Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT1A receptors in rat brain

Chronic treatment with the antidepressant fluoxetine may lead to changes in the properties of pre- and postsynaptic 5-HT(1A) receptors due to modifications in the receptor-G protein coupling process. We have evaluated, in rats, the effect of chronic fluoxetine (10 mg/kg/day) at brain 5-HT(1A) receptors using different techniques. The density of 5-HT(1A) receptors was unchanged in fluoxetine-treated rats vs. vehicle group. Stimulation of [(35)S]GTPgammaS binding induced by (+/-)8-OH-DPAT was significantly attenuated in dorsal raphe nucleus after fluoxetine (+3.7 vs. +31.2% in vehicle). The inhibition of dorsal raphe firing by (+/-)8-OH-DPAT (ED(50) in vehicle = 2.1 microg/kg, i.v.) was also attenuated in rats treated with fluoxetine (ED(50)=4.7 microg/kg). In contrast, a significant increase on (+/-)8-OH-DPAT-induced stimulation of [(35)S]GTPgammaS binding was observed in CA(1) (+53.4 vs.+20.2% in vehicle) and dentate gyrus (+105.7 vs. +52.6% in vehicle) but not in entorhinal cortex. Our data demonstrate that fluoxetine-induced desensitization of 5-HT(1A) autoreceptors occurs at G protein level. Moreover, a relevant finding is the region-specific hypersensitivity of postsynaptic 5-HT(1A) receptors, in the hippocampus but not in entorhinal cortex, following chronic fluoxetine. These differential adaptive changes in brain 5-HT(1A) receptors could underlie the mechanism of action of antidepressants and also contribute to their clinical effects.     

Decreased G-protein coupling of serotonin 5-HT(1A) receptors in the brain of 5-HT(1B) knockout mouse

The firing of central serotonin (5-hydroxytryptamine, 5-HT) neurons and their capacity to release 5-HT are subjected to a receptor-mediated auto-control via 5-HT(1A) and 5-HT(1B) receptors respectively located on the somata/dendrites (5-HT(1A) autoreceptors) and preterminal axon arborizations (5-HT(1B) autoreceptors) of these neurons. To further characterize mutual adaptations of these two receptor subtypes in the absence of one of them, activation of G-protein coupling by agonist was measured and compared to wild-type (WT) in 5-HT(1A) and 5-HT(1B) homozygous knockout (KO) mice. As expected, in WT, the non-selective 5-HT(1A/1B) receptor agonist 5-carboxyamidotryptamine (5-CT) stimulated guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTP(gamma)S) incorporation in many brain regions endowed with one and/or the other receptor. In the respective KOs, no stimulation was measured in regions known to express only or mainly the deleted receptor. In the 5-HT(1A) KOs, the amplitude of G-protein activation in regions endowed with 5-HT(1B) receptors was unchanged by comparison to WT. In the 5-HT(1B) KOs, the magnitude of the 5-CT stimulation was the same as WT in all regions containing 5-HT(1A) receptors, except in the amygdala, where it was significantly lower, even if this region was one of the most strongly activated in the WT. A similar result was obtained in the amygdala of 5-HT(1B) KOs after activation by the selective 5-HT(1A) receptor agonist R-(+)8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Under these conditions, however, there was in addition a significant lowering of the stimulated (but not basal) [(35)S]GTP(gamma)S incorporation by comparison to WT in all regions endowed with 5-HT(1A) receptors, including the dorsal raphe nucleus. Thus, eventhough agonist radioligand binding to either 5-HT(1A) or 5-HT(1B) receptors is unchanged in the reciprocal KOs, it appears that a compensatory decrease in the efficiency of G-protein coupling to 5-HT(1A) receptors has developed in the 5-HT(1B) mutant. This could represent the first indication of a cross-talk between these two 5-HT receptor subtypes, at least in brain regions where they are co localized in the same neurons.     

Autoradiographic characterisation of [35S]GTPgammaS binding stimulation mediated by 5-HT1B receptor in postmortem human brain

G-protein activation mediated by 5-HT1B receptors was studied in human brain by [35S]GTPgammaS autoradiographic methods. 5-HT (10 microM) increased [35S]GTPgammaS binding in caudate-putamen nucleus, globus pallidus, dentate gyrus, CA1, entorhinal cortex and substantia nigra. In basal ganglia and midbrain, this effect was blocked by GR 127935 (5-HT(1B/1D) antagonist). In contrast, WAY 100635 (selective 5-HT1A antagonist) reversed the effect of 5-HT in hippocampus and entorhinal cortex. Therefore, a detailed pharmacological study was carried out in basal ganglia and substantia nigra using 5-HT and the 5-HT(1B/1D) agonists GTI and CP 93129. In these areas, these agonists stimulated [35S]GTPgammaS binding in a concentration-dependent manner, with no significant differences in the potency for a given structure. Furthermore, GTI was more potent in the putamen than in globus pallidus. In caudate-putamen, the three agonists showed the same efficacy, while in globus pallidus and substantia nigra the efficacy of 5-HT was higher than GTI and CP 93129. The selective 5-HT1B antagonist SB-224289 inhibited GTI- and CP 93129-stimulated [35S]GTPgammaS binding in basal ganglia and substantia nigra, while coincubation with BRL 15572 (selective 5-HT1D antagonist) did not result in any significant change. Here we report the anatomical pattern of distribution of 5-HT1B-dependent functionality by using specific pharmacological tools in human brain sections.     

WAY100635 prevents the changes induced by fluoxetine upon the 5-HT1A receptor functionality

5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPgammaS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPgammaS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPgammaS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 +/- 0.32 microg/kg and 4.34 +/- 0.09 microg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 +/- 0.58 microg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPgammaS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination.     

Drug inhibition indicates a single-site model of the 5-HT uptake site/antidepressant binding site in rat and human brain

Drug inhibition against [³H]paroxetine binding to rat cortex and human putamen was investigated in saturation experiments. The addition of 5-HT, imipramine, citalopram and clomipramine all produced changes in apparent binding affinity (K_d) without changes in the number of binding sites (B_max). These data suggest that there is no heterogeneity of specific [³H]paroxetine binding, supporting a single site model of the 5-HT uptake site and antidepressant binding site.     

Allosteric modulation of 5-HT(1A) receptors by zinc: Binding studies

5-HT_1A receptors were studied via [³H]WAY-100635 and [³H]8-OH-DPAT binding to rat brain cortical membranes. We characterized the effect of zinc (Zn²⁺) on the binding properties of the 5-HT_1A receptor. The allosteric ternary complex model was applied to determine the dissociation constant (K_A) of Zn²⁺ and their cooperativity factors (α) affecting the dissociation constants (K_D, K_i) of [³H]WAY-100635, [³H]8-OH-DPAT, and serotonin (5-HT), the endogenous neurotransmitter. Zn²⁺ (5 μM-1 mM) inhibited the binding of agonist/antagonist to 5-HT1A receptors, mostly by decreasing both the ligands' affinity and the maximal number of sites. In [³⁵S]GTPγS binding assays Zn²⁺ behaved as insourmountable antagonist of 5-HT1A receptors, in agreement with radioligand binding assays. The residues involved in the formation of the inhibitory binding site on the 5-HT1A receptor were assessed by using N-ethyl-maleimide (NEM) or diethylpyrocarbonate (DEPC) which modify preferentially cysteine and histidine residues, respectively. Exposure to both agents did not block the negative allosteric effects of Zn²⁺ on agonist and antagonist binding. Our findings represent the first quantitative analysis of allosteric binding interactions for 5-HT_1A receptors. The physiological significance of Zn²⁺ modulation of 5-HT_1A receptors is unclear, but the colocalization of 5-HT_1A receptors and Zn²⁺ in the nervous system (e.g. in the hippocampus and cerebral cortex) suggests that Zn²⁺ released at nerve terminals may modulate signals generated by the 5-HT_1A receptors in vivo. Finally, these findings suggest that synaptic Zn²⁺ may be a factor influencing the effectiveness of therapies that rely on 5-HT_1A receptor activity.     

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.     

Differential effects of the novel antidepressant agomelatine (S 20098) versus fluoxetine on 5-HT1A receptors in the rat brain

Agomelatine (S 20098) is a novel antidepressant drug with melatonin receptor agonist and 5-HT(2C) receptor antagonist properties, but actual mechanisms underlying its antidepressant action are unknown. Because functional desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) occurs after chronic administration of several classes of antidepressants, we investigated whether this adaptive change could also be induced by agomelatine. Neither acute nor chronic treatment with agomelatine (10 mg/kg i.p. for 14 days or 50 mg/kg i.p. for 21 days) changed the density of 5-HT(1A) receptors and their coupling with G proteins in the DRN and the hippocampus in rats. Moreover, these treatments did not affect the basal electrophysiological characteristics and the responses to 5-HT(1A) receptor stimulation of DRN and hippocampal neurons in brain slices. Parallel experiments with melatonin (10 mg/kg i.p. for 14 days) and fluoxetine (5 mg/kg i.p. for 14 days) as reference compounds showed that the former was unable to affect 5-HT(1A) receptors whereas the latter decreased both the 5-HT(1A) receptor-mediated [(35)S]GTP-gamma-S binding and the potency of ipsapirone, a 5-HT(1A) receptor agonist, to inhibit neuronal firing in the DRN. These data indicate that the antidepressant action of agomelatine is not mediated through the same mechanisms as SSRIs or tricyclics.     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.     

Effect of acute and prolonged tianeptine administration on the 5-HT transporter: electrophysiological,biochemical and radioligand binding studies in the rat brain

In the present study, in vivo extracellular unitary recordings, in vitro [³H]5-HT uptake and [³H]cyanoimipramine binding assays were used to assess the effect of acute and prolonged administration of the putative antidepressant tianeptine, on the 5-hydroxytryptamine (5-HT) transporter. Microiontophoretic application of tianeptine onto dorsal hippocampus CA₃ pyramidal neurons, as well as its intravenous administration (2 mg/kg), increased their firing frequency. Following intracerebroventricular administration of 5,7-dihydroxytryptamine, the activation induced by the microiontophoretic application of tianeptine remained unchanged, thus suggesting that the 5-HT carrier is not involved in this effect. Furthermore, in spite of its activating effect on CA₃ pyramidal neuron firing frequency, the intravenous administration of tianeptine did not alter the time of recovery of these neurons from microiontophoretic applications of 5-HT, an index of 5-HT uptake activity. In keeping with this observation, the acute administration of tianeptine did not change the effectiveness of the 5-HT reuptake blocker paroxetine (1 mg/kg, i.v.) in prolonging the suppressant effect of microiontophoretically-applied 5-HT. However, in rats that had received tianeptine for 14 days (20 mg/kg/day, s.c.), the recovery time from the suppressant effect of microiontophoretic applications of 5-HT was reduced by 40% and the effectiveness of paroxetine (1 mg/kg, i.v.) was decreased. These effects were no longer observed following a 48 h washout period. In a second series of experiments, the ability of tianeptine to interfere with the uptake blocking capacity of paroxetine was assessed in vitro, using hippocampal slices obtained from rats that had been treated with tianeptine for 14 days (20 mg/kg/day, s.c.; by minipump). The effectiveness of paroxetine to block [³H]5-HT uptake was unchanged in slices obtained from rats still bearing the osmotic minipump at the time of the sacrifice, as well as from those which had undergone a 48 h washout period. To assess whether prolonged administration of tianeptine would induce adaptive changes on 5-HT uptake sites, [³H]cyanoimipramine-binding parameters were measured following a 48 h washout period. Affinity values remained unchanged while density values were significantly increased in cortex (+22%) but not in hippocampus (+12%). It is concluded that, i) the activation of CA₃ pyramidal neurons observed following acute tianeptine administration cannot be attributed to its 5-HT uptake enhancing properties and ii) the prolonged administration of tianeptine induces adaptive changes on cortical but not on hippocampal 5-HT transporters.Deceased 10 May 1994     

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Effects of long-term biogenic amine transporter blockade on receptor/G-protein coupling in rat brain

This study examines the effect of long-term elevation of brain monoamine levels on receptor/G-protein coupling by chronic administration of a highly potent tropane analog, WF-23 (2beta-propanoyl-3beta-(2-naphthyl) tropane). WF-23 blocks dopamine, serotonin and norepinephrine transporters with high affinity in vitro, and blocks transporters for at least two days following a single in vivo administration. Rats were chronically treated for 15 days with 1mg/kg WF-23, injected i.p. every two days. Receptor activation of G-proteins was determined by [35S]GTPgammaS autoradiography in brain sections for D2, 5-HT1A and alpha2-adrenergic receptors, as well as mu opioid receptors as a non-monoamine receptor control. Chronic treatment with WF-23 produced significant reductions in D2, 5-HT1A, and alpha2-adrenergic receptor-stimulated [35S]GTPgammaS binding in caudate/putamen, hippocampus and amygdala, respectively. There were no effects of WF-23 treatment on mu opioid-stimulated [35S]GTPgammaS binding. Additionally, there was no effect of WF-23 treatment on D2 receptor binding, as determined by [3H]spiperone autoradiography. These data show that chronic blockade of monoamine transporters produces specific uncoupling of receptors and G-proteins in specific brain regions in the absence of receptor downregulation.     

Autoradiographic localisation of [3H]2-BFI imidazoline I2 binding sites in mouse brain

Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain.     

Differential regulation of adenosine A(1) and A(2A) receptors in serotonin transporter and monoamine oxidase A-deficient mice.

The serotonin (5HT) transporter (5HTT) removes 5HT from the synaptic cleft and is thus critical to the control of serotonergic neurotransmission. Mice with a targeted inactivation of the 5HTT represent a novel and unique tool to study serotonergic system functioning. Because the release of 5HT is regulated by adenosine, we investigated 5HTT-deficient mice for possible adaptive changes of adenosine A₁ and A_2A receptors. A₁ and A_2A receptors were studied by means of quantitative autoradiography using the radioligands [³H]8-cyclopentyl-1,3-dipropylxanthine and [³H]CGS 21680, respectively. A comparison of 5HTT knockout versus control mice revealed upregulation of A₁ receptors in the dorsal raphe nucleus (DRN, +21%), but not in any of the serotonergic projection areas, and downregulation of A_2A receptors in basal ganglia. The adaptive changes of A₁ and A_2A receptors in 5HTT-deficient mice are likely to represent a compensatory neuroprotective effect mediated by the adenosinergic modulatory system. For comparison, these receptors were also studied in monoamine oxidase A (MAOA) knockout mice and in 5HTT/MAOA double knockout mice. 5HTT/MAOA double knockout mice showed adaptive changes of adenosine A₁ and A_2A receptors similar to 5HTT knockout mice, while investigation of MAOA-deficient mice revealed an upregulation of A_2A receptors, which may relate to a role of both MAOA and adenosine A_2A receptors in anxiety.     

Influence of eicosanoids on serotonin release in the rat brain: inhibition by prostaglandins E1 and E2

Summary Cardiac alpha- and beta-adrenoceptor sensitivities were examined after chronic pretreatment of rats with reserpine. Increases in sensitivity would indicate that the receptor is under the influence of the sympathetic innervation, removal by catecholamine depletion with reserpine of the tonic effect of neurotransmitter release would permit receptor upregulation. The positive inotropic responses of paced left atria and papillary muscles and the positive chronotropic responses of spontaneously beating right atria were recorded. A concentration-response curve to isoprenaline (beta-adrenoceptor-mediated) was followed, in the presence of beta-blockade, by one to methoxamine (alpha-adrenoceptor-mediated). Methoxamine exerted positive inotropy of left atria and papillary muscles, the maxima being 43.2 ± 2.7 and 26.8 ± 4.4% of the isoprenaline maxima. A small positive chronotropy (16.5 ± 5.6% maximum) of right atria occurred. After pretreatment with reserpine (1.0 mg kg^–1 i.p. daily) for 7 days, the three preparations displayed supersensitivity to isoprenaline, revealed as a significant displacement (P < 0.05) of the concentration-response curves to the left of those for control rats. Reserpine pretreatment, however, had no effect on the sensitivity to methoxamine. The increase in beta-adrenoceptor sensitivity to isoprenaline after reserpine pretreatment was accompanied by a significant 41.3% increase (P < 0.05) in the number of [³H]-dihydroalprenolol ([³H]-DHA) binding sites (B _max) in ventricular membranes, although the dissociation constant (_{K D}) was unaffected. There were more alpha-adrenoceptor [³H]-prazosin binding sites in ventricular than atrial membranes. However, there was no difference in K _D or B _max between reserpine-pretreated and control tissues. It is proposed that the increase in beta-adrenoceptor sensitivity and binding sites arises from the depletion-induced loss of neuronal noradrenaline release onto the beta-adrenoceptors which are therefore directly under the influence of the neurotransmitter. The failure of cardiac alpha-adrenoceptors to exhibit supersensitivity and increased numbers suggests that they are not directly affected by the sympathetic innervation. Send offprint requests to K. J. Broadley at the above address     

Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.     

Time course of the effects of adrenalectomy and corticosterone replacement on 5-HT1A receptors and 5-HT uptake sites in the hippocampus and dorsal raphe nucleus of the rat brain: an autoradiographic analysis

Previous studies have shown that adrenalectomy (ADX) increases the binding of³H-DPAT to 5-HT_1A receptors in the hippocampus (HIP) and this effect is partially overcome by corticosterone (CORT) replacement. The present study investigated the time course of the effects of ADX with or without CORT replacement on serotonin (5-HT) pre- and postsynaptic systems in the HIP and dorsal raphe nucleus (DR) by quantitative autoradiography. In the HIP, ADX for 7, 10 or 14 days caused a significant increase in³H-DPAT binding in the CA₁ region (pyramidal layer), CA_2,3 region (molecular and pyramidal layers) and in the dentate gyrus (molecular and granular layers) which returned to control levels when measurements were made 35 days post-ADX. A decrease in³H-DPAT binding was observed 14 days after ADX in the DR but not in the median raphe nucleus (MR). Although replacement with CORT did not lead to a reversal in³H-DPAT binding at early time points, binding was restored to control levels 7–28 days after CORT replacement in all regions of the HIP. In the DR, CORT did not cause a reversal in³H-DPAT binding at any of the time points examined. In contrast to the effects seen on the 5-HT_1A receptor subtype, no significant change was noted on the binding of³H-CN-IMI to uptake sites for 5-HT in the HIP or DR after ADX or CORT replacement. The results of this study indicate that long-term alterations in the HPA axis lead to changes in the 5-HT_1A receptor system that are both region-specific and time-dependent.