Agata Faron-Górecka

Effect of antidepressant drugs in mice lacking the norepinephrine transporter.

One of the main theories concerning the mechanism of action of antidepressant drugs (ADs) is based on the notion that the neurochemical background of depression involves an impairment of central noradrenergic transmission with a concomitant decrease of the norepinephrine (NE) in the synaptic gap. Many ADs increase synaptic NE availability by inhibition of the reuptake of NE. Using mice lacking NE transporter (NET−/−) we examined their baseline phenotype as well as the response in the forced swim test (FST) and in the tail suspension test (TST) upon treatment with ADs that display different pharmacological profiles. In both tests, the NET−/− mice behaved like wild-type (WT) mice acutely treated with ADs. Autoradiographic studies showed decreased binding of the β-adrenergic ligand [3H]CGP12177 in the cerebral cortex of NET−/− mice, indicating the changes at the level of β-adrenergic receptors similar to those obtained with ADs treatment. The binding of [3H]prazosin to α1-adrenergic receptors in the cerebral cortex of NET−/− mice was also decreased, most probably as an adaptive response to the sustained elevation of extracellular NE levels observed in these mice. A pronounced NET knockout-induced shortening of the immobility time in the TST (by ca 50%) compared to WT mice was not reduced any further by NET-inhibiting ADs such as reboxetine, desipramine, and imipramine. Citalopram, which is devoid of affinity for the NET, exerted a significant reduction of immobility time in the NET−/− mice. In the FST, reboxetine, desipramine, imipramine, and citalopram administered acutely did not reduce any further the immobility time shortened by NET knockout itself (ca 25%); however, antidepressant-like action of repeatedly (7 days) administered desipramine was observed in NET−/− mice, indicating that the chronic presence of this drug may also affect other neurochemical targets involved in the behavioral reactions monitored by this test. From the present study, it may be concluded that mice lacking the NET may represent a good model of some aspects of depression-resistant behavior, paralleled with alterations in the expression of adrenergic receptors, which result as an adaptation to elevated levels of extracellular NE.

Norepinephrine transporter knockout‐induced up‐regulation of brain alpha2A/C‐adrenergic receptors

The norepinephrine transporter (NET) is responsible for the rapid removal of norepinephrine released from sympathetic neurons; this release is controlled by inhibitory α2‐adrenergic receptors (α2ARs). Long‐term inhibition of the NET by antidepressants has been reported to change the density and function of pre‐ and postsynaptic ARs, which may contribute to the antidepressant effects of NET inhibitors such as desipramine. NET‐deficient (NET‐KO) mice have been described to behave like antidepressant‐treated mice. By means of quantitative real‐time PCR we show that mRNAs encoding the α2A‐adrenergic receptor (α2AAR) and the α2C‐adrenergic receptor (α2CAR) are up‐regulated in the brainstem, and that α2CAR mRNA is also elevated in the hippocampus and striatum of NET‐KO mice. These results were confirmed at the protein level by quantitative autoradiography. The NET‐KO mice showed enhanced binding of the selective α2AR antagonist [3H]RX821002 in several brain regions. Most robust increases (20–25%) in α2AR expression were observed in the hippocampus and in the striatum. Significant increases (16%) were also seen in the extended amygdala and thalamic structures. In an ‘in vivo’ test, the α2AR agonist clonidine (0.1 mg/kg) caused a significantly greater reduction of locomotor activity in NET‐KO mice than in wild‐type mice, showing the relevance of our findings at the functional level.

Mesolimbic dopamine D2 receptor plasticity contributes to stress resilience in rats subjected to chronic mild stress

RationaleFew studies have investigated neurobiological and biochemical differences between stress-resilient and stress-vulnerable experimental animals.ObjectivesWe investigated alterations in mesolimbic dopamine D2 receptor density and mRNA expression level in stressed rats at two time points, i.e. after 2 and 5 weeks of chronic mild stress (CMS).MethodsWe used the chronic mild stress paradigm because it is a well-established animal model of depression. Two groups of stressed rats were distinguished during CMS experiments: (1) stress reactive (70 %), which displayed a decrease in the drinking of a palatable sucrose solution during the stress regimen, and (2) stress resilient (30 %), which exhibited an unaltered drinking profile when compared with the unchallenged control group. [3H]Domperidone was used as a ligand to label dopamine D2 receptors, and a mixture of three specific oligonucleotides was used to evaluate dopamine D2 receptor mRNA changes in various regions of the rat brain.ResultsCMS strongly affected the mesolimbic dopamine circuit in stress-resilient group after 2 weeks and stress-reactive group of rats after 5 weeks which exhibited a decrease in the level of dopamine D2 receptor protein without alterations in D2 mRNA expression. Stress-resilient animals, but not stress-reactive animals, effectively adapted to the extended stress and coped with it. The increase in D2 mRNA expression returned the dopamine D2 receptor density to control levels in stress-resilient rats after 5 weeks of CMS, but not in stress-reactive animals.ConclusionsThese results clearly demonstrate that, despite earlier blunting, the activation of dopamine receptor biosynthesis in the dopamine mesoaccumbens system in stress-resilient rats is involved in active coping with stressful experiences, and it exhibits a delay in time.

The role of D1-D2 receptor hetero-dimerization in the mechanism of action of clozapine.

Clozapine is effective although still not perfect drug used to treat schizophrenia. The precise mechanism of its action is not known. Here we show that there are two binding sites for clozapine at the dopamine D1 and D2 receptors, and the affinity of D1R strongly depended on whether the receptor was present alone or together with D2R (or its genetic variant D2Ser311Cys) in the cell membrane, pointing to the role of receptor hetero-dimerization in the observed phenomenon. The use of fluorescence resonance energy transfer (FRET) technology, observed via fluorescence lifetime microscopy of the single cell, indicated that low concentration of clozapine (10(-9) M), sufficient to saturate the high affinity site, uncoupled the D1R-D2R hetero-dimers. Therefore it has been concluded that clozapine might antagonize the effect of concomitant stimulation of both dopamine receptors, which has been shown previously to enhance the formation of hetero-dimers and to stimulate the calcium signaling pathway.

Effects of tramadol on α2-adrenergic receptors in the rat brain

Abstract In recent years, it has been postulated that tramadol, used mainly for the treatment of moderate to severe pain, might display a potential as an antidepressant drug. The present study investigated the effects of acute and repeated tramadol administration on the binding of [3H]RX 821002, a selective α2-adrenergic receptor ligand, in the rat brain. Male Wistar rats were used. Tramadol (20 mg/kg, i.p.) administered acutely (single dose), at 24 h after dosing, induced a significant decrease in the α2-adrenergic receptors in all brain regions studied. The most pronounced effects were observed in all subregions of the olfactory system, nucleus accumbens and septum, thalamus, hypothalamus, amygdala, and cerebral cortex. Repeated treatment with tramadol (20 mg/kg, i.p., once daily for 21 days) also induced statistically significant downregulation of [3H]RX 821002 binding sites in the rat brain. However, the effect—although statistically significant—was less pronounced than in the group treated acutely with the drug. Since drugs such as mianserin and mirtazapine are potent antagonists of central α2-adrenergic receptors and are effective antidepressants, it is tempting to suggest that, in addition to other alterations induced by tramadol, downregulation of these receptors may represent a potential antidepressant efficacy. On the other hand, one should be careful to avoid the treatment of chronic pain with tramadol in patients already receiving antidepressant drugs. Tramadol-induced downregulation of α2-adrenergic receptors—when combined with ongoing antidepressant therapy with drugs, which themselves inhibit serotonin reuptake or are antagonists of α2-adrenergic receptors—might cause threatening complications.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C‐terminus of the dopamine D1 receptor (404EE405) on the receptors’ localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D1–D2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D1–cyan fluorescent protein, D2–yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg‐rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein‐tagged D2R and cyan fluorescent protein‐tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of D1–D2 receptor heterodimerization. However, we find that the acidic epitope from the C‐terminus of the dopamine D1 receptor is engaged in the heterodimerization process.

Effect of clozapine on the dimerization of serotonin 5-HT2A receptor and its genetic variant 5-HT2AH425Y with dopamine D2 receptor

Oligomerization of G protein-coupled receptors has become a very important issue in a present molecular pharmacology. In the present study the level of the serotonin 5-HT(2A) and the dopamine D(2) receptor interactions have been studied since it may have a key significance in understanding the mechanism of action of drugs used to treat schizophrenia. With the use of fluorescence resonance energy transfer we demonstrated that the serotonin 5-HT(2A) receptors form homo- and hetero-dimers with the dopamine D(2) receptors and polymorphism H452Y within the 5-HT(2A) receptor, implicated as a cause of altered response to antipsychotic treatment, disturbs both processes. Clozapine affected the hetero-dimers (5-HT(2A)H452Y/D(2)) complexes and increased the otherwise weakened dimerization to the value observed for combination of both wild type receptors, and had no effect on the serotonin receptor homo-dimers (5-HT(2A)H452Y/5-HT(2A)), while haloperidol has restored the weakened interaction within homo-complexes and did not effect the hetero-complexes. The obtained data suggest that H452Y polymorphism has an influence not only on the level of constitutive oligomerization of investigated receptors but also it changes their pharmacological properties within both homo- and hetero-complexes.

Prolactin and its receptors in the chronic mild stress rat model of depression.

Prolactin (PRL) exhibits many physiological functions with wide effects on the central nervous system including stress responses. Our study aimed to investigate the effect of chronic unpredictable mild stress (CMS) - which is a good animal model of depression - on PRL receptor (PRLR) expression in the rat brain. Rats were exposed to CMS for two weeks and subsequently to CMS in combination with imipramine (IMI) treatment for five consecutive weeks. Behavioral deficit measured in anhedonic animals is a reduced intake of sucrose solution. Two weeks of CMS procedure allowed the selection of animals reactive to stress and displaying anhedonia, and the group which is considered as stress-non-reactive as far as behavioral measures are concerned. In this group the elevated level of PRL in plasma was observed, decrease in dopamine release in the hypothalamus, increase in [(125)I]PRL binding to PRLR in the choroid plexus, increase of mRNA encoding the long form of PRLR in the arcuate nucleus and the decrease of mRNA encoding its short form, and decrease in the mRNA encoding dopamine D2 receptor. All these alterations indicate these parameters as involved in the phenomenon of stress-resilience. The prolongation of the CMS procedure for additional five weeks shows the form of habituation to the stressful conditions. The most interesting result, however, was the up-regulation of PRLR in the choroid plexus of rats subjected to full CMS procedure combined with treatment with IMI, which may speak in favor of the role of this receptor in the mechanisms of antidepressant action.

Research reportEffects of tramadol on α2-adrenergic receptors in the rat brain

Abstract In recent years, it has been postulated that tramadol, used mainly for the treatment of moderate to severe pain, might display a potential as an antidepressant drug. The present study investigated the effects of acute and repeated tramadol administration on the binding of [3H]RX 821002, a selective α2-adrenergic receptor ligand, in the rat brain. Male Wistar rats were used. Tramadol (20 mg/kg, i.p.) administered acutely (single dose), at 24 h after dosing, induced a significant decrease in the α2-adrenergic receptors in all brain regions studied. The most pronounced effects were observed in all subregions of the olfactory system, nucleus accumbens and septum, thalamus, hypothalamus, amygdala, and cerebral cortex. Repeated treatment with tramadol (20 mg/kg, i.p., once daily for 21 days) also induced statistically significant downregulation of [3H]RX 821002 binding sites in the rat brain. However, the effect—although statistically significant—was less pronounced than in the group treated acutely with the drug. Since drugs such as mianserin and mirtazapine are potent antagonists of central α2-adrenergic receptors and are effective antidepressants, it is tempting to suggest that, in addition to other alterations induced by tramadol, downregulation of these receptors may represent a potential antidepressant efficacy. On the other hand, one should be careful to avoid the treatment of chronic pain with tramadol in patients already receiving antidepressant drugs. Tramadol-induced downregulation of α2-adrenergic receptors—when combined with ongoing antidepressant therapy with drugs, which themselves inhibit serotonin reuptake or are antagonists of α2-adrenergic receptors—might cause threatening complications.

Involvement of prolactin and somatostatin in depression and the mechanism of action of antidepressant drugs

Neuropeptides have been implicated in the physiology and pathophysiology of stress responses and therefore may play an important role in the pathogenesis of affective disorders such as Major Depression Disorder (MDD). The data presented in this mini-review demonstrate the role of prolactin (PRL) and somatostatin (STT) in the pathology and pharmacotherapy of MDD, focusing particularly on the response to antidepressant treatment, and compare the available data with the results obtained in our laboratory using the well-validated chronic mild stress (CMS) animal model of MDD. Despite the availability of many pharmacological therapies for depression, ca. 35% patients remain treatment resistant. This clinical situation is also true for rats subjected to CMS; some animals do not respond to antidepressant therapy and are considered treatment resistant. The most interesting results presented in this mini-review concern the changes in PRL and SST receptors in the brains of rats subjected to the full CMS procedure and IMI treatment and demonstrate the role of these receptors in the mechanisms of antidepressant action. The possible interaction between SST and PRL, the involvement of the D2 dopamine receptor, and their direct protein-protein interactions are also discussed, with the conclusion that these two neurohormones play an important role in the mechanism of resilience after stress as well as in the mechanism of action of antidepressant drugs.