Distribution of inositol 1,4,5-trisphosphate receptors in rat osteoclasts

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are Ca2+ channels that localize to intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Recently, IP3Rs were found to participate in the formation of the cytoskeleton and cellular adhesions. In this study, we examined the cellular localization of type I, II, and III IP3Rs to assess their role in cellular adhesion in rat osteoclasts. Rat bone marrow cells were cultured in alpha-MEM with 10% fetal bovine serum, M-CSF, RANKL, and 1,25(OH)2D3 for 1 week to promote osteoclast formation. Type I, II, and III IP3R expression in the osteoclasts was then examined by RT-PCR. Double-staining was performed using antibodies against type I, II, and III IP3Rs and DiOC6, an ER marker, or TRITC-phalloidin, an actin filament marker. Expression of all three IP3Rs was detected in the newly formed osteoclasts; however, the localization of the type I and II IP3Rs was predominantly close to nuclear, and possibly colocalized with the ER, while the type III IP3Rs were localized to the ER and podosomes, actin-rich adhesion structures in osteoclasts. These findings suggest that type III IP3Rs are associated with osteoclast adhesion.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

Characterization of spontaneous inhibitory postsynaptic currents in cultured rat retinal amacrine cells

Spontaneous postsynaptic current is a reflection of spontaneous neurotransmitter release that plays multiple roles in a variety of neurobiological activities. In the present study, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) by patch-clamp techniques in cultured rat retinal GABAergic amacrine cells (ACs), which provide inhibitory inputs to both bipolar and ganglion cells in the inner retina, and examined if and how Ca²⁺ was involved in the induction of spontaneous GABA release from the terminals of these cells. sIPSCs were completely blocked by application of either 10 μM bicuculline or 10 μM gabazine, and the reversal potential of sIPSCs was close to E_Cl−, indicating that these events were exclusively mediated by GABA_A receptors. Increase of external Ca²⁺ concentrations from 2 to 5 mM significantly enhanced the frequency, but did not change the amplitude of sIPSCs. In contrast, perfusion of Ca²⁺-free external solution greatly reduced the events of sIPSCs and decreased the amplitude of sIPSCs. Consistently, the non-selective voltage-gated calcium channel blocker CdCl₂ (200 μM) considerably suppressed both the frequency and the amplitude of sIPSCs. Furthermore, the ryanodine receptor (RyR) antagonist dantrolene (10 μM) failed to affect sIPSCs, while the inositol 1,4,5-trisphosphate (IP₃) receptor antagonists 2-aminoethyl diphenylborinate (2-APB, 20 μM) and xestospongin C (XeC, 1 μM) significantly decreased the frequency of sIPSCs. In the presence of SKF96365 (10 μM), a non-specific transient receptor potential channel (TRP) blocker, 2-APB persisted to show its effect on sIPSCs. These results suggest that spontaneous GABA release from the terminals of GABAergic ACs is Ca²⁺-dependent, and both extracellular calcium influx through presynaptic calcium channels and Ca²⁺ release through activation of the IP₃-sensitive pathway, but not the ryanodine-sensitive one, from intracellular stores are responsible for the generation of sIPSCs under our experimental conditions.     

Inositol 1,4,5-trisphosphate mediates adrenaline activation of K+ conductance in mouse peritoneal macrophages

In mouse peritoneal macrophages, ₁-adrenoceptor stimulation evokes a Ca²⁺-dependent K⁺ current [I ₀(Adr)] [Hara et al. (1991) Pflügers Arch 419:371–379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP ₃) and a GTP-binding protein (G protein) in I ₀(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lnsP ₃ (5–50 M) evoked transient outward currents [I ₀(InsP ₃)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5-[3-thio]triphosphate (GTP[S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10–90 s. I ₀(InsP ₃) and the GTP[S]-induced current {I ₀(GTP[S])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K⁺ concentration. Fura-2 measurements revealed that I ₀(InsP ₃) and I ₀(GTP[S]) result from a rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Removal of extracellular Ca²⁺ did not abolish I ₀(InsP ₃) and I ₀(GTP[S]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP ₄, 50 M) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP ₃(2,4,5), 20 M] elicited an outward current at -40 mV. I₀(InsP ₃) was completely blocked by prior dialysis with the InsP ₃ receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5-[2-thio] diphosphate (GDP[S], 2 mM) or heparin (5 mg/ml) together with GTP[S] in the patch pipette solution completely blocked I ₀(GTP[S]). These results indicate that intracellular injection of InsP ₃ or GTP[S] mimic I ₀(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[S] (2 mM) greatly accelerated a run-down of I ₀(Adr). On the other hand, I ₀(Adr) was markedly prolonged in a cell dialysed with GTP[S] (0.2 mM). Therefore, it is concluded that I ₀(Adr) results from stimulation of ₁-adrenoceptor and InsP ₃ formation via a G protein.     

Thyrotropin-releasing hormone inhibits long-term potentiation in synaptic systems of rat hippocampus

The effects of thyrotropin-releasing hormone (TRH) on long-term potentiation of field responses in mossy fibers—CA3 and Shaffer collaterals—CA1 synaptic systems were studied on rat hippocampal slices. Incubation with micromolar concentrations of TRH inhibited the development of long-term potentiation in both synaptic systems. It is suggested that this phenomenon underlies the antiamnesic effect of TRH. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 12, pp. 690–693, December, 1999     

Prostaglandin E2 receptor expression in the rat trigeminal-vascular system and other brain structures involved in pain

Prostaglandin E₂ (PGE₂) is considered to be a key mediator in migraine pathophysiology. PGE₂ acts via four receptors (EP₁-EP₄) but their distribution in the brain districts implicated in migraine has yet to be delineated. We quantified amount of mRNA and protein expression for the EP receptors in both peripheral and central structures involved in pain transmission and perception in migraine: dura mater, cerebral arteries, trigeminal ganglion, trigeminal nucleus caudalis, periaqueductal grey, thalamus, hypothalamus, cortex, pituitary gland, hippocampus and cerebellum. In the trigeminal-vascular system (TVS) we found highest expression of EP₁ and EP₂ protein in the trigeminal nucleus caudalis. EP₃ and EP₄ mRNA expression were highest in the trigeminal ganglion. Within intracranial structures EP₁ mRNA and protein expression were significantly higher in pituitary gland and cerebellum than in dorsal root ganglia (peripheral control), whereas the EP₂ mRNA and protein were highly abundant in the pituitary gland. EP₃ mRNA was mainly found in thalamus and hypothalamus. The most robust mRNA and protein expression for EP₄ receptor was seen in the dorsal root ganglion. In conclusion, all four receptors are located in areas implicated in migraine supporting the possible involvement of PGE₂ in this disease.     

Adenosine A(3) receptor mediated coronary vasodilation in the rat heart: changes that occur with maturation

Adenosine A(2B) and A(3) receptors (ADOR) have been reported to induce coronary vasodilation in the rat. This study investigated the effect of age on ADORA(3) mediated coronary responses using hearts from rats aged 6-8 weeks (immature), 16-18 weeks (young) and 52-54 weeks (mature) perfused in Langendorff mode. APNEA (ADORA(3)>ADORA(1) agonist) was observed to activate at least two receptor subtypes to mediate a biphasic vasodilator response in hearts from immature rats. The potency of APNEA at the high affinity site was enhanced by alloxazine (ADORA(2B) antagonist) and reduced when combined with MRS1191 (ADORA(3) antagonist). This indicates that the high affinity phase is the ADORA(3), and ADORA(2B) signalling is likely to play a negative regulatory role towards the ADORA(3) mediated response. The activity at this site was also reduced with maturation. The low affinity site was inhibited by alloxazine but not MRS1191, indicating that this response is mediated by the ADORA(2B) or another receptor subtype. The response at this site did not alter with age. Cl-IB-MECA (ADORA(3) agonist) produced monophasic responses that were inhibited by alloxazine but remained unaffected by MRS1191 in all age groups. In addition the potency of Cl-IB-MECA does not change in hearts from PTX-treated rats. However, the maximal responses increased, indicating G(i) protein independent and dependent signalling. Q-PCR analysis of rat hearts indicated the presence of an ADORA(3) splice variant (ADORA(3i)), which increased in mRNA expression with age. Cl-IB-MECA responses may be mediated by this ADORA(3i). In conclusion, APNEA mediates coronary vasodilation in the rat heart via at least two receptor sites, the ADORA(3) and ADORA(2B). ADORA(3) responses are reduced while ADORA(2B) remain unchanged with maturation. In addition, the splice variant ADORA(3i) may contribute to coronary responses in the rat heart.     

Hypoxia-induced desensitization and internalization of adenosine A1 receptors in the rat hippocampus

Activation of A1 adenosine receptors is important for both the neuromodulatory and neuroprotective effects of adenosine. However, short periods of global ischemia decrease A1 adenosine receptor density in the brain and it is not known if a parallel loss of functional efficiency of A1 adenosine receptors occurs. We now tested if hypoxia leads to changes in the density and efficiency of A1 adenosine receptors to inhibit excitatory synaptic transmission in rat hippocampal slices. In control conditions, the adenosine analog 2-chloroadenosine, inhibited field excitatory post-synaptic potentials with an EC50 of 0.23 microM. After hypoxia (95% N2 and 5% CO2, for 60 min) and reoxygenation (30 min), the EC50 increased to 0.73 microM. This EC50 shift was prevented by the presence of the A1 adenosine receptor antagonist 8-phenyltheophyline, but not by the A(2A)R antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine, during the hypoxic period. This decreased efficiency of A1 adenosine receptors was not paralleled by a global change of A1 adenosine receptor density or affinity (as evaluated by the binding parameters obtained in nerve terminal membranes). However, the density of biotinylated A1 adenosine receptors at the plasma membrane of nerve terminals was reduced by 30% upon hypoxia/reoxygenation, in a manner prevented by the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine and mimicked by prolonged (60 min) supra-maximal activation of A1 adenosine receptors with 2-chloroadenosine (10 microM). These results indicate that hypoxia leads to a rapid (<90 min) homologous desensitization of A1 adenosine receptor-mediated inhibition of synaptic transmission that is likely due to an internalization of A1 adenosine receptors in nerve terminals.     

Effects of calcium-sensing receptors on apoptosis in rat hippocampus during hypoxia/reoxygenation through the ERK1/2 pathway

Objectives: To explore the effects of calcium-sensing receptors (CaSR) on apoptosis in rat hippocampus during hypoxia/reoxygenation (H/R). Methods: After rat hippocampus was isolated, the cultures were subjected to H/R, and meanwhile gadolinium chloride (GdCl₃, agonist of CaSR) and NPS 2390 (antagonists of CaSR) were added to reperfusion solution. The number of hippocampal neuron, cell viability and apoptosis rate were determined by inverted microscope, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometer (FCM), respectively. Besides, caspase-3, Bax, cytochrome C (Cyt-c), extracellular signal-regulated protein kinase (ERK) 1/2, pERK1/2, P38 and pP38 were analyzed by Western blotting. Results: The hippocampal neuron number and cell viability were significantly decreased during H/R, and were further significantly reduced when co-treatment with CaSR agonist GdCl₃. But the effects of GdCl₃ were attenuated by NPS-2390. Whereas, apoptosis rate, the expression level of caspase-3, Bax and Cyt-c were all significantly increased under H/R condition, and was further significantly increased by GdCl₃, but were reversed by NPS-2390 (P < 0.05). Moreover, there were no significant differences in expression of ERK1/2, P38 and pP38 among different groups. However, the expression of pERK1/2 was significantly increased during H/R, but was significantly reduced by NPS 2390 (P < 0.05). Conclusion: The results suggest that CaSR might play significant roles in the induction of hippocampus apoptosis in rat during H/R through phosphorylation of ERK1/2.     

Selective decline of 5-HT1A receptor binding sites in rat cortex, hippocampus and cholinergic basal forebrain nuclei during aging

The effect of aging on 5-HT_1A receptor binding in several forebrain areas associated with the basal forebrain cholinergic system was investigated in rats of 3-, 24- and 30-months-old by receptor autoradiography and biochemical binding assay using [³H]8-OH-DPAT as a ligand. Autoradiographic measurements demonstrated a marked region-specific decline of ligand binding in: (i) regions of the basal forebrain cholinergic cell groups, i.e. the medial septum, diagonal band nuclei and magnocellular nucleus basalis, (ii) the frontal and parietal neocortex and (iii) the dentate gyrus of the hippocampus. No change or only a slight decrease of the 5-HT_1A receptor density was found in other areas investigated: the CA1 and CA3 sectors of hippocampus, the cingular and perirhinal cerebral cortex and the lateral septum. The autoradiographic findings were substantiated by the biochemical binding assay, which revealed a comparable loss of 5-HT_1A receptor in the hippocampus and neocortex at the age of 30 months. The results clearly show that with increasing age the decrement of 5-HT_1A receptor binding in the rat forebrain is remarkably region-selective and particularly affects the cholinergic cell groups that innervate cortex and hippocampus. This phenomenon appears to be especially significant in relation to the neuronal substrates underlying the age-related alterations of mood and cognition.     

Exposure to novelty and forced swimming evoke stressor-dependent changes in extracellular GABA in the rat hippocampus

In the hippocampus, a brain structure critically important in the stress response, GABA controls neuronal activity not only via synaptic inhibition, but also via tonic inhibition through stimulation of extrasynaptic GABA receptors. The extracellular level of GABA may represent a major determinant for tonic inhibition and, therefore, it is surprising that its responsiveness to stress has hardly been investigated. To clarify whether hippocampal extracellular GABA levels change in response to acute stress, we conducted an in vivo microdialysis study in rats. We found that dialysate GABA levels respond to various neuropharmacological manipulations such as reuptake inhibition, elevated concentrations of K(+), tetrodotoxin and baclofen, indicating that a large proportion of hippocampal extracellular GABA depends on neuronal release and that GABA re-uptake plays a role in determining the extracellular levels of this neurotransmitter. Next, rats were exposed to a novel cage or to forced swimming in 25 degrees C water. Interestingly, these two stressors resulted in opposite effects. Novelty caused a fast increase in GABA (120% of baseline), whereas forced swimming resulted in a profound decrease (70% of baseline). To discriminate between the psychological and physical aspects (i.e. the effects on body temperature) of forced swimming, another group of animals was forced to swim at 35 degrees C. This stressor, like novelty, caused an increase in hippocampal GABA, suggesting a stimulatory effect of psychological stress. The effects of novelty could not be blocked by the corticotropin-releasing factor receptor antagonist D-Phe-CRF(12-41). These results are the first to demonstrate stressor-dependent changes in hippocampal extracellular GABA; an observation which may be of particular significance for GABAergic tonic inhibition of hippocampal neurons.     

Distribution of the myocardial tissuePO2 in the rat and the inhomogeneity of the coronary bed

The structural inhomogeneity of the myocardial capillary bed is simulated by microcirculatory units (MCU's) in a diffusion model. This simulation is based on MCU's in which the arrangement of the capillary ends (concurrent structure, partial and total countercurrent structure, helical structure) as well as the structure and supply parameters are varied. The variation of these parameters is based on own measurements of the intracapillary HbO₂ saturation as well as on the following parameters from the literature: frequency distribution of capillary distance and capillary radius, mean capillary length or capillary section length respectively, arterial and mean venousPO₂, mean coronary blood flow, mean O₂ consumption and diffusion conductivity. The analysis of O₂ supply of the normoxic rat heart shows that an O₂ diffusion shunt is obligatory except for MCU's with an extremely large capillary distance or with a concurrent capillary structure. Therefore the minimal tissuePO₂ lies at the level of the capillary venousPO₂ of a MCU. The maximum of the totalPO₂ frequency distribution in the normoxic rat myocardium lies at 25±5 mm Hg, i.e. above the mean venousPO₂ (20 mm Hg). TissuePO₂ values between 0 and 5 mm Hg amount to 0.5%, i.e. they are extremely rare. TissuePO₂ values of 0–1 mm Hg represent less than 0.2%.List of Symbols a arterial capillary end - a branching point of a capillary near the arterial capillary end (branching point of an anastomosis) - A maximal O₂-consumption - A(P) O₂-consumption dependent uponPO₂ - AVDO₂ arterio-venous difference - AVDO₂; AVDO_{2 c} ^(j) arterio-venous difference of a capillary - c _j weighting factor of the capillary distance - c _Hb hemoglobin concentration in the blood - d, d ^j capillary distance - mean capillary distance - i index for the different MCU's (i=1...8) - i.e. PO₂ intracapillaryPO₂ - j index for the parameters of an MCU with the capillary distanced ^(j) (j=1...7) - K diffusion conductivity - l capillary length - l _s capillary section length - MCU microcirculatory unit - P, P(x,y,z), PO₂ O₂ partial pressure - P _a arterialPO₂ - P _a; P_a ^(j) PO₂ at the branching pointa - P _i; P_v ^(j) venousPO₂ - mean venousPO₂ - P ₅₀ PO₂ at half maximal O₂ consumption - P _min,P _min ^(j) minimal tissuePO₂ - r _c, r_c ^(j) capillary radius - mean capillary radius - s(P) relative HbO₂ saturation (HbO₂ dissociation curve) - s ^–1 inverse function of the HbO₂ dissorciation curve - S _v, S_v ^(j) capillary venous HbO₂ saturation - mean venous HbO₂ saturation - v venous capillary end - V volume of the tissue fragment of a MCU - V _c, V_c ^(j) capillary supply volume - W _c, W_c ^(j) blood flow of the supply volume of a capillary (local blood flow) - mean blood flow - x,y,z cartesian coordinates of thePO₂ in a MCU - Bunsen's solubility coefficient - _c . _c ^(J) capilary blood flow - , ^(j) blood flow of an MCU - _i ^(j) (P) relative frequency distribution of thePO₂ in thei-th MCU - (P) relative frequency distribution of thePO₂ of all MCU's, total frequency distribution of the myocardial tissuePO₂ - Laplace operator Supported by the DFG     

Cellular and subcellular distributions of beta1- and beta2-adrenoceptors in the CA1 and CA3 regions of the rat hippocampus

Beta-adrenoceptors (ARs) in the hippocampus play an important role in regulating synaptic plasticity and memory consolidation. However, little is known about the distributions of beta-ARs in the hippocampus, especially in the cornu ammonis (CA)1 and CA3 regions of Sprague-Dawley rats. Here, we report that beta1- and beta2-ARs in the CA1 and CA3 regions have differential subcellular distributions. Using double immunofluorescence labeling and confocal laser scanning microscopy, we found that almost all of the neuronal nuclei positive cells express beta1- and beta2-ARs, while few glial fibrillary acidic protein positive cells express them. Interestingly, beta1-ARs are predominantly distributed in the cell membrane and cytoplasm, whereas beta2-ARs are predominantly distributed not only in the membrane and cytoplasm, but also in the nucleus. The differential subcellular distribution of beta1- and beta2-ARs may have functional significance.     

Cloning of serotonin 5-HT1 receptor subtypes from the chimpanzee, gorilla and Rhesus monkey and their agonist-induced guanosine 5′γS triphosphate binding

5-HT₁ receptor subtypes (_1B, _1D and _1F) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT_1D, and here 5-HT_1B and 5-HT_1F receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT_1B receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT_1F receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT_1D, 5-HT_1B and monkey 5-HT_1F receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5′gamma ³⁵S triphosphate (GTPγ³⁵S) binding through activation of G proteins containing Gγ_i subunits. Moreover, pronounced inhibition of basal GTPγ³⁵S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT_1D. Overall, ligand binding and GTPγ³⁵S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.     

Strain-selective effects of nicotine on electrophysiological responses evoked in hippocampus from DBA/2Ibg and C3H/2Ibg mice

We used the hippocampal slice to examine extracellular electrophysiological responses to nicotine and the difference in sensitivity to nicotine-induced electrophysiological effects between the DBA and C3H mouse strains. Nicotine enhanced CAj population spikes (PS) evoked by Schaffer collateral stimulation in a concentration-dependent manner (100 μM to 3.2 mM). This enhancement was slow to appear, achieving a maximum after 15 min. The enhanced PS was accompanied by the development of epileptiform activity (multiple PS's) at nicotine concentrations ≥400μM. Slices from DBA mice were significantly more sensitive than those from the C3H strain to these electrophysiological effects of nicotine. Mecamylamine (400 μM) blocked both the nicotine-induced PS enhancement and secondary population spikes in both strains, suggesting the involvement of nicotinic cholinergic receptors.     

Activation of the 5-HT6 receptor attenuates long-term potentiation and facilitates GABAergic neurotransmission in rat hippocampus

The 5-HT₆ receptor is predominantly expressed in the CNS and has been implicated in the regulation of cognitive function. Antagonists of the 5-HT₆ receptor improve cognitive performance in a number of preclinical models and have recently been found to be effective in Alzheimer's disease patients. Systemic administration of 5-HT₆ antagonists increases the release of acetylcholine and glutamate in the frontal cortex and dorsal hippocampus. In contrast, the selective 5-HT₆ agonist, WAY-181187, can elicit robust increases in extracellular levels of GABA. The reported behavioral and neurochemical effects of 5-HT₆ receptor ligands raise the possibility that the 5-HT₆ receptor may modulate synaptic plasticity in the hippocampus. In the present study, selective pharmacological tools were employed to determine the effect of 5-HT₆ receptor activation on long-term potentiation (LTP) in brain slices containing area CA1 of the hippocampus. While having no effect on baseline synaptic transmission, the results demonstrate that the selective 5-HT₆ agonist, WAY-181187, attenuated LTP over a narrow dose range (100–300 nM). The increase in the slope of the field excitatory post synaptic potential (fEPSP) caused by theta burst stimulation in brain slices treated with the most efficacious dose of WAY-181187 (200 nM) was 80.1±4.0% of that observed in controls. This effect was dose-dependently blocked by the selective 5-HT₆ antagonist, SB-399885. WAY-181187 also increased the frequency of spontaneous GABA release in area CA1. As assessed by measuring and evaluating spontaneous inhibitory postsynaptic currents (sIPSCs), 200 nM WAY-181187 increased sIPSC frequency by 3.4±0.9 Hz. This increase in GABA sIPSCs was prevented by the selective 5-HT₆ antagonist SB-399885 (300 nM). Taken together, these results suggest that the 5-HT₆ receptor plays a role in the modulation of synaptic plasticity in hippocampal area CA1 and that the regulation of GABAergic interneuron activity may underlie the cognition enhancing effects of 5-HT₆ antagonists.     

Role of 1,25-Dihydroxy Vitamin D3 and Parathyroid Hormone in Urinary Calcium Excretion in Calcium Stone Formers

Purpose

To find out the possible role of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] and parathyroid hormone (PTH) as intrinsic factors in urinary calcium stone formers (SFs), we investigated their relationship with serum and urinary biochemical parameters.

Materials and Methods

A total of 326 calcium SFs (male: 204, female: 122) were enrolled and underwent outpatient metabolic evaluations including 1,25(OH)₂D₃ and PTH as well as serum and 24-hour urinary biochemical parameters. As control, 163 age- and sex-matched (2:1) individuals (non-SFs) who have never urinary stone episode were included.

Results

1,25(OH)₂D₃ level was positively correlated with urinary calcium excretion (r=0.347, p<0.001). The hypercalciuric group and recurrent SFs had higher serum 1,25(OH)₂D₃ levels than the normocalciuric group (p<0.001) and first SFs (p=0.050). In the adjusted multiple linear regression analysis, serum 1,25(OH)₂D₃ level (β=0.259, p<0.001) and serum PTH level (β=-0.160, p<0.001) were significantly correlated with urinary calcium excretion. The patients in highest tertile of 1,25(OH)₂D₃ had a more than 3.1 fold risk of hypercalciuria than those in the lowest tertile (odds ratio=3.14, 95% confidence interval: 1.431-6.888, p=0.004). No correlation was observed between PTH and 1,25(OH)₂D₃ (R=0.005, p=0.929) in calcium SFs, while a negative correlation was found in controls (R=-0.269, p=0.001).

Conclusion

1,25(OH)₂D₃ was closely correlated with urinary calcium excretion, and high 1,25(OH)₂D₃ levels were detected in the hypercalciuric group and in recurrent SFs. However, 1,25(OH)₂D₃ was not correlated with PTH in calcium SFs. These findings suggest that 1,25(OH)₂D₃ might be important intrinsic factor for altered calcium regulation in SFs.     

In vivo 5-HT(6) receptor occupancy by antipsychotic drugs in the rat brain

The 5-HT₆ receptor subtype is predominantly expressed in the central nervous system, and preclinical evidence suggests that it plays a critical role in the regulation of molecular pathways underlying cognitive function. Patients with schizophrenia show cognitive impairment as a fundamental symptom, and it is proposed that the procognitive properties of some antipsychotics such as olanzapine and clozapine would be, in part, due to the central blockade of 5-HT₆ receptors. In this study, we characterized the brain 5-HT₆ receptor occupancy of olanzapine, clozapine and chlorpromazine in relation to their pharmacokinetic profiles using in vivo [³H]GSK215083 binding assay in rat brain. Oral administration of olanzapine (3 mg/kg), clozapine (30 mg/kg) and chlorpromazine (30 mg/kg) produced significant 5-HT₆ receptor occupancy in the brain, inhibiting radioligand binding by 88, 97 and 81%, respectively. The blood concentrations required to achieve significant occupancy were clinically achievable (9.6, 26.9 and 98.6 nM for olanzapine, clozapine and chlorpromazine, respectively). This data provides preclinical evidence to support the hypothesis that brain 5-HT₆ antagonism contributes to the procognitive properties of antipsychotic drugs such as olanzapine and clozapine.