Manipulations of synaptic serotonin: discrepancy of effects on serotonin S1 and S2 sites

The effect of repeated treatment (28 day) with D-fenfluramine, a serotonin (5HT) releaser, L-tryptophan, a 5HT precursor, or fluoxetine, a 5HT uptake inhibitor, on 3H-5HT and 3H-spiperone binding in the rat cerebral cortex was investigated. Treatment with fenfluramine and fluoxetine caused a significant decrease in the number of 3H-5HT binding sites (Bmax). Fenfluramine also decreased binding of 3H-spiperone in the cortex, but fluoxetine treatment increased this binding. Treatment with L-tryptophan produced no change in the binding of either 3H-5HT or of 3H-spiperone significantly. The data show that manipulation of synaptic 5HT concentration does not always result in parallel changes in S1 and S2 receptors. This suggests that the 5HT S1 and S2 receptors may be subject to different regulatory mechanisms.     

Binding of 3H-spiperone in the mouse brain after intraperitoneal injection

In experiments on mice 3H-spiperone binding after intraperitoneal injection was studied. The binding of 3H-spiperone was saturable in the frontal cortex and subcortical structures, whereas in the cerebellum, the amount of radioactivity increased in a linear manner and was referred to as nonspecific binding. The neuroleptics haloperidol, chlorpromazine and sulpiride given 0.5 h before 3H-spiperone displaced 3H-spiperone in the subcortical structures in a dose-related manner. Although the level of the specific 3H-spiperone binding after intraperitoneal injection was lower than after intravenous injection, the intraperitoneal method is simpler, well reproduced and given results comparable with the intravenous method.     

Serotonin receptors in the brain of animals selected for their domesticated type of behavior

Participation was studied of central serotonin receptors of the first and second types in behaviour change of animals selected by the character of defensive reaction to man. Serotonin receptors were determined by radioligand method by binding of the brain preparations 3H-serotonin and 3H-spiperone. An increase of C2 receptors number was found in the frontal brain cortex of the tame brown rats in comparison with the aggressive ones. Differences were not found in specific C1-receptor binding in the frontal brain cortex of tame and aggressive brown rats, silver foxes and American minks in various relatively early selection stages. It is supposed that disappearance of aggressive reaction to man at domestication is connected with an increase of C2 receptors number.     

In vivo binding of 3H-N-methylspiperone to dopamine and serotonin receptors

3H-N-methylspiperone (3H-NMSP) was used to label dopamine-2 and serotonin-2 in vivo in the mouse. The striatum/cerebellum binding ratio reached a maximum of 80 eight hours after intravenous administration of 3H-NMSP. The frontal cortex/cerebellum ratio was 5 one hour after injection. The binding of 3H-NMSP was saturable in the frontal cortex and cerebellum between doses of 10 and 1,000 micrograms/kg. Between 0.01 and 10 micrograms/kg the ratio total/nonspecific binding increased from 14 to 21. Inhibition of 3H-NMSP binding in the frontal cortex and striatum by ketanserin, a selective serotonin-2 antagonist, demonstrated that 20% of the total binding in the striatum was to serotonin-2 receptors and 91% of the total binding in the frontal cortex was to serotonin-2 receptors. Compared to 3H-spiperone, 3H-NMSP results in a much higher specific/nonspecific binding ratio in the striatum and frontal cortex and displays more than a two-fold higher brain uptake.     

Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2-receptors of rat brain frontal cortex

Using radio-receptor analysis, it has been demonstrated that human beta-casomorphin-7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human beta-casomorphin-7 was 8 microM. These data suggest that one of the mechanisms of neurotropic action of beta-casomorphin-7 is might be associated with its influence on the serotoninergic system.     

Chronic neuroleptic treatment specifically alters the number of dopamine receptors in rat brain

Trifluoperazine dihydrochloride (2.8–4.0 mg/kg/day) was administered continuously to rats in drinking water for six months. Animals killed at this time exhibited an increase in the number of dopamine receptors in the striatum and mesolimbic area, with a corresponding decrease in affinity (increase in the dissociation constant) for ³H-spiperone binding. In frontal cortex, ³H-spiperone binding to 5-HT receptors indicated no apparent change in numbers of receptors, but a slight increase in the dissociation constant. There was no obvious alteration in ³H-apomorphine binding in the striatum and mesolimbic area, but the individual results were very variable. The number and binding affinity of muscarinic receptors in striatum, mesolimbic area and cerebral cortex as identified by ³H-dexetemide were unchanged. Nor was there any alternation in the number or binding affinity of H-1 receptors identified by ³H-mepyramine, or of α-noradrenergic receptors identified by ³H-WB 4101, in cerebral cortex. The number and binding affinity of GABA receptors in the cerebellum identified by ³H-muscimol also was not altered.Chronic neuroleptic administration to rats appears to alter specifically the number of cerebral dopamine receptors.     

Different kinetic properties of neuroleptic receptor binding in the rat striatum and frontal cortex.

³H-spiperone binding in the striatum is 80% stereospecific and 6 % non-stereospecific. In the frontal cortex stereospecific sites account for 60 % of the total binding and non-stereospecific sites for 25 %. Stereospecific sites are of different nature in both brain regions : dopaminergic in the striatum and serotonergic in the frontal cortex. The non-stereospecific sites are saturable and can only be detected by the use of appropriate blanks.Release experiments demonstrate the occurrence of positive coöperative effects in the dissociation of spiperone from striatal receptors while this is not detectable in the frontal cortex. In both brain regions a rapidly and a slowly dissociating component have been observed. In the frontal cortex the rapid component is ascribed to the dissociation of spiperone from the stereospecific sites and the slow component to the dissociation from non-stereospecific sites. The nature of these sites is yet to be identified.     

Altered serotonin, dopamine and norepinepherine levels in 15q duplication and angelman syndrome mouse models

Childhood neurodevelopmental disorders like Angelman syndrome and autism may be the result of underlying defects in neuronal plasticity and ongoing problems with synaptic signaling. Some of these defects may be due to abnormal monoamine levels in different regions of the brain. Ube3a, a gene that causes Angelman syndrome (AS) when maternally deleted and is associated with autism when maternally duplicated has recently been shown to regulate monoamine synthesis in the Drosophila brain. Therefore, we examined monoamine levels in striatum, ventral midbrain, frontal cerebral cortex, cerebellar cortex and hippocampus in Ube3a deficient and Ube3a duplication animals. We found that serotonin (5HT), a monoamine affected in autism, was elevated in the striatum and cortex of AS mice. Dopamine levels were almost uniformly elevated compared to control littermates in the striatum, midbrain and frontal cortex regardless of genotype in Ube3a deficient and Ube3a duplication animals. In the duplication 15q autism mouse model, paternal but not maternal duplication animals showed a decrease in 5HT levels when compared to their wild type littermates, in accordance with previously published data. However, maternal duplication animals show no significant changes in 5HT levels throughout the brain. These abnormal monoamine levels could be responsible for many of the behavioral abnormalities observed in both AS and autism, but further investigation is required to determine if any of these changes are purely dependent on Ube3a levels in the brain.     

Characterization of specific in vivo binding of neuroleptic drugs in rat brain

$\text{In vivo}$ binding of ³H-spiperone is saturable in the striatum, the limbic system and the frontal cortex but not in the cerebellum. A specific binding is different in all the brain regions thus the amount of labelling in the cerebellum may not be considered as a blank value.³H-spiperone binding revealed a specific subcellular distribution only when a very low dose was injected into rats.$\text{Ex vivo}$ experiments allow the assessment of biochemical profiles of neuroleptic drugs according to their relative affinity for dopamine or serotonin receptors.     

[3H]Ketanserin (Serotonin Type 2) Binding Increases in Rat Cortex Following Basal Forebrain Lesions with Ibotenic Acid

The response of the serotonergic system following injury to the basal forebrain cholinergic system was investigated in rats. The density of 5-hydroxytryptamine (serotonin) type 2 (S2) receptor sites in the frontal cortex and hippocampus was determined 1 week and 4 months after production of lesions by injections of ibotenic acid into the medial septum and nucleus basalis magnocellularis. One week later, the number of S2 receptor sites in the frontal neocortex, as defined by [3H]ketanserin binding, was unchanged. Four months later, the number of [3H]ketanserin binding sites (and Bmax) was increased and high-affinity [3H]serotonin uptake was decreased in the frontal neocortex, but not in the hippocampus, relative to unlesioned controls. Choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase; EC 2.3.1.6) activity was decreased significantly in the frontal neocortex and hippocampus 1 week and 4 months after surgery. The change in frontal neocortical S2 receptor site density was inversely related to the level of choline acetyltransferase activity, was specific for cholinergic denervation associated with the cortex but not the hippocampus, and may represent a localized denervation supersensitivity due to degeneration of median raphe cortical afferents.     

The Dissociation Rate of Unlabelled Drugs from Receptor Sites: A Poorly Investigated,Yet Important Aspect in Receptor Research. Studies on the Serotonin-S2 Receptor

Abstract

The labelling by ³H-spiperone of serotonin-S₂ receptors in rat frontal cortex tissue adsorbed to glass fibre filters was investigated. For 12 unlabelled serotonin antagonists the dissociation time from serotonin-S₂ receptors was measured using rat frontal cortex tissue preparations adsorbed to glass fibre filters. The dissociation half-time varied from 4.8 min for pipamperone to 160 min for ritanserin. The drug-receptor dissociation time was not related to a particular class of chemical structure, or to the lipophilicity or the acid dissociation constant of the drugs. The essential requirement of experimental determination of the drug-receptor dissociation time for each drug individually is illustrated. The possible applications of the knowledge of the drug-receptor dissociation time in in vitro and in vivo receptor studies, in pharmacological and pharmaco-kinetic studies and in drug design and receptor modelling is discussed. For various serotonin-S₂ antagonists, the type of inhibition produced by the drug on ³H-ketanserin binding to serotonin-S₂ receptors was determined using suspensions of rat frontal cortical tissue. The observed patterns of inhibition were clearly related to the drug-receptor dissociation times: rapidly dissociating drugs produced competitive inhibition, drugs with dissociation half-times between 15-30 min produced mixed type inhibition, and the very slowly dissociating ritanserin produced non-competitive inhibition.     

Low dose of the gamma acute radiation syndrome (1.5 Gy) does not significantly alter either cognitive behavior or dopaminergic and serotoninergic metabolism.

The aim of this study was to evaluate the early-delayed effects of a low dose of the gamma acute radiation syndrome (1.5 Gy) on memory and on dopaminergic and serotoninergic metabolism in Swiss albino CD1 mice, of various ages (6, 10 and 20 weeks). At different times after irradiation (from 24 hr to three months), the mice were trained in a single-trial passive avoidance task and tested for retention either 24 hr or 5 days later. Their performance was compared to that of mice that were sham-irradiated. At the end of the behavioral test (days 3, 9, 30 and 93), the concentrations of dopamine (DA) and serotonin (5HT) and their metabolites were determined in hippocampus, anterior cortex and striatum of mice irradiated at the age of six weeks. No significant behavioral effect was observed whichever the age of the animals or the delay of observation. On the contrary at the moderate dose of 4.5 Gy we observed a significant memory deficit 9 days after the exposure. Considering the neurochemical study, in the striatum or in the frontal cortex, no significant modification was observed whichever the delay or the molecule. In the hippocampus slight modifications were noted: an increase (+144%, p = 0.002) in DA level on day 3 after exposure, and a decrease (-27%, p = 0.028) of 5HT level on day 30 post-irradiation. These modifications were only transient and not associated to modifications of the catabolites. This study demonstrates that total-body exposure to gamma radiation at low dose seems to induce only slight effects on the central nervous system.     

Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

Association of upregulated Ras/Raf/ERK1/2 signaling with autism

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. BTBR mouse is currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism. Growing evidence suggests that Ras/Raf/ERK1/2 signaling plays death-promoting apoptotic roles in neural cells. Recent studies showed a possible association between neural cell death and autism. In addition, two studies reported that a deletion of a locus on chromosome 16, which includes the MAPK3 gene that encodes ERK1, is associated with autism. We thus hypothesized that Ras/Raf/ERK1/2 signaling could be abnormally regulated in the brain of BTBR mice that models autism. In this study, we show that expression of Ras protein was significantly elevated in frontal cortex and cerebellum of BTBR mice as compared with B6 mice. The phosphorylations of A-Raf, B-Raf and C-Raf were all significantly increased in frontal cortex of BTBR mice. However, only C-Raf phosphorylation was increased in the cerebellum of BTBR mice. In addition, we further detected that the activities of both MEK1/2 and ERK1/2, which are the downstream kinases of Ras/Raf signaling, were significantly enhanced in the frontal cortex. We also detected that ERK1/2 is significantly over-expressed in frontal cortex of autistic subjects. Our results indicate that Ras/Raf/ERK1/2 signaling is upregulated in the frontal cortex of BTBR mice that model autism. These findings, together with the enhanced ERK1/2 expression in autistic frontal cortex, imply that Ras/Raf/ERK1/2 signaling activities could be increased in autistic brain and involved in the pathogenesis of autism.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Effect of carbidine on the dopamine and serotonin receptors of the central nervous system

It has been shown in experiments on mice and rats that unlike haloperidol and sulpiride, carbidine does not influence the intensity and even increases the duration of apomorphine stereotypy. However, similarly to haloperidol carbidine decreases head twitches in mice, induced by administration of 5-hydroxytryptophan. In radioligand-binding experiments in vitro and in vivo carbidine displaced 3H-spiperone but in the brain cortex without having any effect on the binding in the striatum. Carbidine did not affect the dopamine-sensitive adenylate cyclase in rat striatum. Based on these data it is suggested that in contrast to haloperidol and sulpiride, carbidine selectively inhibits serotonin receptors of the brain.     

Selective reduction by isolation rearing of 5-HT1A receptor-mediated dopamine release in vivo in the frontal cortex of mice

Serotonin (5-HT)1A receptors modulate in vivo release of brain monoaminergic neurotransmitters which may be involved in isolation-induced aggressive behavior. The present study examined the effect of isolation rearing on the 5-HT1A receptor-mediated modulation of dopamine (DA), 5-HT and noradrenaline (NA) release in the frontal cortex of mice. The selective 5-HT1A receptor agonist (S)-5-[-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole HCl (MKC-242) increased the release of DA and NA and decreased the release of 5-HT in the frontal cortex of mice. The effect of MKC-242 on DA release was significantly less in isolation-reared mice than in group-reared mice, while effects of the drug on NA and 5-HT release did not differ between both groups. The effect of the other 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin on cortical DA release was also less in isolation-reared mice than in group-reared mice, and that of the drug on cortical 5-HT release did not differ between both groups. In contrast to MKC-242-induced DA release, amphetamine-induced increase in cortical DA release in vivo was greater in isolation-reared mice. The present findings suggest that isolation rearing enhances the activity of cortical dopaminergic neurons and reduces selectively the 5-HT1A receptor-mediated release of DA in the cortex.     

Different effects of serotonin antagonists on 3H-mianserin and 3H-ketanserin recognition sites

In minces prepared from the frontal cortex of rats treated with ketanserin (10 mg/kg i.p.) or mianserin (5 mg/kg i.p.) twice daily for 21 days, the Vmax of the adenylate cyclase stimulated by NE (100 microM) is attenuated, suggesting that ketanserin and mianserin share with a number of antidepressants the ability to attenuate the adenylate cyclase stimulation by NE. Ketanserin, given with the above mentioned dose schedule for 7 consecutive days, reduced the Bmax of 5HT2 recognition sites but failed to change either the Bmax or the apparent Kd of H-mianserin binding. A significant decrease in the Bmax of 5HT2 binding sites is elicited also by a single injection of mianserin (1). This drug also down-regulates its own binding when given twice daily for 3 weeks. From this and other information (2,3), it is concluded that ketanserin and mianserin bind to distinct recognition sites. The possibility that 5HT2 and mianserin recognition sites are functionally related and that serotonergic synapses are modulated by multiple chemical signals might be considered.     

Changes in the sensitivity of brain dopamine and serotonin receptors during the prolonged administration of carbidine

Male Wistar rats were treated chronically with either carbidine (10 mg/kg/day) or haloperidol (1 mg/kg/day) for 23 consecutive days. On days 4-5 after the treatment discontinuation the animals were challenged with apomorphine HCl (0.5 mg/kg) or 5-OTP (150 mg/kg i. p) in combination with pargiline (40 mg/kg i. p) and stereotype responses were scored. In carbidine-treated rats the intensity of stereotype sniffings was increased after apomorphine treatment. In contrast, animals treated with haloperidol exhibited more intensive gnawing after apomorphine in comparison to vehicle-treated rats. 5-OTP-induced head twitches were increased only in carbidine-treated rats. Prolonged carbidine treatment in contrast to haloperidol induced a decrease in 5H-spiperone and 3H-LSD binding in the frontal cortex, with the density of D-2 receptors in the striatum practically unchanged. It is concluded that neuroleptic carbidine in contrast to classical neuroleptics has a more selective effect in serotonin (S-2) receptors and antidepressive properties of this compound may be due to the down-regulation of S-2 receptors in the brain.     

Allosteric modulation of the NMDA receptor by neurosteroids in rat brain and the impact of long term morphine administration

This study examined the allosteric modulation of the NMDA receptor by nanomolar concentrations of neurosteroids in rats treated long term with morphine. The neurosteroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PS) and pregnanolone sulfate (3α5βS) are important mediators in the central nervous system. They induce rapid responses by non-classical steroidal mechanisms, e.g. via interaction with the N-methyl-d-aspartate (NMDA) receptor, and are known to modify the binding of ifenprodil to the NMDA receptor subunit NR2B. The NMDA receptor is involved in several processes, including memory, learning, synaptic plasticity and neuronal development. Morphine, a μ-opioid receptor agonist, has an important role in the clinical treatment of pain. The main drawback of morphine treatment is the associated development of dependence and tolerance. The mechanisms behind these phenomena are still to be elucidated, but several reports suggest the involvement of the NMDA receptor. The results of the present study indicate that the allosteric modulation induced by the neurosteroids DHEAS, PS and 3α5βS was similar in all tested brain regions. This suggests that the NR2B receptor subunit behaves independently of its site of expression. Moreover, the NR2B subunit was up-regulated in the frontal cortex but not in the hippocampus or hypothalamus. It is concluded that morphine does not affect the neurosteroid modulatory effect on ifenprodil binding in the rat hippocampus or hypothalamus but does significantly affect both the expression of the NR2B subunit and the 3α5βS modulatory effect on ifenprodil binding in the frontal cortex. It is suggested that the observed effect of long term morphine on the properties of NR2B in the frontal cortex may be associated with the mechanism underlying the development of opiate dependence.