐HT did not seem to affect either the mRNA level of the SERT or the protein transporter measured in either the membrane or the cell lysate. The 5‐HT treatment effect was additive to the inhibitory effect of treatment with a low concentration of citalopram and fluoxetine. Nevertheless, 5‐HT did not increase the inhibition yielded by treatment with high concentration citalopram. Conclusion: The chronic increase in serotonin in the extracellular medium diminishes the function of the SERT. This effect seems to be due to an effect on the transporter molecule itself in the membrane, without altering protein synthesis, intracellular traffic, or its availability.     

Itch-associated response induced by intradermal serotonin through 5-HT2 receptors in mice

Serotonin (5-HT) is pruritogenic in humans and suggested to be involved in some pruritic diseases. Our experiments were carried out to determine whether an intradermal injection of 5-HT would elicit itch-associated response in mice and to elucidate the 5-HT receptor subtypes involved in this 5-HT action. 5-HT (14.1-235 nmol site(-1)) injected intradermally into the rostral back elicited scratching of the injected site, with bell-shaped dose-response relationship. The scratching induced by 5-HT (100 nmol site(-1), peak effective dose) was suppressed by capsaicin (repeated administration) and the opioid antagonist naloxone, features being similar to human itching. Scratching was also elicited by the 5-HT2 receptor agonist alpha-methylserotonin, but not by the 5-HT1A receptor agonist R(+)-8-hydroxy-N,N-dipropyl-2-aminotetralin nor the 5-HT3 receptor agonists 2-methylserotonin and 1-phenylbiganide. Scratching induced by 5-HT and alpha-methylserotonin was inhibited by peroral pretreatment with 5-HT1/2 receptor antagonists methysergide and cyproheptadine. 5-HT-induced scratching was also inhibited by intradermal injection of methysergide. Peroral pretreatment with 5-HT3 receptor antagonists ondansetron and 3-tropanyl-3, 5-dichrobenzoate did not significantly suppress 5-HT-induced scratching. The results suggest that scratching induced by intradermal injection of 5-HT is itch-associated response. The 5-HT action may be mediated at least partly by cutaneous 5-HT2 receptors.     

Buspirone affects hippocampal rhythmical slow activity through serotonin1A rather than dopamine D2 receptors.

Buspirone reduces anxiety clinically but, unlike classical anxiolytics, is not muscle relaxant, sedative, anticonvulsant or effective in increasing GABA function. The basis for its clinical action is not known, but action at both dopamine D2 and serotonin1A receptors has been suggested. Buspirone, like classical anxiolytics, produces a general reduction in the frequency of hippocampal rhythmical slow activity elicited by stimulation of the midbrain in the rat. Methysergide (3 mg/kg i.p.), GR38032F (0.3 mg/kg i.p.) and haloperidol (0.2 mg/kg and 2.0 mg/kg i.p.) failed to block this effect of buspirone (10 mg/kg i.p.). Apomorphine (0.3 mg/kg i.p.) had minor effects, but did not produce a general reduction in frequency. Pindolol (2 mg/kg i.p.) produced a small reduction in frequency itself. In the presence of pindolol, buspirone was without effect, while the effect of chlordiazepoxide (5 mg/kg i.p.) was potentiated. These results show that: (a) the similar effects of buspirone and classical anxiolytics such as chlordiazepoxide on reticular-elicited hippocampal rhythmical slow activity are achieved through different mechanisms; (b) the effects of buspirone in this particular test are more likely to depend on its interaction with serotonin1A receptors than its interaction with D2 receptors; and (c) that, as in other tests, buspirone does not act via serotonin2 or serotonin3 receptors.     

Histamine excites rat cerebellar Purkinje cells via H2 receptors in vitro

Recent neuroanatomical studies have revealed a direct hypothalamocerebellar histaminergic pathway. However, the functional significance of the histaminergic fibers in the cerebellum is not yet clear. In this study, the effects of histamine on the firing of cerebellar Purkinje cells (PCs) were investigated in vitro. Histamine predominantly produced excitatory (106/111, 95.5%) and in a few cases inhibitory (5/111, 4.5%) responses in PCs. The histamine-induced excitation was not blocked by perfusing the slice with low Ca2+ high/Mg2+ medium (n = 8), supporting a direct postsynaptic action of histamine. The histamine H2 receptor antagonist ranitidine effectively blocked the excitatory response of PCs to histamine (n = 20), but triprolidine, an H1 receptor antagonist, could not significantly block the histamine-induced excitation, or only very slightly decreased the excitatory effect of histamine on the cells (n = 13). On the other hand, the highly selective H2 receptor agonist dimaprit mimicked the excitatory effect of histamine on PCs and this dimaprit-induced excitation was also blocked by ranitidine (n = 20), but not triprolidine (n = 8). However, the H1 receptor agonists betahistine and 2-thiazolylethylamine did not show any effect on the PCs (n = 9 and 14). These results reveal that histamine excites cerebellar PCs via H2 receptors and suggest that the hypothalamocerebellar histaminergic fibers may play an important role in functional activities of the cerebellum.     

Serotonin1A autoreceptor activation by S 15535 enhances circadian activity rhythms in hamsters: evaluation of potential interactions with serotonin2A and serotonin2C receptors

Mammalian circadian activity rhythms are generated by pacemaker cells in the suprachiasmatic nucleus (SCN). As revealed by the actions of diverse agonists, serotonergic input from raphe nuclei generally inhibits photic signaling in the suprachiasmatic nucleus. In contrast, the serotonin (5HT)1A partial agonist, 4-(benzodioxan-5-yl)1-(indan2-yl)piperazine (S 15535), was found to enhance the phase-shifting influence of light on hamster circadian rhythms [Gannon, Neuroscience 119 (2003) 567]. Herein, we extend this observation in showing that S 15535 (5.0 mg/kg, i.p.) markedly (275%) enhanced the light-induced phase shift in circadian activity rhythms: further, this action was dose-dependently abolished by the highly-selective 5HT1A receptor antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]N-2-pyridinyl-cyclohexane-carboxamide maleate) (0.1-0.5 mg/kg, i.p.). WAY 100,635, which was inactive alone, shares the antagonist actions of S 15535 at postsynaptic 5HT1A sites, yet blocks its effects at their presynaptic counterparts. Thus, 5HT1A autoreceptor activation must be involved in this effect of S 15535 which contrasts with the opposite, inhibitory influence upon phase shifts of the "full" agonist, 8-OH-DPAT, which acts by stimulation of postsynaptic 5HT1A receptors [Rea et al., J Neurosci 14 (1994) 3635]. Despite the occurrence of 5HT2A and 5HT2C receptors in the (rat) suprachiasmatic nucleus, their influence on circadian rhythms is unknown since actions of selective ligands have never been evaluated. This issue was investigated with the most selective agents currently available. However, the 5HT2A agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.25 and 0.5 mg/kg), and the 5HT2C agonist, alphaS-6-chloro-5-fluoro-a-methyl-1H-indole-1-ethanamine fumarate (Ro-60-0175) (1.0 and 5.0 mg/kg), failed to affect light-induced phase shifts in hamsters. Moreover, even over broad dose-ranges, the 5HT2A antagonist, (+)-(2,3-dimethoxy-phenyl)-[1-[2-(4-fluoro-phenyl)-ethyl]-piperidin-4-yl]methanol (MDL 100,907) (0.1-1.0 mg/kg), and the 5HT2C antagonist, 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl carbamoyl]indoline (SB 242,084) (1.0-10.0 mg/kg), were likewise inactive. In view of evidence that 5HT2A and 5HT2C sites functionally interact with 5HT1A receptors, we also examined the influence of these agents upon the actions of S 15535, but no significant alteration was seen in its enhancement of rhythms. In conclusion, S 15535 elicits a striking enhancement of light-induced phase shifts in circadian rhythms by specifically recruiting 5HT1A autoreceptors, which leads to suppression of serotonergic input to the suprachiasmatic nucleus. Surprisingly, no evidence for a role of 5HT2A or 5HT2C sites was found, though comparable functional studies remain to be undertaken in rats. Indeed, the present work underlines the importance of comparative studies of circadian rhythms in various species, as well as the need for further study of potential interactions among 5HT receptor subtypes in their control.     

Serotonin1 and serotonin2 receptors hyperpolarize and depolarize separate populations of medial pontine reticular formation neurons in vitro.

The action of serotonin on medial pontine reticular formation neurons was examined using intracellular electrophysiological methods in rat brainstem slices in vitro. A hyperpolarization associated with a decrease in input resistance was elicited by serotonin in 34% of the neurons, and a depolarization associated with an increase in input resistance was produced in 56% of the neurons. Both responses persisted in the presence of tetrodotoxin. The hyperpolarization resulted from a steady-state increase in outward current which varied with the external potassium concentration in a manner consistent with a conductance increase primarily to this ion. This response was mimicked by the serotonin1 agonist, 5-carboxamidotryptamine, as well as by the serotonin1a agonist, 8-hydroxy-dipropyl aminotetralin hydrobromide, and was blocked by spiperone, an antagonist of serotonin1 sites. The depolarization resulted from a steady-state decrease in outward current which varied with external potassium. The depolarization was mimicked by the serotonin2 agonist, alpha-methyl-5-hydroxytryptamine, and was blocked by the serotonin2 antagonist, ketanserin. Neither of these agents had any effect upon serotonin-induced hyperpolarizations. In conclusion, the excitability of medial pontine reticular formation neurons is influenced by serotonin acting to increase or decrease potassium conductance(s). These opposing effects reflect actions on distinct serotonin receptor subtypes that are segregated to distinct populations of medial pontine reticular formation neurons.     

Thrombopoietin enhances rapid current responses mediated by P2X1 receptors on megakaryocytic cells in culture

The effect of thrombopoietin (TPO), a magakaryocytopoietic cytokine, on the functional maturation of megakaryocytes was studied by using cell culture and patch-clamp techniques focusing on purinergic 2X(1) (P2X(1))-receptors, which are expressed specifically on platelets and their progenitors. Meg-01 cells, one of the typical human megakaryocytic cell lines, were cultured and studied by using a whole-cell patch electrode. In control cells cultured in RPMI1640 medium, an application of adenosine nucleotide (ADP, 40 microM) evoked transient inward currents with amplitudes of 45 +/- 19 pA (at -43 mV). Based on kinetic, ionic, and pharmacological properties as well as on previously reported findings, these currents were thought to be mediated by P2X(1) receptors. When Meg-01 cells were cultured for 7-9 d in a medium to which the differentiation-inducing agent phorbol ester (PMA; 10 nM) or TPO (100 ng/ml) had been added, the responses of the cells to ADP increased to about 150% of the control with PMA and to about 200% of the control value with TPO. A combination of the two agents enhanced the response of the cells to ADP to about 570% of the control value. These results suggest that phorbol ester and TPO cause cellular differentiation of Meg-01 cells and enhance the level of expression of P2X(1)-receptors on cell membranes in a synergetic manner. The effect of TPO on the induction of P2X(1)-receptors on mouse megakaryocytes in culture was more obvious.     

Regulation of hematopoiesis through adhesion receptors

Normal steady-state hematopoiesis takes place in the bone marrow microenvironment. Soluble factors as well as contact interactions between the hematopoietic cells and the marrow microenvironment dictate the fate of hematopoietic stem cells and progenitors. Over the last decade it has become clear that cell-cell and cell-extracellular matrix interactions through adhesion receptors play a major role in the hematopoietic process. They are required for the residence of stem cells and progenitors in the marrow, as well as for homing of stem and progenitor cells to the marrow in the setting of stem cell transplantation. Furthermore, adhesion receptors play an important role in regulation of cell behavior, either through direct activation of signal pathways important for cell survival, cell growth, and cell fate decision-making processes, or by modulating responses to growth factors. Insights in the abnormalities seen in these interactions in diseases of the hematopoietic system will help to develop better therapeutic strategies based on the pathogenesis of these diseases.     

Modulatory role of serotonin on GABA-elicited inhibition of cerebellar Purkinje cells

The present study was designed to directly examine the postsynaptic actions of serotonin on GABA-mediated inhibition of cerebellar Purkinje cells. The findings indicate that serotonin at currents that produced minimal effects on the spontaneous firing rates of Purkinje cells modified GABA effects in a biphasic manner. Serotonin initially decreased GABA-mediated inhibitions followed secondarily by either continued inhibition or, in the majority of cases, augmentation of GABA responses. When a comparison was made of the secondary effects of serotonin on GABA-mediated inhibition with the initial spontaneous firing rates of the Purkinje cells, the group in which serotonin augmented GABA actions had a significantly higher initial firing frequency than the group in which serotonin attenuated GABA-mediated inhibition. Furthermore, with increasing firing rates, the proportion of cells showing augmentation of GABA inhibition increased, and the proportion of cells displaying attenuation of GABA effects decreased. Serotonin affected beta-alanine-mediated inhibitions in a manner similar to that seen with GABA, whereas glycine was differentially altered. This study identifies another neuromodulatory role of serotonin on Purkinje cells in the cerebellum. Furthermore, the effects of serotonin on GABA inhibition seem to be governed by some intrinsic property of the Purkinje cell, which is apparently related to the firing rate of the cell.     

Voltage-activated calcium channels in rat Purkinje cells maintained in culture

Cell-attached patch recordings were used to study calcium channels on the dendritic membrane of rat cerebellar Purkinje cells maintained in culture. Experiments were performed with isotonic BaCl₂ (110 mM) in the pipette and isotonic potassium gluconate in the bath to zero the cell membrane potential. Two distinct types of voltage-activated calcium channels were identified. The first one had a small conductance (9 pS), was activated at a low threshold ( –50 mV) and could be inactivated by holding the membrane potential at –30 mV. This channel had the same characteristics as the T channel described in other neuronal preparations. The second type of Ca channel activated at a high threshold (–30 or +10 mV depending on whether BAY K 8644 was added or not to the pipette solution) and was still activatable even when the membrane was held at –40 mV. In the presence of BAY K 8644 this channel had a conductance of 21 pS with long openings. All these characteristics are similar to those of the S (L) Ca channel described in many preparations. The present study is in agreement with our previous experiments on Purkinje dendrites, where we identified low and high threshold Ca currents using the whole-cell configuration. Up to now, no channel corresponding to the N current has been observed but we cannot exclude us presence.Present Andress: Centre CNRS-INSERM de Pharmacologie-Endocrinologie, rue de la Cardonille, B.P. 5055, 34033 Montpellier, France     

Climbing-fibre activation of NMDA receptors in Purkinje cells of adult mice

Among principal neurons, adult Purkinje cells have long been considered unusual in lacking functional NMDA receptors. This view has emerged largely from studies on rats, where NMDA receptors are expressed in Purkinje cells of newborn animals, but are lost after 2 weeks. By contrast, immunolabelling data have shown that Purkinje cells from adult mice express multiple NMDA receptor subunits, suggesting a possible species difference. To investigate the presence of functional NMDA receptors in Purkinje cells of mice, and to explore the contribution of different receptor subunits, we made whole-cell and single-channel patch-clamp recordings from Purkinje cells of wild-type and NR2D−/− mice of different ages. Here we report that multiple NMDA receptor subtypes are indeed expressed in Purkinje cells of young and adult mice; in the adult, both NR2A- and NR2B-containing subtypes are present. Furthermore, we show that NMDA receptor-mediated EPSCs can be evoked by climbing fibre stimulation, and appear to be mediated mainly by NR2A-containing receptors.     

The metabolism of serotonin in neuronal cells in culture and platelets

The aim of this study is to find a relationship between serotonin (5-HT) and its metabolite 5-hydroxy indol acetic acid (5-HIAA) in hippocampus, frontal neocortex and platelets. Serotonin and 5-HIAA were measured in cultured neurons and compared with those produced by human platelets. The cortical neuronal 5-HIAA/serotonin ratio was 4.7 and for hippocampal neurons it was 3.2. In human platelets, this ratio was 1.35 suggesting that the highest serotonin metabolism occurs in the frontal neocortex followed by the hippocampus and platelets. In the presence of 0.3 μM of p-chlorophenylalanine both cultured neurons and platelets exhibited an approximately 50% decrease in serotonin and 5-HIAA concentration suggesting similarities in the metabolic profile in both preparations. In addition, we found that serotonin by itself does not play any role in platelet aggregation but potentiates this phenomenon in the presence of calcium ionophore A23187. This synergistic interaction between serotonin (2–5 μM) and A23187 (0.5–2 μM) was inhibited by serotonin receptor blockers [methysergide (IC₅₀ = 18 μM) and cyproheptadine (IC₅₀, 20 μM)] and calcium channel blockers (verapamil and diltiazem, IC₅₀ = 20 and 40 μM, respectively) that indicate both mechanisms are receptor mediated. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect of serotonin and ionophore at an IC₅₀ value of 9.2 μM. Wortmannin, a phosphoinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC₅₀ = 2.6 μM) by inhibiting respiratory burst. However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C (PKC) inhibitor, affected aggregation. Our results are strongly suggestive of a synergistic interaction between serotonin type-2 and Ca-ionophore via a PLC/Ca signalling pathway.     

The contribution of serotonin 1A receptors to kappa opioid immunosuppression

Activation of kappa opioid receptors (kappa-OR) with the selective agonist rimorphin (0.1 mg/kg) produced marked suppression of the immune response in CBA mice. This effect was not seen on administration of rimorphin on the background of a reduction in the activity of the serotoninergic (5-HTergic) system resulting from stimulation of presynaptic (8-OH-DPAT, 0.1 mg/kg) or blockade of postsynaptic (WAY-100635, 1.0 mg/kg) 5-HT_1A receptors. These data led to the conclusion that 5-HTergic mechanisms involving preand postsynaptic 5-HT_1A receptors have a role in kappa-opioid-mediated immunosuppression. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 7, pp. 807–813, July, 2008.     

Effects of metaphit on phencyclidine and serotonin2 receptors

We have investigated whether metaphit, a derivative of phencyclidine (PCP) which irreversibly binds to a population of PCP receptor sites in rat brain, blocks PCP-induced head-twitch response which is produced through serotonin2 (5-HT2) receptors, and also whether metaphit decreases the capacity of 5-HT2 receptors. Metaphit (1 mumol/rat) had decreased the intensity of PCP-induced head-twitch response and had depleted both PCP and 5-HT2 receptors by 24 h after administration, but it failed to block 5-HT agonist 5-methoxy-N,N-dimethyltryptamine-induced 5-HT1A receptor-dependent behaviors. These results reconfirmed our hypothesis that PCP and 5-HT2 receptors may have very similar binding sites.