Desipramine targets astrocytes to attenuate synaptic plasticity via modulation of the ephrinA3/EphA4 signalling.

Long-term potentiation (LTP), a major cellular correlate of memory storage, depends on activation of the ERK/MAPK signalling pathway, but the cell type-specific localization of activated MAPKs remains unknown. We found that in the CA1 field of the hippocampus, shortly after LTP induction, an increase in the number of MAPK-positive cells occurred specifically among astrocytes of the stratum radiatum, suggesting a putative role of astrocytes for LTP. Desipramine (DMI) is an antidepressant which is used to treat major depressive disorder, but also other pathologies such as neuropathic pain or attention-deficit/hyperactivity disorder. Tricyclic antidepressants such as DMI may cause memory impairment as a side effect. However, biological underpinnings of this effect still remain unclear. Here, we show that DMI inhibited the astrocytic MAPK activation and thereby hindered synaptic potentiation. These effects correlated with a reduced neuronal activation in the stratum pyramidale, thereby prompting us to analyse a regulator of LTP located at the astrocyte-neuron interface in the stratum radiatum, namely the ephrinA3/EphA4 signalling pathway. DMI enhanced EphA4 clustering, which favoured an increased ephrinA3-mediated EphA4 phosphorylation and elevated EphA4 forward signalling. The co-administration of DMI with the Src inhibitor SU6656, which blocks EphA4 forward signalling, could partially reverse the LTP attenuation, further supporting the targeting of the ephrinA3/EphA4 pathway by DMI. Thus, our findings suggest a putative novel mechanism for DMI to modulate LTP through the regulation of the ephrinA3/EphA4 signalling pathway. A further exploration of the molecular and behavioral consequences of targeting ephrinA3/EphA4 might help to improve the clinical use of DMI.

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-ß-cyclodextrine (MßCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Impact of lipid raft integrity on 5-HT3 receptor function and its modulation by antidepressants.

Because of the biochemical colocalization of the 5-HT(3) receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT(3) receptor function and its modulation by antidepressants. Treatment with methyl-beta-cyclodextrine (MbetaCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MbetaCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT(3) receptor were retained. Although both the 5-HT(3) receptor and flotillin-1 were predominantly found in raft-like domains in western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT(3) receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT(3) receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT(3) receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel.

Identification of a domain which affects kinetics and antagonistic potency of clozapine at 5-HT3 receptors.

The widely used atypical antipsychotic clozapine is a potent competitive antagonist at 5-HT(3) receptors which may contribute to its unique psychopharmacological profile. Clozapine binds to 5-HT(3) receptors of various species. However, the structural requirements of the respective binding site for clozapine remain to be determined. Differences in the primary sequences within the 5-HT(3A) receptor gene in schizophrenic patients may result in an alteration of the antipsychotic potency and/or the side effect profile of clozapine. To determine these structural requirements we constructed chimeras with different 5-HT(3A) receptor sequences of murine and human origin and expressed these mutants in human embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant mouse 5-HT(3A) receptors with higher potency compared to recombinant human 5-HT(3A) receptors. 5-HT activation curves and clozapine inhibition curves yielded the parameters EC(50) and IC(50) for all receptors tested in the range of 0.6 - 2.7 microM and 1.5 - 83.3 nM, respectively. The use of the Cheng-Prusoff equation to calculate the dissociation constant K(b) values for clozapine revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 strongly determines the binding affinity of clozapine. K(b) values of clozapine were significantly lower (0.3-1.1 nM) for receptors containing the murine sequence and higher when compared with receptors containing the respective human sequence (5.8-13.4 nM). Thus, individual differences in the primary sequence of 5-HT(3) receptors may be crucial for the antipsychotic potency and/or the side effect profile of clozapine.