Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.

Activity of aromatic substituted phenylpiperazines lacking affinity for dopamine binding sites in a preclinical test of antipsychotic efficacy.

Generally, antipsychotic agents are dopamine receptor blocking agents that also block conditioned avoidance responding (CAR) in the rat. Recently, however, both (Q-methoxyphenyl)piperazine (OMPP, 1h) and (m-chlorophenyl)piperazine (MCPP, 1o) have been reported to block conditioned avoidance responding in the rat although neither has dopamine receptor blocking properties. The present paper examines the behavioral and biochemical profile of a number of additional substituted phenylpiperazines. None of the phenylpiperazines tested demonstrated high affinity for either dopamine D-1 or D-2 receptor sites, yet many were effective in blocking CAR. The results suggest that the phenylpiperazines may be effective antipsychotic agents without blocking dopamine receptors. Moreover, the active compounds did demonstrate activity in displacing ligand binding to serotonin receptors. Receptor binding profiles were determined for 5-HT-1A and 5-HT-1B binding sites as well as for 5-HT-2 sites. The data from this preclinical test suggest these phenylpiperazines might be effective antipsychotic agents acting via a nondopaminergic mechanism of action.

Pyrrole mannich bases as potential antipsychotic agents.

Recently, we reported on a series of arylpiperazines 4 which exhibit high affinity for the serotonin 5-HT-1A and 5-HT-1B binding sites. Although these compounds interact weakly with dopamine D-1 and D-2 receptors, they are reasonably potent in inhibiting conditioned avoidance responding (CAR) in the rat, an indication of potential antipsychotic activity. Conversion of these arylpiperazines to pyrrole Mannich bases has provided a series of compounds (10-44) which exhibit potent inhibition of CAR when given po and have strong affinity for both the D-2 and 5-HT-1A binding sites. Some of these agents also fail to produce catalepsy. The D-2 binding data and the block of CAR suggest that they are potential antipsychotic agents and the lack of cataleptogenic potential suggests some might possess less liability for producing extrapyramidal side effects and tardive dyskinesias in man.

Prolactin implants in the hypothalamus inhibit prolactin surges during pregnancy and alter prolactin release in response to dopamine receptor blockade.

Abstract The purpose of this study was to determine whether prolactin (PRL) could exert a negative feedback on it's own secretion during pregnancy and following ovariectomy, and to determine the possible mechanism by which this feedback acts. Implantation of ovine PRL into the arcuate nucleus-median eminence area of the hypothalamus completely inhibited the nocturnal PRL surge during pregnancy in the rat, and lowered baseline PRL to almost undetectable levels in the ovariectomized rat. When ovariectomized rats implanted with ovine PRL in the hypothalamus were injected with the dopamine receptor blocker domperidone the PRL response was significantly lower than in control rats implanted with albumin. Pregnancy increased the ability of domperidone to cause PRL release following ovine PRL implantation compared to what occurred in ovariectomized rats. These results suggest that PRL implantation increases the secretion of dopamine from the tuberoinfundibular neurons, resulting in a decrease in PRL secretion.

Central administration of serotonin decreases tyrosine hydroxylase catalytic activity and messenger ribonucleic Acid signal levels in the hypothalamus of female rats.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating prolactin (PRL) levels. Ovariectomized rats were treated with either vehicle or 5-HT through a lateral ventricular cannula in one of two dose paradigms: 1) a bolus of 20 µg, with tissues taken at 30 min, or 2) the same bolus immediately followed by 20 µg/30 min via a syringe pump for 120 min, and tissues taken at 120 min. Blood samples were taken throughout experiments and plasma PRL determined by radioimmunoassay. Under both paradigms, NSD 1015, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (25 mg/kg intraarterially) was injected 10 min before decapitation and brain excision followed by stalk-median eminence dissection. The rate of DOPA accumulation, determined by measuring DOPA levels in the stalk-median eminence by high-performance liquid chromatography with electrochemical detection was used as a measure of tyrosine hydroxylase (TH) catalytic activity. Stalk-median eminence DOPA accumulation in control rats was 29.9 ± 4.2 and 28.8 ± 4,4 ng/mg protein (30 and 120 min experiments, respectively). DOPA accumulation in 5-HT-treated rats was significantly reduced (P<0.05) after 30 min to 17.8 ± 1.2 ng/mg protein, but it was similar (21.7 ± 3.9) to controls after 120 min of 5-HT infusion. 5-HT levels in the stalk-median eminence of rats treated with 5-HT were 13- to 17-fold greater than controls (16.9 to 18.5 ng/mg protein). Plasma PRL levels in both groups increased 10-fold after 5-HT treatment with a peak at 5 min, returning to baseline by 120 min. TH mRNA levels were determined by in situ hybridization in a second group of rats which were treated with the 20µg bolus and subsequent 120 min infusion of 5-HT. TH mRNA signal levels in the arcuate nucleus of control rats averaged 144 ± 21 grains/cell. After treatment with 5-HT, TH mRNA levels in the arcuate nucleus were significantly lower (P<0.0001) with 69±14 grains/cell. In a third group of rats, the effects of the 30 min 5-HT treatment on TH catalytic activity and circulating PRL levels was challenged with two 5-HT(2) receptor antagonists, LY53857 (5 mg/kg intraperitoneally) or ketanserin (10 mg/kg intraperitoneally). Neither the 5-HT-induced decrease in TH catalytic activity nor the increase in PRL was altered by pretreatment (120 min) with 5-HT(2) antagonists. These data suggest that central 5-HT is capable of decreasing TH activity and TH mRNA levels in the tuberoinfundibular dopamine neurons and that the decrease in dopaminergic neuronal activity may contribute to the 5-HT-induced PRL rise. The changes in TH catalytic activity and PRL after intracerebroventricular administration of 5-HT do not appear to be mediated by 5-HT(2) receptors.

Blockade of conditioned avoidance responding by trazodone, etoperidone, and MCPP.

Phenylpiperazines, such as meta-chlorophenylpiperazine (MCPP) a serotonin agonist, have recently been reported to block conditioned avoidance responding (CAR) in the rat, which is an indication of possible antipsychotic utility. Since MCPP is a major metabolite of both antidepressant drugs trazodone (TZ) and etoperidone (ET), both were examined for activity in blocking CAR in a single-trial lever press task in Fisher 344 rats. Both TZ and ET produced dose-related falls in CAR with ED50 values (95% confidence limits) of 13.3 (9.6, 18.5) and 10.4 (8.5, 13.2) mg/kg IP, respectively. In contrast, MCPP had an ED50 value of 2.5 (1.8, 3.6) mg/kg IP TZ, ET, and MCPP were also examined for the production of catalepsy and the blockade of amphetamine-induced stereotypy to determine whether each was acting to block CAR via a dopaminergic mechanism of action. None, however, was found highly active. On the other hand, the serotonin receptor blocker metergoline (1.0 mg/kg IP) significantly reduced the CAR block produced by each, suggesting a serotonergic mechanism of action. Since TZ and ET are both less potent than MCPP, the data also suggest TZ and ET may block CAR via formation of MCPP.

[3H][D-Ala2,NMePhe4,Gly-ol5]-enkephalin (mu-opioid) binding in beige-J mice.

Tritiated [D-Ala2,NMePhe4,Gly-ol5]-enkephalin ([3H]DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on [3H]DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.

Mu-, but not delta-, opioid receptor-mediated antinociception in mice is attenuated by gamma-irradiation.

Mice were exposed to whole-body irradiation (500 rads) from a 137Cs gamma-source and tested 2 h later for antinociception (tail-flick test) produced by intracerebroventricular administration of morphine or the more delta-selective opioid peptide, [D-Pen2,L-Pen5]enkephalin (DPLPE). Irradiation significantly attenuated the antinociception produced by morphine, but not by DPLPE. These results demonstrate a differential sensitivity of mu- and delta-opioid receptors to gamma-irradiation and, in addition, may be of clinical relevance for cancer patients receiving concurrent radiation therapy and opioid analgesics.

[D-Pen2,L-Pen5]enkephalin induced analgesia in the jimpy mouse: in vivo evidence for delta-receptor mediated analgesia.

Jimpy (B6CBA-A W-J/A-Ta jp) mice, which are known to be deficient in neuronal cerebroside sulfate (a putative component of the mu opioid receptor), were non-responsive in the tail flick test (compared to littermate controls and a standard Swiss strain of mouse) to analgesic doses of two mu opioid receptor agonists, morphine sulfate and [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO). However, the jimpy mice responded normally (compared to controls) to the analgesic effects of the selective delta opioid receptor agonist [D-Pen2,L-Pen5]enkephalin (DPLPE). These results suggest (1) that delta opioid receptors can mediate analgesia and (2) that cerebroside sulfate is not necessary for delta opioid receptor activation.

Central administration of p-octopamine to mice: assessment of antinociception.

Administration of p-octopamine by intracerebroventricular (i.c.v.) or intrathecal (i.t.) routes, but not orally, produced antinociception in the acetylcholine-induced abdominal constriction test (ED50 = 24.8 and 3.6 micrograms, respectively). Likewise, i.c.v. and i.t., but not peripheral (up to 200 mg/kg s.c.), administration increased latency in the 48 degrees C hot-plate test (ED50 = 11.5 micrograms i.c.v. and 0.2 micrograms i.t.). These actions were relatively long-lasting and not blocked by naloxone. Antinociception following i.c.v. administration was abolished in reserpinized mice or by pretreatment with i.t. phentolamine (2 micrograms). These results suggest a moderate antinociceptive action of p-octopamine involving non-opioid, reserpine-sensitive, central pathways.

Restoration of analgesic response of C57BL/6J-bgJ (beige-J) mice to morphine by carbachol.

C57BL/6J-bgJ (beige-J) mice respond poorly to the analgesic effects of centrally administered (intracerebroventricular; i.c.v.) morphine in the tail-flick test. In the present study C57BL/6J-bgJ mice, their littermate (heterozygous) controls, and normal Swiss mice were given carbachol (200 micrograms/ml) in their drinking water for three weeks. Carbachol had no effect on control animals. However, the carbachol-treated beige-J mice responded to i.c.v.-administered morphine (1 microgram) significantly better than untreated beige-J mice and nearly as well as the normal mice.

Block of conditioned avoidance responding in the rat by substituted phenylpiperazines.

Ortho-methoxyphenylpiperazine (OMPP) and meta-substituted chlorophenylpiperazine (MCPP) blocked conditioned avoidance responding (CAR) in the rat (ED50 values = 5.6 (4.6, 7.3) and 2.4 (1.9, 2.9) mg/kg i.p. (95% confidence limits), respectively) without markedly altering escape responding. Since this test predicts antipsychotic efficacy, the piperazines were examined in radioligand binding assays and found to have no affinity for dopamine (DA) binding sites, but were active at serotonin binding sites. OMPP displaced ligands for the 5-HT1A binding site with high affinity (Ki = 9.5 (5.4, 17.9) nM) but was inactive at 5-HT2 sites (Ki greater than 1000 nM). MCPP, on the other hand, displaced ligands for 5-HT1, 5-HT1A and 5-HT2 binding sites with similar potencies (Ki values = 25 (3, 67), 23 (14, 40) and 40 (33, 48) nM, respectively). Pretreatment with metergoline (1.0 mg/kg i.p. -30 min) reduced MCPP- but not OMPP-induced block of CAR. OMPP, on the other hand, acted as a DA receptor antagonist in vivo blocking amphetamine-induced stereotyped behavior, whereas MCPP did not. Neither produced catalepsy even given in doses 8-10 times those required to block CAR. Insofar as these compounds lack antidopaminergic activity in vivo, yet are active in a test (CAR) predictive of antipsychotic activity in which DA receptor antagonists are active, they may be novel antipsychotic agents, or, perhaps, false positives in the CAR paradigm.

The effect of Phe-Met-Arg-Phe-NH2 (FMRFamide) on morphine-induced inhibition of colonic propulsive motility in mice.

Morphine and the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) were administered to mice alone or in combination intracerebroventricularly (i.c.v.) and the effect on colonic propulsive motility was measured. Both morphine (1.0 microgram, i.c.v.) and FMRFamide (10 and 50 micrograms, i.c.v.) delayed expulsion of a 3 mm glass bead placed in the distal colon of mice compared to vehicle-treated controls. The inhibitory effects of morphine and FMRFamide on expulsion time were additive at the doses used and individually blocked by naloxone. These data suggest that FMRFamide does not antagonize this nonanalgesic effect of morphine, but appears to have opioid agonist properties.

The analgesic defect of C57BL/6J-bgJ/bgJ (beige-J: Chediak-Higashi syndrome) mice transmitted by adoptive transfer of spleen cells to normal littermates.

We recently discovered and reported that C57BL/6J-bgJ/bgJ (beige-J) mice have a deficiency in their analgesic response to intracerebroventricularly-administered morphine in the tail-flick test. Postulating a link between these findings and the known immunological defect of beige-J mice (Chediak-Higashi syndrome), we examined the effect of splenectomy on beige-J mice and the adoptive transfer of their mononuclear spleen cells to normal littermate controls (2 x 10(7) cells via tail vein). Eight days after these interventions, the splenectomized beige-J mice responded nearly as well as normal mice to centrally administered morphine in the tail-flick test. The adoptive transfer recipients, in contrast, nearly completely lost their response to the analgesic action of morphine in this test. From the combined results, the spleen appears to be a significant factor in the analgesic defect of beige-J mice and, furthermore, mononuclear splenocytes appear to be the source of a substance that can transfer this defect to otherwise normal animals.

Colonic bead expulsion time in normal and mu-opioid receptor deficient (CXBK) mice following central (ICV) administration of mu- and delta-opioid agonists.

A method utilizing the insertion of a 3 mm glass bead into the distal colon was used to evaluate the activity of intracerebroventricularly (ICV) administered mu- and delta-opioid agonists on colonic bead expulsion time in mice. Specifically, the ability of two mu-opioid receptor agonists, morphine and [D-Ala2,NMePhe4, Gly-ol5]-enkephalin (DAGO) and a selective delta-opioid receptor agonist, [D-Pen2,L-Pen5]-enkephalin (DPLPE), to inhibit colonic bead expulsion time was measured in normal (Swiss) and mu-opioid deficient (CXBK) mice. All three compounds maximally inhibited colonic bead expulsion time in normal mice. All three compounds also inhibited colonic bead expulsion time in CXBK mice, but none maximally. These results are in contrast to previous work in which clear differential analgesic sensitivity of CXBK mice to centrally administered mu- and delta-opioid receptor agonists was observed in the tail-flick test. Taken together, the results suggest (a) that mu-, and possibly delta-, opioid receptors can mediate supraspinal inhibition of colonic bead expulsion in mice and (b) that the genetic deficits of mu-receptor number or genetically-induced alteration in receptor function in CXBK mice do not equally affect inhibition of colonic bead expulsion and tail-flick antinociception.

C57BL/6J-bgJ (beige) mice: differential sensitivity in the tail flick test to centrally administered mu- and delta-opioid receptor agonists.

The antinociceptive effects of two mu-opioid receptor agonists, morphine and [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO), and a selective delta-receptor agonist, [D-Pen2, L-Pen5]enkephalin (DPLPE), were determined in C57BL/6J-bgJ (beige) and control mice (CRS-CDl and C57BL/6By) using a standard tail-flick assay. The antinociceptive response of C57BL/6J-bgJ mice to intracerebro-ventricularly administered morphine and DAGO was significantly reduced compared to controls, but there was no difference in the antinociceptive response to DPLPE. These results suggest that there is a genetic deficit of mu-opioid receptor number or a genetically-induced alteration in receptor function in regions of C57BL/6J-bgJ brains involved in antinociception, that delta-opioid receptors can mediate antinociception in mice, and that the C57BL/6J-bgJ strain may offer a practical new animal model for studying the function of opioid receptor subtypes.

Evidence that reciprocal hindlimb scratching elicited in mice by intrathecal administration of muscarinic agonists is mediated through M1 type receptors.

Intrathecal (IT) administration of pilocarpine to mice produces a vigorous and dose-related reciprocal hindlimb scratching (RHS) response (ED50 = 0.6 microgram) that is potently blocked by simultaneous IT administration of atropine (ID50 = 0.002 microgram). We now report that RHS is (1) also elicited by the more selective M1 agonist McN-A-343-11 (ED50 = 11.6 micrograms), (2) blocked by the selective M1 antagonist pirenzepine (ID50 = 0.001 microgram), and (3) is not blocked by the selective M2 antagonist AF-DX 116 BS at a dose up to 100 times the ID50 dose of pirenzepine. These results extend our earlier findings and suggest that the RHS elicited in mice by IT injection of muscarinic agonists is mediated through pirenzepine-sensitive (presumably M1) receptors and that RHS may be a convenient in vivo centrally mediated M1 endpoint.

The combined immunological and antinociceptive defects of beige-J mice: the possible existence of a 'mu-repressin'.

A selective non-responsiveness to the analgesic effects of opioid mu receptor-, but not opioid delta receptor-, mediated antinociception in the tail-flick test has been identified in C57BL/6J-bgJ (beige-J) mice. The beige-J mutation is also known to give rise to multiple immunological disorders and immune cell dysfunctions. A link between these apparently disparate manifestations has been examined in a series of studies using, for example, adoptive transfer of spleen cells. The findings appear to have broad implications for the link between the immune and opioid systems.

Differential regulation of the nocturnal and diurnal prolactin surges in pregnant rats revealed by dopamine receptor antagonism.

We have explored temporal changes in the magnitude of dopamine (DA) interaction (DA tone) at the anterior pituitary lactotrophs related to both the nocturnal and diurnal prolactin (PRL) surges on day 8 of pregnancy, by utilizing a competitive DA D2 antagonist, domperidone (DOM). After withdrawal of blood from pregnant rats on day 7 in order to demonstrate the presence of a PRL surge, experimental rats received DOM (100 micrograms/kg i.v. or i.a.) at various times on day 8. Blood samples were taken immediately before and following injection of DOM at 5, 15, 30 and 60 min. The peak PRL response to DOM occurred 15 min after injection. Comparisons were made between circulating PRL levels immediately prior to and at several times following DOM administration for the various times of the day, and represented as incremental increases in PRL following DOM. During times on day 8 when PRL levels were normally low (24:00, 06:00, 12:00 and 16:00 h), pregnant rats exhibited a substantial PRL response to DOM. However, during the nocturnal PRL surge (02:00, 04:00 h) the peak PRL response to DOM was significantly lower. In sharp contrast, the PRL response to DOM administered during the diurnal PRL surge (18:00 h) was significantly higher than all other times of the day tested. In a dose-response study in which 10, 100 and 1,000 micrograms/kg DOM was administered at the two critical times when the response to DOM differed greatly, 02:00 and 18:00 h, there was a significantly reduced PRL response to DOM at 02:00 h compared to 18:00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of the involvement of supraspinal and spinal mu and delta opioid receptors in analgesia using the mu receptor deficient CXBK mouse.

The role of mu and delta opioid receptors in the spinal and supraspinal analgesic actions of morphine and [D-Pen2, L-Pen5] enkephalin were examined in the tail-flick test utilizing the mu opioid receptor deficient CXBK mouse and BALB/cBy and C57BL/6By, the progenitor strains of CXBK. The analgesic effects of i.c.v. administered morphine were equivalent in the CRS-CD1 (Swiss) standard laboratory mice and the progenitor strains of CXBK. Morphine did not, however, produce analgesia in the CXBK mice at doses greater than 10 times the ED50 dose in the progenitor strains. Similarly, the analgesic effect of i.c.v. [D-Ala2, NMePhe4, Gly-ol]enkephalin, a highly selective mu receptor peptide agonist, also was reduced greatly in the CXBK mice. These data are consistent with the deficiency in mu opioid receptors observed autoradiographically in this strain. In contrast, the highly selective delta opioid receptor peptide agonist [D-Pen2, L-Pen5]enkephalin was equipotent i.c.v. in the CXBK mice and in the progenitor strains of CXBK. In contrast to the effects produced by i.c.v. administration, the analgesic effects of intrathecally administered morphine were similar between CRS-CD1 and CXBX strains of mice. These results suggest that 1) both mu and delta opioid receptors can mediate supraspinal analgesia and 2) that the receptor(s) involved in spinally mediated analgesia is (are) quite distinct from those involved supraspinally.