Time Course of Dopamine-D2 and Serotonin-5-HT2 Receptor Occupancy Rates by Haloperidol and Clozapine In Vivo

Abstract: In vivo occupancy of dopamine-D₁, D₂ and serotonin-5-HT₂ receptors by haloperidol 10 mg/kg and clozapine 20 mg/kg were studied. Rats were injected intravenously with [³H]-YM-09151-2, [³H]-SCH23390, or [³H]-ketanserin 10 min after the administration of the tested drugs. Fifteen to 240 min after the ligand injection, the receptor occupancy rates of the drugs in the striatum and frontal cortex were calculated. Clozapine demonstrated the higher 5-HT₂ and lower D₂ occupancies in the respective regions. A dose-response analysis of D₂ and 5-HT₂, receptor occupancy by the drugs consolidated the higher 5-HT₂ binding affinity of clozapine in comparison with haloperidol. The present methodology may serve as an accurate tool to evaluate the peculiarity of various antipsychotics.     

Expression of the α1, α2 and α3 isoforms of the GABAA receptor in human alcoholic brain

The expression of the α₁, α₂ and α₃ isoforms of the GABA_A receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the α class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because α₁ expression was raised in both groups of alcoholics cf controls. The two groups of alcoholics could be differentiated on the basis of regional variations in α₁ expression. In frontal cortex, α₁ mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, α₁ expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform. © 1997 Elsevier Science B.V. All rights reserved.     

Dopamine D1 and D2 Receptor Antagonists Suppress Acute Stimulant Action of Cocaine, but Enhance Cocaine Sensitization

Abstract: The ambulation increase caused by the repeated dosing of cocaine (10 mg/kg s. c.) was dose-dependently reduced by the simultaneous administration of the selective dopamine D₁ and D₂ receptor antagonists, SCH 23390 (0.01, 0.03 and 0.1 mg/kg s. c.) and YM-09151-2 (nemonapride) (0.01, 0.03 and 0.1 mg/kg s. c.), respectively. However, the mice given cocaine with SCH 23390 (0.03 mg/kg) and cocaine with YM-09151-2 (0.03 and 0.1 mg/kg) 5 times at 3 to 4-day intervals showed significantly higher sensitivity than the mice given cocaine alone to the challenge cocaine. The present results suggest that, although the blockade of either dopamine D₁ or D₂ receptor is effective for a reduction in the stimulant action of cocaine, such treatment enhances the induction of cocaine sensitization.     

Localization of the mRNAs for Two Dopamine D2 Receptor Isoforms in the Rat Brain

Abstract: Two molecular forms of the dopamine D., receptor were generated by alternative RNA splicing. To investigate the relative distributions of the two mRNAs encoding the D2 receptor isoforms, D2(415) and D2(444), we performed in situ hybridization histochemistry in the rat brain with the two oligonucleotide probes. An insert probe complementary to an additional fragment of the D, receptor mRNA cloned from the rat brain, and a spanning probe complementary to its contiguous sequence were used. These 48 base probes were 3'-end labeled with [35S]dATP. The brains were dissected from male SD rats and frozen in dry ice and acetone. Cryostat sections (16 um) were collected on gelatin coated slides and stored at – 20^oC. In situ hybridization studies were conducted with a probe concentration of 1 × 10e dpm/100 ^1 of buffer per brain slice at 37^oC for 18–20 h in a humid chamber. The slides were washed, dried and exposed to tritium sensitive film for one week. The autoradiograph showed that both mRNA were present at high levels in the corpus striatum, accumbens nucleus and substantia nigra (pars compacta). Identical patterns of labeling were obtained in the rat brain using both the insert and spanning probes, although the optical densities detected with the insert probe were higher than those with the spanning probe in the corpus striatum. This suggests that both D2 receptor mRNAs are expressed similarly in each region of the rat brain and D2(14) expressed dominantly in the corpus striatum.     

Involvement of M2 muscarinic receptors in relaxant response of circular muscle of mouse gastric antrum

Our previous study showed that atropine significantly inhibited the sustained relaxation induced by electrical field stimulation (EFS) in the circular muscle strips prepared from the mouse antrum, and that pituitary adenylate cyclase activating peptide (PACAP) partially mediated the sustained relaxation. The muscarinic receptor subtype associated with the sustained relaxation was examined in the present study by using each muscarinic receptor subtype of knockout (KO) mice. EFS-induced sustained relaxation in the antrum prepared from M(2) receptor KO mice was significantly less than that of wild-type mice. Atropine failed to inhibit this relaxation. On the other hand, similar sustained relaxation and inhibitory effects of atropine to those of wild-type mice were observed in M(1), M(3) and M(4) receptor KO mice. Exogenously added PACAP-27 relaxed the antral strips of wild-type and M(2) receptor KO mice to a similar extent. Immunohistochemical study revealed that M(2) receptor immunoreactivity was localized with PACAP-immunoreactivity in enteric neurons within the antrum of wild-type mice. In contrast, atropine did not affect the EFS-induced sustained relaxation in the gastric fundus. These results suggest that M(2) receptors modulate the sustained relaxation, probably through the regulation of PACAP release, in the mouse antrum.     

Localization of Dopamine D2 Receptor in Rat Spinal Cord Identified with Immunocytochemistry and In Situ Hybridization

In the present study the distribution of dopamine D₂ receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D₂ receptor and in situ hybridization (ISH) using a [³⁵S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D₂ receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D₂ receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D₂ receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D₂ receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Neuroleptic Drugs and 5HT1 Receptor:Different Potencies of Various Neuroleptic Drugs on 5HT1 Receptors in Discrete Regions of the Rat Brain

Abstract: The potencies of various neuroleptic drugs (zotepine, chlorpromazine-HCl, haloperidol and spiperone) on serotonin, (5HT₁) receptors were examined in discrete rat brain regions using the radio receptor assay. The potencies of the neuroleptic drugs on 5HT₁ receptors were clearly differentiated in the discrete brain regions:zotepine was the most potent in the frontal cortex, striatum and brain stem; spiperone was the most potent in the hippocampus. Furthermore, zotepine and chlorpromazine-HCl produced no great differences among the various regional 5HT₁ receptors, while butyrophenones, haloperidol and spiperone showed remarkable differences. These findings demonstrate that the neuroleptic drugs can be differentiated according to their different affinities for regionally discrete 5HT, receptors in the brain. This suggests that 5HT₁ receptors may be able to be classified into two subtypes:the zotepine and chlorpromazine-HCl group having a high affinity for one subtype and butyrophenones a high affinity for the other.     

A Role for the Mineralocorticoid Receptor in a Rapid and Transient Suppression of Hippocampal 5-HT1A Receptor mRNA by Corticosterone

The interaction between corticosterone and hippocampal 5-HT_1A receptor mRNA expression was investigated in male rats. Two days after adrenalectomy, 5-HT_1A receptor mRNA was increased in the granule cell layer of the dentate gyrus. Injection of corticosterone led to a significant dose-dependent and transient suppression of 5-HT_1A receptor mRNA. The effect of a low dose of corticosterone (50 μg/kg) could be blocked by RU 28318, a specific mineralocorticoid receptor antagonist, but not by RU 38486, a glucocorticoid antagonist. The effect of a higher dose of corticosterone (300 μg/kg) could be partially blocked by RU 28318, whereas there was no additional effect of RU 38486. These results point to a stringent regulation of hippocampal 5-HT_1A receptors by corticosterone that is predominantly mediated by the mineralocorticoid receptor.     

Evaluation of a Possible Role for the Dopamine D1 and D2 Receptors in the Steroid-Dependent Suppression of Luteinizing Hormone Secretion in the Seasonally Anoestrous Ewe

This study was undertaken to determine whether dopaminergic suppression of pulsatile luteinizing hormone (LH) secretion during seasonal anoestrus in the ewe is mediated via the dopamine D₁ or D₂ receptor. This was tested by 1) assessing the response to dopamine D₁ and D₂ antagonists during seasonal anoestrus, and 2) determining the ability of D₁ and D₂ agonists to suppress pulsatile LH secretion during the breeding season. In seasonally anoestrous ewes the D₂ antagonist pimozide increased LH pulse frequency although this effect did not reach significance (P = 0.07). The D₁ antagonist SCH 23390 had no effect on LH pulse frequency. LH pulse amplitude and mean LH were not affected by either treatment. During the breeding season, ovariectomized oestradiol-implanted ewes were injected intracerebroventricularly with vehicle, LY 171555 (dopamine D₂ agonist) and SKF 38393 (D₁ agonist) with each drug tested at 50 μg and 200 μg. At the higher dose, LY 171555 significantly (P<0.05) reduced LH pulse frequency in the 2 h period immediately after treatment. Mean LH declined at both doses but only in the first hour after treatment. SKF 38393 did not affect LH pulse frequency, pulse amplitude or mean LH. These results suggest that the D₁ receptor is not involved in the suppression of pulsatile LH secretion during seasonal anoestrus. Dopaminergic suppression of pulsatile LH secretion is mediated via the D₂ receptor but the significance of this neurotransmitter in the seasonal suppression of LH remains to be elucidated.     

Possible Relationship between Antimanic Effect and Activity of Zotepine to 5HT1 Receptor

Abstract: Zotepine (ZTP), synthesized by Fujisawa Pharmaceutical Co., Ltd. for possible use as an antipsychotic drug, clinically features a very rapid and potent antimanic effect. To elucidate the psychopharmacological mechanisms of zotepine, we have attempted to measure the potency of ZTP compared with other neuroleptic drugs in competing for binding sites in the brain associated with dopamine, serotonin (5–HT₁, 5–HT₂), noradrenaline (NA) and acetyl-choline. Zotepine was found to have the most potent activity to the 5HT1 receptor among the test drugs. Chlorpromazine and thioridazine, which belong to phenothiazines and clinically have less potent antimanic effect, shared ZTP's potent activity to the NA receptor, while they were less potent than ZTP in activity to the 5HT₁ receptor. These results show that the activity of the drugs to the 5HT₁ receptor may be associated with the antimanic effect.     

Use of positron emission tomography to measure the effects of nalmefene on D1 and D2 dopamine receptors in rat brain

Positron emission tomography (PET) has been used in humans and in non-human primates to image and measure radioligand binding to neuroreceptors. The present study evaluated the feasibility of performing high-resolution PET experiments in a rodent model to measure receptor kinetics. The effects of acute and chronic administration of the opioid antagonist, nalmefene, on the binding activity of [¹¹C]SCH23390 and [¹¹C]N-methylspiperone at D₁ and D₂ dopamine receptors, respectively, was investigated in the rat. The interaction between central opioid and dopaminergic systems has been the focus of much attention due to their interactive role in mediating reinforcement and locomotor activity. In the present study, adult male Sprague–Dawley rats received either a single injection of 10 mg/kg of nalmefene or control vehicle solution 1 h prior to the PET scan or were chronically administered 10 mg/kg/day of nalmefene or vehicle for 7 days by an osmotic minipump. Following acute administration of nalmefene, the binding potential of [¹¹C]SCH23390 in the striatum was significantly increased. No changes in [¹¹C]N-methylspiperone binding were found. Following chronic nalmefene administration, no significant change in either [¹¹C]SCH23390 binding potential or [¹¹C]N-methylspiperone binding was detected. These results suggest that nalmefene administration produces transient changes in the binding potential of D₁-receptors in the striatum that are normalized after 1 week of steady-state administration.     

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

Hypothyroidism Alters the Expression of Prostaglandin D2 Synthase/β- Trace in Specific Areas of the Developing Rat Brain

Lipocalin-type prostaglandin D₂ synthase is the enzyme responsible for the synthesis of prostaglandin D₂, a major prostaglandin in the central nervous system. We analysed the effects of thyroid hormone deprivation on prostaglandin D₂ synthase gene expression in the developing rat brain. By in situ hybridization, the strongest prostaglandin D₂ synthase mRNA signal was detected in the leptomeninges and choroid plexus. The signal was greatly reduced in the cerebellar interlaminar meninges of hypothyroid rats aged 15 and 25 days. lmmunohistochemical studies defined changes in the location of the prostaglandin D₂ synthase protein. In control but not in hypothyroid animals, Cajal-Retzius neurons of cortical layer 1, and pyramidal cortical plate neurons were intensely stained on postnatal day 5. Conversely, prostaglandin D₂ synthase protein levels were higher in neurons of the CA1 and CA3 regions and the dentate gyrus of the hippocampus of hypothyroid animals on postnatal days 5, 15 and 25, and also in subplate neurons on postnatal days 15 and 25. In agreement with the in situ hybridization and northern blotting data, the major difference was found in the cerebellar interlaminar meninges of hypothyroid animals, where the protein was clearly down-regulated on postnatal days 15 and 25. These results show that hypothyroidism causes both age- and region-specific alterations in the expression and location of the prostaglandin D₂ synthase during postnatal brain development, probably reflecting a cell-specific regulatory effect of thyroid hormone on the prostaglandin D₂ synthase.     

Expression of the Neural Recognition Molecule L1 by Cultured Neural Cells is influenced by K+ and the Glutamate Receptor Agonist NMDA

To investigate the influence of neuronal activity on the expression of neural recognition molecules, cultures of neural cell lines and dissociated cells of early postnatal mouse cerebellum were maintained in the presence of elevated concentrations of K+ and the glutamate agonist N-methyl-d-aspartate (NMDA). Levels of expression of the neural adhesion molecules L1 and N-CAM at the cell surface were measured by an enzyme-linked immunosorbent assay. Expression of L1 was up-regulated in neuroblastoma N2A cells after 1 day of maintenance in 40 and 60 mM K+, but not in phaeochromocytoma PC12 cells. Expression levels of N-CAM and antigens recognized by the monoclonal antibody A2B5 or by polyclonal antibodies to crude membrane fractions of liver were not significantly altered by elevated K+ concentrations in these two cell lines. In monolayer cultures of early postnatal mouse cerebellum, an increase of 60% in expression of L1, but not N-CAM or A2B5, was seen at 20 and 40 mM K+. This increase in L1 expression was specifically inhibitable by the Ca2+ channel blocker nicardipine. NMDA at a concentration of 100 microM increased levels of L1, but not of N-CAM. This increase was inhibitable by the NMDA antagonists 2-amino-5-phosphonovalerate and MK-801, but not significantly by the kainate/quisqualate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The increase in L1 expression at higher K+ concentrations was not inhibitable by the NMDA antagonists, indicating that the K+-mediated increase in L1 expression is not due to release of glutamate by cerebellar neurons. These observations indicate that compounds influencing neuronal membrane properties, and thus neuronal excitability, are capable of regulating the expression of L1. In a more general context, these findings suggest that previously observed changes in synaptic connectivity in situ, resulting from activity-dependent fine tuning of neuronal morphology, may be mediated by alterations in the expression of recognition molecules.     

Divergent role for CRF1 and CRF2 receptors in the modulation of visceral pain

Both anti- and pro-nociceptive effects of corticotropin-releasing factor (CRF) treatment on visceral pain have been reported. Here, this dual action of CRF was differentiated by selective (in)activation of the CRF1 and CRF2 receptor prior to a visceral pain stimulus. Visceral pain was evaluated out of behavioural and visceromotor (abdominal electromyogram) responses to duodenal distension in the freely moving rat. Intraperitoneal (i.p.) CRF (50 microg kg-1) increased the distension-induced visceromotor and behavioural pain response. The pro-nociceptive effects of CRF on the behavioural response were attenuated by a selective CRF1 (CP-154526; 20 mg kg-1) but not a selective CRF2 [antiSauvagine30 (aSVG30); 100 microg kg-1] antagonist. Selective activation of the CRF2 receptor by stresscopin-related peptide (SRP; i.p. 25 microg kg-1) reduced the distension-induced visceromotor and behavioural response. Intrathecal injection of CRF (2 microg 10 microL-1) or SRP (20 microg 10 microL-1) decreased the distension-induced visceromotor and behavioural response. The antinociceptive effects of intrathecal CRF on the behavioural response were attenuated by aSVG30 (20 microg 10 microL-1) but not with CP-154526 (10 microg 10 microL-1). These findings indicate that the CRF1 receptor is involved in pro-nociception of visceral pain, whereas the CRF2 receptor is mainly involved in antinociception. This divergent role of the CRF subreceptors may explain the bimodal effects of CRF treatment on visceral nociception.