Álvaro Díaz

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.

Sensorineural hearing loss in insulin-like growth factor I-null mice: a new model of human deafness

It has been reported that mutations in the gene encoding human insulin‐like growth factor‐I (IGF‐I) cause syndromic hearing loss. To study the precise role of IGF‐I in auditory function and to hypothesize the possible morphological and electrophysiological changes that may occur in the human inner ear, we have analysed the auditory brainstem response in a mouse model of IGF‐I deficiency. We show here that homozygous Igf‐1–/– mice present an all‐frequency involved bilateral sensorineural hearing loss. Igf‐1–/– mice also present a delayed response to acoustic stimuli; this increases along the auditory pathway, indicating a contribution of the central nervous system to the hearing loss in Igf‐1–/– mice. These results support the use of the Igf‐1–/– mouse as a new model for the study of human syndromic deafness.

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.

Long-term fluoxetine treatment modulates cannabinoid type 1 receptor-mediated inhibition of adenylyl cyclase in the rat prefrontal cortex through 5-hydroxytryptamine 1A receptor-dependent mechanisms.

Increasing data indicate that brain endocannabinoid system plays a role in the effects of antidepressant medications. Here we examined the effect of in vivo exposure to the selective serotonin uptake inhibitor fluoxetine on cannabinoid type 1 (CB1) receptor density and functionality in the rat prefrontal cortex (PFC) and cerebellum. Long-term treatment with fluoxetine (10 mg/kg/day) enhanced CB1 receptor inhibition of adenylyl cyclase (AC) in the PFC and reduced it in the cerebellum without altering receptor density and agonist stimulation of guanosine 5′-O-(3-[35S]thio) triphosphate ([35S]GTPγS) in either area. Analysis of [35S]GTPγS-labeled Gα subunits allowed for the detection of up-regulated CB1 receptor coupling to Gαi2, Gαi3 in the PFC, and reduced coupling to Gαi3 in the cerebellum of fluoxetine-treated rats. Concomitant administration of the 5-HT1A receptor antagonist N-[2-[4- (2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY100635; 0.1 mg/kg/day) reduced fluoxetine-induced modulation of CB1 receptor coupling to Gα subunits and AC in the PFC but not in the cerebellum. These results indicate that increased CB1 receptor signaling at the Gαi-AC transduction level is a long-term adaptation induced by fluoxetine in the PFC and point to a role for 5-HT1A receptors in this effect. Basal AC activity, protein kinase A (PKA) catalytic subunit expression, and phospho-cAMP response element-binding protein (pCREB)/CREB ratio were also up-regulated in the PFC of fluoxetine-treated animals, whereas no differences were detected in the cerebellum. It is interesting that long-term Δ9-tetrahydrocannabinol treatment did not elicit antidepressant-like effects or modulated behavioral responses of fluoxetine in an animal model of depression (olfactory bulbectomy). These data suggest that altered signal transduction through CB1 receptors in the PFC may participate in the regulation of the AC-PKA-CREB cascade induced by fluoxetine in this brain area.

Regulation of μ-opioid receptors, G-protein-coupled receptor kinases and β-arrestin 2 in the rat brain after chronic opioid receptor antagonism

The aim of this study was to analyse the biochemical and behavioural consequences of chronic treatment with opioid receptor antagonists in rats. We have evaluated the respiratory depressant and antinociceptive effects of the mu-opioid agonist sufentanil, the density of brain mu-opioid receptors, and the expression of G-protein-coupled receptor kinases and beta-arrestin 2 in cerebral cortex and striatum, following sustained opioid receptor blockade. Our results demonstrate that 24 h after interruption of 7 days chronic infusion of naltrexone (120 microg/h), the respiratory depressant potency of the mu-opioid receptor agonist sufentanil was increased to a similar extent as the antinociceptive potency (about three-fold). This was accompanied by mu-opioid receptor up-regulation in several areas of the rat brain associated with opioid control of pain perception and breathing. Moreover, chronic treatment with either naltrexone (120 microg/h) or naloxone (120 microg/h) caused significant increases in the expression levels of G-protein-coupled receptor kinases types 2, 3, and 6, and of beta-arrestin 2 in brain cortex and striatum. Together our data suggest an increased constitutive receptor activity secondary to mu-opioid receptor up-regulation following chronic antagonist treatment.

Neural Plasticity and Proliferation in the Generation of Antidepressant Effects: Hippocampal Implication

It is widely accepted that changes underlying depression and antidepressant-like effects involve not only alterations in the levels of neurotransmitters as monoamines and their receptors in the brain, but also structural and functional changes far beyond. During the last two decades, emerging theories are providing new explanations about the neurobiology of depression and the mechanism of action of antidepressant strategies based on cellular changes at the CNS level. The neurotrophic/plasticity hypothesis of depression, proposed more than a decade ago, is now supported by multiple basic and clinical studies focused on the role of intracellular-signalling cascades that govern neural proliferation and plasticity. Herein, we review the state-of-the-art of the changes in these signalling pathways which appear to underlie both depressive disorders and antidepressant actions. We will especially focus on the hippocampal cellularity and plasticity modulation by serotonin, trophic factors as brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) through intracellular signalling pathways—cAMP, Wnt/β-catenin, and mTOR. Connecting the classic monoaminergic hypothesis with proliferation/neuroplasticity-related evidence is an appealing and comprehensive attempt for improving our knowledge about the neurobiological events leading to depression and associated to antidepressant therapies.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-beta family in pain modulation.

Transforming growth factors-β (TGF-βs) signal through type I and type II serine–threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-β family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-β signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI−/− mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI−/− mice also appeared attenuated through a mechanism involving δ-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI−/−. Transcript levels of TGF-βs and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-β family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.

Modulation of neuroplasticity pathways and antidepressant-like behavioural responses following the short-term (3 and 7 days) administration of the 5-HT4 receptor agonist RS67333

It has been recently suggested that activation of 5-HT₄ receptors might exert antidepressant-like effects in rats after 3 d treatment, suggesting a new strategy for developing faster-acting antidepressants. We studied the effects of 3 d and 7 d treatment with the 5-HT₄ receptor partial agonist RS67333 (1.5 mg/kg.d) in behavioural tests of chronic efficacy and on neuroplastic-associated changes, such as adult hippocampal neurogenesis, expression of CREB, BDNF, β-catenin, AKT and 5-HT₄ receptor functionality. RS67333 treatment up-regulated hippocampal cell proliferation, β-catenin expression and pCREB/CREB ratio after 3 d treatment. This short-term treatment also reduced immobility time in the forced swim test (FST), together with a partial reversion of the anhedonic-like state (sucrose consumption after chronic corticosterone). Administration of RS67333 for 7 d resulted in a higher increase in the rate of hippocampal cell proliferation, a significant desensitization of 5-HT₄ receptor-coupled adenylate cyclase activity and a more marked increase in the expression of neuroplasticity-related proteins (BDNF, CREB, AKT): these changes reached the same magnitude as those observed after 3 wk administration of classical antidepressants. Consistently, a positive behavioural response in the novelty suppressed feeding (NSF) test and a complete reversion of the anhedonic-like state (sucrose consumption) were also observed after 7 d treatment. These results support the antidepressant-like profile of RS67333 with a shorter onset of action and suggest that this time period of administration (3-7 d) could be a good approximation to experimentally predict the onset of action of this promising strategy.

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α2, β1 and β2‐adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [3H]Bromoxidine (α2‐adrenoceptor‐), [3H]CGP 12177 (β‐adrenoceptor) and [1251]cyanopindolol (β‐adrenoceptor) were used as radioligands. In both species, [3H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α2‐adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α2‐adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β‐adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β2 subtype, in contrast to what has been reported in the mammalian brain, where the β1 subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [1251]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.

Transforming Growth Factor-β in Normal Nociceptive Processing and Pathological Pain Models

SummaryThe transforming growth factor-β (TGF-β) superfamily is a multifunctional, contextually acting family of cytokines that participate in the regulation of development, disease and tissue repair in the nervous system. The TGF-β family is composed of several members, including TGF-βs, bone morphogenetic proteins (BMPs) and activins. In this review, we discuss recent findings that suggest TGF-β function as important pleiotropic modulators of nociceptive processing both physiologically and under pathological painful conditions. The strategy of increasing TGF-β signaling by deleting “BMP and activin membrane-bound inhibitor” (BAMBI), a TGF-β pseudoreceptor, has demonstrated the inhibitory role of TGF-β signaling pathways in normal nociception and in inflammatory and neuropathic pain models. In particular, strong evidence suggests that TGF-β1 is a relevant mediator of nociception and has protective effects against the development of chronic neuropathic pain by inhibiting the neuroimmune responses of neurons and glia and promoting the expression of endogenous opioids within the spinal cord. In the peripheral nervous system, activins and BMPs function as target-derived differentiation factors that determine and maintain the phenotypic identity and circuit assembly of peptidergic nociceptors. In this context, activin is involved in the complex events of neuroinflammation that modulate the expression of pain during wound healing. These findings have provided new insights into the physiopathology of nociception. Moreover, specific members of the TGF-β family and their signaling effectors and modulator molecules may be promising molecular targets for novel therapeutic agents for pain management.