A. Rodríguez-Gaztelumendi

Altered CB1 receptor‐signaling in prefrontal cortex from an animal model of depression is reversed by chronic fluoxetine

Bilateral olfactory bulbectomy in the rat (OBX) induces behavioral, neurochemical, and structural abnormalities similar to those observed in human depression that are normalized after chronic, but not acute, treatment with antidepressants. In our study, OBX animals exhibited significant increases in both CB1 receptor density ([3H]CP55490 binding) and functionality (stimulation of [35S]GTPγS binding by the cannabinoid (CB) agonist WIN 55212‐2) at the prefrontal cortex (PFC). After chronic treatment with fluoxetine (10 mg/kg/day, 14 days, s.c.), OBX‐induced hyperactivity in the open‐field test was fully abolished. Interestingly, chronic fluoxetine fully reversed the enhanced CB1‐receptor signaling in PFC observed following OBX. The CB agonist Δ9‐tetrahydrocannabinol (5 mg/kg, i.p., 1 day) did not produce any behavioral effect in sham‐operated animals but returned locomotor activity to control values in OBX rats. As both acute administration of Δ9‐tetrahydrocannabinol and chronic fluoxetine elicited a similar behavioral effect in the OBX rat, it is not unlikely that the regionally selective enhancement of CB1 receptor‐signaling in the PFC could be related with the altered OBX behavior. Our findings reinforce the utility of this animal model to further investigating the implication of the endocannabinoid system in the modulation of emotional processes and its potential role in the adaptive responses to chronic antidepressants.

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.

Differential adaptive changes on serotonin and noradrenaline transporters in a rat model of peripheral neuropathic pain

Serotonin and noradrenaline reuptake inhibitors have shown to produce antinociceptive effects in several animal models of neuropathic pain. In the present work, we have analyzed the density of brain and spinal serotonin and noradrenaline transporters (5-HTT and NAT) in a rat model of neuropathic pain, the spinal nerve ligation (SNL). Quantitative autoradiography revealed a significant decrease in the density of 5-HTT ([(3)H]citalopram binding) at the level of the lumbar spinal cord following 2 weeks of neuropathic surgery (lamina V, -40%: 6.01±0.64 nCi/mg tissue in sham-animals vs 3.59±1.56 in SNL-animals; lamina X, -30%: 9.10±2.00 vs 6.40±1.93 and lamina IX, -22%: 12.01±2.41 vs 9.42±1.58). By contrast, NAT density ([(3)H]nisoxetine binding) was significantly increased (lamina I-II, +34%: 2.20±0.45 vs 2.96±0.65; lamina V, +57%: 1.34±0.28 vs 2.11±0.66; and lamina IX, +58%: 2.39±0.71 vs 3.78±1.10). At supraspinal structures, SNL induced adaptive changes only in the density of 5-HTT (septal nuclei, +33%: 10.18±2.03 vs 13.53±1.14; CA3 field of hippocampus, +18%: 6.94±1.01 vs 8.21±0.81; paraventricular thalamic nucleus, +21%: 15.18±1.88 vs 18.35±2.08; lateral hypothalamic area, +40%: 12.68±1.90 vs 17.8±2.55; ventromedial hypothalamic nuclei, +19%: 7.16±0.92 vs 8.55±0.40; and dorsal raphe nucleus, +15%: 35.22±3.88 vs 40.68±3.11). Thus, we demonstrate, in the SNL model of neuropathic pain, the existence of opposite changes in the spinal expression of 5-HTT (down-regulation) and NAT (up-regulation), and the presence of supraspinal adaptive changes (up-regulation) only on 5-HTT density. These findings may help understanding the pathogeny of neuropathic pain and the differential analgesic action of antidepressants targeting 5-HTT and/or NAT transporters.

Chronic fluoxetine regulates both 5-HT1A receptor functionality and hippocampal b-catenin pathway in an animal model of depression

P105 Administration of late endothelial progenitor cells enhances cerebral infarction outcome after transient middle cerebral artery occlusion in rats C Moubarik, JL Codaccioni, L Velly, F Sabatier, F Dignat-George, MD Piercecchi, B Guillet, P Pisano Faculté de Pharmacie, unité INSERM U 608, Marseille, France; Département d’anesthésie réanimation, Marseille, France Endothelial progenitor cells (EPCs) are a novel promising option for treatment of patients with ischemic diseases. We characterized the effect of late outgrowth EPCs transplantation in a model of transient middle cerebral artery occlusion (MCAO) in rat. EPCs were obtained from human umbilical cord blood. Adult male SpragueDawley rats were subjected to 1 h of MCAO and allocated 24 h after MCAO to transplanted (4–5 · 10 late EPCs IV, n = 12) and control (PBS IV, n = 13). Body weight, adhesive-removal dot test and the mNNS test were performed over the 14 days following MCAO. Seven or 14 days after MCAO, brain infarct volumes, human transplanted cells localization, apoptosis and capillary density staining were done. Animal body weights were significantly higher in the transplanted group 7 days after MCAO (P < 0.05). Significantly higher neurological performance was found in transplanted rats compared to control rats (P < 0.05). Immunochemical staining showed that EPCs survived and were preferentially localized in the ischemic boundary zone. No difference has been observed in term of infarct volume (P = 0.98). At day 3, apoptotic cell number was significantly lower in transplanted rats (48.0 ± 18/mm) compared to control rats (130.0 ± 50/mm) (P < 0.05). Capillary densities showed were significantly increased (P= <0.05) at the boundary of the ischemic lesion in transplanted rats (n = 4; 2.80 ± 0.3) compared to control rats (n = 4; 1.72 ± 0.7) 14 days after MCAO. Late EPCs administered intravenously 24 h after MCAO enter the brain, survive, migrate, and improve functional recovery. This may be involved ischemia-induced apoptosis together with angiogenic response modulation.

Effects of spinal administration of amitryptiline, venlafaxine and fluoxetine on C-fibre and A-fibre evoked responses of dorsal horn neurones

P105 Administration of late endothelial progenitor cells enhances cerebral infarction outcome after transient middle cerebral artery occlusion in rats C Moubarik, JL Codaccioni, L Velly, F Sabatier, F Dignat-George, MD Piercecchi, B Guillet, P Pisano Faculté de Pharmacie, unité INSERM U 608, Marseille, France; Département d’anesthésie réanimation, Marseille, France Endothelial progenitor cells (EPCs) are a novel promising option for treatment of patients with ischemic diseases. We characterized the effect of late outgrowth EPCs transplantation in a model of transient middle cerebral artery occlusion (MCAO) in rat. EPCs were obtained from human umbilical cord blood. Adult male SpragueDawley rats were subjected to 1 h of MCAO and allocated 24 h after MCAO to transplanted (4–5 · 10 late EPCs IV, n = 12) and control (PBS IV, n = 13). Body weight, adhesive-removal dot test and the mNNS test were performed over the 14 days following MCAO. Seven or 14 days after MCAO, brain infarct volumes, human transplanted cells localization, apoptosis and capillary density staining were done. Animal body weights were significantly higher in the transplanted group 7 days after MCAO (P < 0.05). Significantly higher neurological performance was found in transplanted rats compared to control rats (P < 0.05). Immunochemical staining showed that EPCs survived and were preferentially localized in the ischemic boundary zone. No difference has been observed in term of infarct volume (P = 0.98). At day 3, apoptotic cell number was significantly lower in transplanted rats (48.0 ± 18/mm) compared to control rats (130.0 ± 50/mm) (P < 0.05). Capillary densities showed were significantly increased (P= <0.05) at the boundary of the ischemic lesion in transplanted rats (n = 4; 2.80 ± 0.3) compared to control rats (n = 4; 1.72 ± 0.7) 14 days after MCAO. Late EPCs administered intravenously 24 h after MCAO enter the brain, survive, migrate, and improve functional recovery. This may be involved ischemia-induced apoptosis together with angiogenic response modulation.

Autoradiographic study of G-protein coupling to cannabinoid CB1-receptors in the brain of rats with peripheral mononeuropathy

P053 Non-genomic effects of testosterone on contraction in the mouse vas deferens J Kennard, J Young, K Brain Department of Pharmacology, University of Oxford, Oxford, UK Several studies have demonstrated non-genomic effects of androgens on a range of tissue preparations, characterised by a short time lag and insensitivity to transcription and translation inhibitors (Falkenstein et al., 2000). It is not known whether there are non-genomic effects of testosterone in the mouse vas deferens, an important model for studying junctional transmission. Vasa deferentia were excised from eightto twelve-week-old male Balb/C mice (20–40 g) which had been humanely killed by an approved Schedule I method. The effects of testosterone on contraction were examined using electrical field stimulation and the exogenous application of the P2 purinoceptor agonist, a,b-methylene ATP; on membrane depolarisation using intracellular microelectrodes; and on whole cell Calcium (Ca) transients using confocal microscopy and Oregon Green BAPTA-1 as a fluorescent indicator of free Ca, delivered to smooth muscle cells as a membranepermeable AM form. Testosterone caused a rapid, concentration-dependent (3– 300 lM) inhibition of neurogenic contraction (5 pulses, 10 Hz, 0.5 ms duration, supramaximal voltage) with an EC50 of 120 lM and 87 ± 8% inhibition at 300 lM (n = 6). Such contractions were resistant to the androgen receptor antagonist, flutamide, and the translation inhibitor, cycloheximide. This inhibitory effect was also observed with contractions elicited by the exogenous application of a,bmetATP. Combined with the observations from the electrophysiology experiments, this indicates a post-junctional site of action for testosterone. These observations might be due to effects on L-type Cachannels as has been shown in vascular smooth muscle cells (Hall et al., 2006). Although the concentrations of testosterone used in our studies are supraphysiological, raised levels of androgens are seen clinically in conditions such as androgen insensitivity syndrome and with abuse of anabolic steroids. References: Falkenstein E et al. Pharmacol Rev. 2000; 52: 513–556. Hall J et al. Endocrinology. 2006; 147: 2675–2680.