Chronic fluoxetine treatment impairs motivation and reward learning by affecting neuronal plasticity in the central amygdala

Background and Purpose The therapeutic effects of fluoxetine (FLX) are believed to be due to its potency for increasing neuronal plasticity and reversing some learning deficits. Nevertheless, a growing amount of evidence shows the adverse effects of the drug on cognition and some forms of neuronal plasticity. EXPERIMENTAL APPROACH To study the effects of chronic FLX treatment we combine an automated assessment of motivation and learning in mice with an investigation of various forms of neuronal plasticity in the central (CeA) and basolateral amygdala (BLA). We use immunohistochemistry to visualize neuronal types and perineuronal nets (PNN), and DI-staining to assess dendritic spine morphology. Gel zymography is used to test FLX's impact on matrix metalloproteinase-9 (MMP-9), an enzyme involved in synaptic plasticity. KEY RESULTS We show that chronic FLX treatment in non-stressed mice increases PNN-dependent plasticity in the BLA, while simultaneously impairing MMP-9-dependent plasticity in the CeA. Further, we illustrate how the latter contributes to anhedonia and deficits of reward learning. Behavioral impairments are accompanied by alterations in morphology of dendritic spines in the CeA towards a more immature state, most likely reflecting animals' inability to adapt. We strengthen the link between the adverse effects of FLX and its influence on MMP-9 by showing that behavior of MMP-9 knock-out animals remains unaffected by the drug. CONCLUSION AND IMPLICATIONS In conclusion, chronic FLX treatment differentially affects various forms of neuronal plasticity, which may explain its contradicting effects on the brain and behavior. Presented findings are of immediate clinical relevance since reported side effects of FLX pose a potential threat to patients

Mitochondrial morphology in control and proband-derived fibroblasts cultured for 72 hours in glucose and glucose-free medium.

<p>Fibroblasts were stained with 50 nM MitoTracker for 30 min in 37°C and visualized using confocal microscopy. Representative images of MitoTracker stained mitochondria are shown. Bar = 10 μm.</p

The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient

<div><p>Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (<i>MFN2</i>). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a <i>de novo</i> Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions.</p></div

GTP-hydrolysis activity of mitofusin 2 obtained from control and proband-derived fibroblasts.

<p>GTPase activity of control and mutated MFN2 presented as HPLC traces (A) and GDP/GTP ratio (B). Endogenous MFN2 was immunoprecipitated from 300 μg PNS lysates obtained from control (IP MFN2 C) and proband-derived fibroblasts (IP MFN2 Arg274Trp) and incubated with GTP at 37°C for 60 minutes, before HPLC analysis as described in Methods. In parallel, immunoprecipitation with nonspecific rabbit antibody was performed with PNS lysates obtained from control (IP IgG C) and proband-derived fibroblasts (IP IgG Arg274Trp). RB–reaction buffer containing 100 μM GTP.</p

Overall MFN2 model structure.

<p>Colors demonstrate domain division: GTPase (blue), HR-1 (green), HR-2 (yellow), and paddle region (orange). A. MFN2 monomer structure with visible GTP showed as Van der Waals spheres. B. Sequence-oriented MFN2 domain composition, with boundary residues. C. MFN2 homodimer embedded in lipid bilayer. Paddle residues 620–662 deeply embedded, residues 438–479 localized above hydrophobic layer.</p

Clinical characterization of the CMT2A patient harboring the p.Arg274Trp mutation.

<p>A. Wasting of distal muscles (upper and lower limbs) in the proband. B. Severe wasting of the small hand muscles and forearms in the proband. C. <i>Pes cavus</i> deformity in the proband.</p

The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient - Fig 2

<p>Morphology, lactate formation and oxygen consumption in proband-derived fibroblasts A. Transmitted light images of control and proband-derived fibroblasts cultured for 24 and 72 hours in glucose and glucose-free medium. Bar = 200 μm. B. Lactate synthesis was measured enzymatically in control and proband-derived fibroblasts cultured in glucose and glucose-free medium for 72 h. Data are expressed as mean amount of lactate (nanomoles per mg of protein) ± S.D.; n = 3, *p < 0.05. C. Oxygen consumption of cells grown in glucose or glucose-free media for 72 h was measured polarographically at 37°C. Data are expressed as pmoles O₂ x s^-1/mg protein and show mean values ± S.D.; n = 2–3.</p

Endoplasmic reticulum morphology and reticulum stress in control and proband-derived fibroblasts cultured for 72 hours in glucose and glucose-free medium.

<p>A. Representative transmission electron microscopy images of ultrastructure of endoplasmic reticulum (ER) are shown. On right, zoomed pictures to highlight distinct ER structures. Bar = 2 μm. B. Immunoreactivity of reticulum stress marker, GRP78/BiP, was analysed by western blot. Densities of GRP78/BiP bands were evaluated and data are expressed as a percentage of β-Tubulin. (mean ± SD, n = 2–3).</p

Mitochondrial DNA content was determined in control and proband-derived fibroblasts cultured for 72 hours in glucose and glucose-free medium by real time PCR.

<p>Quantification was based on mtDNA to nuclear Kir4.1 (nDNA) gene encoding ratio. Mean values and standard deviations were calculated from at least X independent experiments. *p<0.05</p