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Traumatic brain injury (TBI) is a chronic pathology, inducing long-term deficits that remain understudied in pre-clinical studies. In this context, exploration, anxiety-like behavior, cognitive flexibility, and motor coordination were assessed until 5 and 10 months after an experimental TBI in the adult mouse, using two cohorts. In order to differentiate age, surgery, and remote gray and white matter lesions, three groups (unoperated, sham-operated, and TBI) were studied. TBI induced delayed motor coordination deficits at the pole test, 4.5 months after injury, that could be explained by gray and white matter damages in ipsilateral nigrostriatal structures (striatum, internal capsule) that were spreading to new structures between cohorts, at 5 versus 10 months after the injury. Further, TBI induced an enhanced exploratory behavior during stressful situations (active phase during actimetry test, object exploration in an open field), risk-taking behaviors in the elevated plus maze 5 months after injury, and a cognitive inflexibility in the Barnes maze that persisted until 9 months after the injury. These behavioral modifications could be related to the white and gray matter lesions observed in ipsi- and contralateral limbic structures (amygdala, hilus/cornu ammonis 4, hypothalamus, external capsule, corpus callosum, and cingular cortex) that were spreading to new structures between cohorts, at 5 months versus 10 months after the injury. The present study corroborates clinical findings on TBI and provides a relevant rodent chronic model which could help in validating pharmacological strategies against the chronic consequences of TBI.

Psychedelic drugs, often referred to as hallucinogens, are quite distinct from other classes of psychotropic drugs. Although the subjective and behavioral effects they induce are quite dramatic, they possess little addictive potential when compared to nicotine, alcohol or opiates. Since the discovery of ketamine antidepressant effects, there has been growing interest for these molecules. Serotonergic psychedelics such as psilocybin and lysergic acid diethylamide (LSD) are gaining attention as potential treatments for depression and addiction, similarly to 3,4-methylenedioxymethamphetamine (MDMA) for post-traumatic stress disorder (PTSD), and ibogaine for addiction. Although they possess distinct pharmacological profiles, their kinetics of action are quite similar: the therapeutic effects are felt within the hours following administration, and last well beyond drug elimination by the organism. This strongly suggests the induction of neurogenic and plastic mechanisms, including the involvement of trophic factors. This review will explore the literature dealing with the effects of psychedelics on neurotrophins, as well as the plastic adaptations that they induce, in an attempt to understand their surprising therapeutic potential. We will show that although ketamine and serotonergic psychedelics have affinity for very different receptors (NMDA, 5-HT2A), they ultimately initiate similar plastic adaptations in the prefrontal cortex through the involvement of the brain-derived neurotrophic factor (BDNF). We will see that although MDMA uses the same receptors as serotonergic psychedelics to alleviate PTSD symptoms, its effect on BDNF levels seem paradoxical and quite different. Finally, we show how ibogaine could exert its anti-addictive properties through a completely different neurotrophic factor than other psychedelic drugs, the glial cell line-derived neurotrophic factor (GDNF). While the current literature concerning the psychiatric applications of psychedelic therapy is encouraging, it remains to be determined whether their benefits could be obtained without their psychotomimetic effects, or concerns over potential toxicity.

Insulin-like growth factors (IGF) are potent neurotrophic and neurorepair factors that were recently proposed as biomarkers of traumatic brain injury (TBI) and associated psychiatric comorbidities, in particular post-traumatic stress disorder (PSTD). We tested the hypothesis that the IGF system is differentially deregulated in the acute and early chronic stages of TBI, and under acute stress. Plasma and brain IGF1 and IGF2 levels were evaluated in mice 3 weeks and 3 days after a controlled cortical impact (CCI)-induced mild-to-moderate TBI. The effects of conditioned fear on IGF levels and its interaction with TBI (TBI followed, 3 weeks later, by fear-inducing procedures) were also evaluated. In the plasma, IGF1 decreased 3 weeks post-TBI only (−9%), whereas IGF2 remained unaffected. In the brain, IGF1 increased only in the cortex and hippocampus at 3 weeks post-TBI (up to +650%). At 3 days, surpringly, this increase was more diffuse and more important in sham (craniotomized) animals. Additionally, IGF2 immunostaining in brain ventricles was reorganized in TBI animals at both post-TBI stages. Conditioned fear exposure did not influence the effects of early chronic TBI on plasma IGF1 levels, but reduced plasma IGF2 (−6%) levels. It also dampened the effects of TBI on brain IGF systems, but brain IGF1 level and IGF2 tissue distribution remained statistically different from controls under these conditions. In co-exposed animals, DNA methylation increased at the hippocampal Igf1 gene promoter. These results show that blood IGF1 and IGF2 are most reduced in the early chronic phase of TBI and after exposure to a stressful event, and that the brain IGF system is up-regulated after TBI, and more so in the acute phase.

Background and Purpose It has recently been suggested that 5-HT3 receptor blockade enhances the efficacy of selective 5-HT (serotonin) reuptake inhibitor (SSRI) antidepressants and may reverse stress-induced deficits in rodents. Experimental Approach To further explore this hypothesis, we used mice lacking the 5-HT3 receptor (Htr3a KO) and their wild-type (WT) controls to assess their response in behavioural paradigms relevant to anxiety and depression. Mice were studied under basal, antidepressant treatments and chronic social defeat stress (CSDS) conditions. Key Results In basal conditions, Htr3a KO mice displayed anxiolytic- and antidepressant-like behaviours in the elevated plus maze, the social interaction and the forced swim tests (FST), but behaved as WT mice in response to acute citalopram in the FST. However, the effects of fluoxetine were blunted in Htr3a KO mice in these same tests. In an in vitro electrophysiological paradigm, a low-dose citalopram treatment triggered 5-HT1A receptor desensitization only in the dorsal raphe nucleus of Htr3a KO, although a high dose desensitized 5-HT1A autoreceptor function equally in Htr3a KO and WT mice, suggesting that citalopram may become effective at lower doses when 5-HT3 receptors are inactivated. In addition, Htr3a deletion blocked CSDS-induced modification in the cortical expression of two genes involved in oxidative stress, CaMKIIa and SOD1. Conclusions and Implications Taken together, these data show that Htr3a deletion promotes SSRI efficacy and prevents the occurrence of stress-induced deleterious effects, suggesting that the 5-HT3 receptor may represent an interesting target for the treatment of stress-related disorders.

Post-traumatic stress disorder (PTSD) is a trauma- and stress-related disorder with dysregulated fear responses and neurobiological impairments, notably at neurotrophic and inflammation levels. Understanding the mechanisms underlying this disease is crucial to develop PTSD models that meet behavioral and neurobiological validity criteria as well as innovative therapeutic approaches. Serotonin 2C receptors (5-HT2CR) are known for their important role in anxiety, and mice having only the fully edited VGV isoform of 5-HT2CR, which thereby overexpressed brain 5-HT2CR, are of special interest to study PTSD predisposition. Innate and conditioned fear-related behaviors were assessed in VGV and wild-type mice. mRNA expression of brain-derived neurotrophic factor (BDNF), tissue-plasminogen activator (tPA), and pro-inflammatory cytokines (IL-6, IL-1β, and calcineurin) were measured by qRT-PCR. The effect of acute and chronic paroxetine was evaluated on both behavior and gene expression. VGV mice displayed greater fear expression, extensive fear extinction deficits, and fear generalization. Paroxetine restored fear extinction in VGV mice when administered acutely and decreased innate fear and fear generalization when administered chronically. In parallel, Bdnf, tPA, and pro-inflammatory cytokines mRNA levels were dysregulated in VGV mice. Bdnf and tPA mRNA expression was decreased in the hippocampus but increased in the amygdala, and chronic paroxetine normalized Bdnf mRNA levels both in the amygdala and the hippocampus. Amygdalar calcineurin mRNA level in VGV mice was also normalized by chronic paroxetine. VGV-transgenic mice displayed behavioral and neurobiological features that could be accessory to the investigation of PTSD and its treatment. Furthermore, these data point out to the role of 5-HT2CR in neuroplasticity and neuroinflammation.

Growing evidence indicates that impairment of the stress response, in particular the negative feedback regulation mechanism exerted by the hypothalamo-pituitary-adrenal (HPA) axis, might be responsible for the hippocampal atrophy observed in depressed patients. Antidepressants, possibly through the activation of BDNF signaling, may enhance neuroplasticity and restore normal hippocampal functions. In this context, glucocorticoid receptor-impaired (GR-i) mice-a transgenic mouse model of reduced GR-induced negative feedback regulation of the HPA axis-were used to investigate the role of BDNF/TrkB signaling in the behavioral and neurochemical effects of the new generation antidepressant drug, agomelatine. GR-i mice exhibited marked alterations in depressive-like and anxiety-like behaviors, together with a decreased cell proliferation and altered levels of neuroplastic and epigenetic markers in the hippocampus. GR-i mice and their wild-type littermates were treated for 21 days with vehicle, agomelatine (50 mg/kg/day; i.p) or the TrkB inhibitor Ana-12 (0.5 mg/kg/day, i.p) alone, or in combination with agomelatine. Chronic treatment with agomelatine resulted in antidepressant like effects in GR-i mice and reversed the deficit in hippocampal cell proliferation and some of the alterations of mRNA plasticity markers in GR-i mice. Ana-12 blocked the effect of agomelatine on motor activity as well as its ability to restore a normal hippocampal cell proliferation and expression of neurotrophic factors. Altogether, our findings indicate that agomelatine requires TrkB signaling to reverse some of the molecular and behavioral alterations caused by HPA axis impairment.

Mild traumatic brain injury (TBI) is a major risk factor for post-traumatic stress disorder (PTSD), and both disorders share common symptoms and neurobiological defects. Relapse after successful treatment, known as long-term fear resurgence, is common in PTSD patients and a major therapeutic hurdle. We induced a mild focal TBI by controlled cortical impact (CCI) in male C57BL/6 J mice and used fear conditioning to assess PTSD-like behaviors and concomitant alterations in the fear circuitry. We found for the first time that mild TBI, and to a lesser extent sham (craniotomy), mice displayed a spontaneous resurgence of conditioned fear when tested for fear extinction memory recall, despite having effectively acquired and extinguished conditioned fear 6 weeks earlier in the same context. Other characteristic symptoms of PTSD are risk-taking behaviors and cognitive deficits. CCI mice displayed risk-taking behaviors, behavioral inflexibility and reductions in processing speed compared to naive mice. In conjunction with these changes there were alterations in amygdala morphology 3 months post-trauma, and decreased myelin basic protein density at the primary lesion site and in distant secondary sites such as the hippocampus, thalamus, and amygdala, compared to sham mice. Furthermore, activity-dependent brain-derived neurotrophic factor (BDNF) transcripts were decreased in the prefrontal cortex, a key region for fear extinction consolidation, following fear extinction training in both TBI and, to a lesser extent, sham mice. This study shows for the first time that a mild brain injury can generate a spontaneous resurgence of conditioned fear associated with defective BDNF signalling in the prefrontal cortex, PTSD-like behaviors, and have enduring effects on the brain.

This review examines the epigenetic of neurotrophin signaling in anxiety, affective disorders and related symptoms associated with drug addiction, in particular alcoholism. It is first important to understand the epigenetics of aversion memories, as they are so central to anxiety and affective disorders symptomology. The crucial role of neurotrophins in memory formation, in particular the brain-derived nerve growth factor (BDNF), is explored at the physiological and behavioral levels. Numerous studies describe how various epigenetic phenomena, mainly histone acetylation, histone methylation, DNA methylation, but also other less known epigenetic phenomena such as histone poly[ADP]-ribosylation and 5-HT2C receptor pre-mRNA editing, exert significant regulatory roles in aversion memory and fear extinction memory formation. Other models of anxiety and affective disorders, that use stress or transgenic constructs directed at elements of the stress axis or the serotonergic system, are then explored in relation with the epigenetic of neurotrophin signaling. Epigenetic marks differentially change according to brain areas. In the hippocampus for example, anxious or chronically stressed animals tend to show epigenetic changes that are at the opposite of those observed after memory consolidation following a brief aversive stimulus. Behaviorally relevant epigenetic changes have been found to be reversible by drug treatments. Surprisingly, moderate alcohol consumption may trigger, on the long term, changes of BDNF expression and of its epigenetic elements that are somehow similar to those produced by antidepressant drugs. However, alcohol withdrawal associated with anxiety symptoms has not yet been very well explored. Overall, it appears that a multidisciplinary view on the epigenetics of neurotrophin secretion might bring innovative treatments to psychiatric diseases involving stress and fear memories.

The present study was aimed at analyzing the effects of physical exercise on mitochondrial physiology, anxio-depressive-like behaviors and neuroplasticity in mice. Adult C57BL/6J male mice were isolated in home cages equipped or not with free-running wheels. After 6weeks of exercise, mice were tested in various behavioral paradigms to evaluate anxiety- and depressive-like behaviors. The hippocampi were dissected for neurochemical assays, including mitochondrial activity, monoamines content and the expression of genes involved in energy metabolism and brain-derived neurotrophic factor (BDNF) regulation. Exercise decreased anxiety-like behaviors in the open field and elevated plus maze, and exerted antidepressant-like effects in the tail suspension test. Exercise stimulated brain mitochondrial activity and increased resistance against rotenone, an inhibitor of complex I activity. Furthermore, mRNA expression of Bdnf, Gdnf, Tfam (mitochondrial transcription factor A), and Ndufa6 (mitochondrial I subunit) genes, as well as the phosphorylation of cAMP response element-binding protein were increased after exercise. In summary, exercise appears to engage mitochondrial pathways and to potentiate neuroplasticity and might be associated to mood improvement.

Evidence from the various sources indicates alterations in 5-HT2C receptor functions in anxiety, depression and suicide, and other stress-related disorders treated with antidepressant drugs. Although the notion of a 5-HT2C receptor desensitization following antidepressant treatments is rather well anchored in the literature, this concept is mainly based on in vitro assays and/or behavioral assays (hypolocomotion, hyperthermia) that have poor relevance to anxio-depressive disorders. Our objective herein is to provide a comprehensive overview of the studies that have assessed the effects of antidepressant drugs on 5-HT2C receptors. Relevant molecular (second messengers, editing), neurochemical (receptor binding and mRNA levels), physiological (5-HT2C receptor-induced hyperthermia and hormone release), behavioral (5-HT2C receptor-induced changes in feeding, anxiety, defense and motor activity) data are summarized and discussed. Setting the record straight about drug-induced changes in 5-HT2C receptor function in specific brain regions should help to determine which pharmacotherapeutic strategy is best for affective and anxiety disorders.

Serotonin (5-HT)2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5-HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5-HT2C receptor-induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5-HT turnover by a 5-HT2C receptor agonist (RO 60-0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA-A or GABA-B receptors in mice. Neither the GABA-B receptor antagonist phaclofen nor the specific genetic ablation of either GABA-B1a or GABA-B1b subunits altered the inhibitory effect of RO 60-0175, although 5-HT turnover was markedly decreased in GABA-B1a knock-out mice in both basal and stress conditions. In contrast, the 5-HT2C receptor-mediated inhibition of 5-HT turnover was reduced by the GABA-A receptor antagonist bicuculline. However, a significant effect of 5-HT2C receptor activation persisted in mutant mice deficient in the α3 subunit of GABA-A receptors. It can be inferred that non-α3 subunit-containing GABA-A receptors, but not GABA-B receptors, mediate the 5-HT2C -induced inhibition of stress-induced increase in hippocampal 5-HT turnover in mice.

The loss of nigral dopaminergic neurons in Parkinson's disease (PD) is believed to result from interactions between genetic susceptibility and environmental factors. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, the hybrid 129Sv-C57BL/6 parkin-deficient mice did not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to neurotoxicity induced by 6-hydroxydopamine (6-OHDA) or intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. We aimed to re-evaluate the role of parkin in a pure C57BL/6 background after an acute intranasal (i.n.) MPTP administration, a new route of toxin delivery to the brain that mimics environmental exposure to neurotoxins. We found that the deficiency of parkin gene modifies the D-amphetamine-induced locomotion in saline-treated animals. Intranasal MPTP induced Parkinsonism in parkin⁺/⁺ mice, through depletion of striatal dopamine, decreased number of dopaminergic neurons in the substantia nigra, and decreased D-amphetamine-induced hyperlocomotion. Additionally, the deletion of the parkin gene in a pure C57BL/6 background did not lead to increased vulnerability to i.n. MPTP-induced neurotoxicity. Moreover, the i.n. MPTP induced nigral astrogliosis predominantly in the pars reticulata in wild type and parkin⁻/⁻ mice. Taken together, these results showed that the absence of parkin did not modify the vulnerability of nigrostriatal dopaminergic pathway after i.n. MPTP intoxication, suggesting that independently of mouse strain, the endogenous parkin is not required for protection of this system. These findings also suggest that the development of familial parkin-linked PD is not associated with exposure to environmental factors that specifically affects the dopaminergic system.

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson's disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

Stress is known to activate the central 5-hydroxytryptamine (5-HT) system, and this is probably part of a coping response involving several 5-HT receptors. Although 5-HT(2C) receptors are well known to be implicated in anxiety, their participation in stress-induced changes had not been investigated in parallel at both behavioral and neurochemical levels. We show here that the preferential 5-HT(2C) receptor agonist, m-chlorophenylpiperazine, as well as restraint stress increased anxiety in the mouse social interaction test. The selective 5-HT(2C) receptor antagonist, SB 242,084, prevented both of these anxiogenic effects. Restraint stress increased 5-HT turnover in various brain areas, and this effect was prevented by the 5-HT(2B/2C) receptor agonist RO 60-0175 (1 mg/kg), but not the preferential 5-HT(2A) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (1 mg/kg), and in contrast potentiated by SB 242,084 (1 mg/kg), which also blocked the effect of RO 60-0175. Using microdialysis, RO 60-0175 was shown to inhibit cortical 5-HT overflow in stressed mice when 5-HT reuptake was blocked locally. Chronic paroxetine prevented both the anxiogenic effect of m-chlorophenylpiperazine and the inhibitory effect of RO 60-0175 on locomotion and stress-induced increase in 5-HT turnover. The anxiolytic action of chronic paroxetine might be associated with an enhancement of 5-HT neurotransmission caused by a decreased 5-HT(2C) receptor-mediated inhibition of stress-induced increase in 5-HT release.

Many studies have shown that deficits in olfactory and cognitive functions precede the classical motor symptoms seen in Parkinson's disease (PD) and that olfactory testing may contribute to the early diagnosis of this disorder. Although the primary cause of PD is still unknown, epidemiological studies have revealed that its incidence is increased in consequence of exposure to certain environmental toxins. In this study, most of the impairments presented by C57BL/6 mice infused with a single intranasal (i.n.) administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (1 mg/nostril) were similar to those observed during the early phase of PD, when a moderate loss of nigral dopamine neurons results in olfactory and memory deficits with no major motor impairments. Such infusion decreased the levels of the enzyme tyrosine hydroxylase in the olfactory bulb, striatum, and substantia nigra by means of apoptotic mechanisms, reducing dopamine concentration in different brain structures such as olfactory bulb, striatum, and prefrontal cortex, but not in the hippocampus. These findings reinforce the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD. These results also provide new insights in experimental models of PD, indicating that the i.n. administration of MPTP represents a valuable mouse model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

The effects on latent inhibition (LI; a delay in conditioning when a CS has been pre-exposed without consequences) of repeated restraint stress and the anxiolytic drug diazepam were examined in C57BL/6 mice to know whether previous aversive events or anxiolysis are factors determining the expression of LI. The LI model was optimized for this strain particularly sensitive to stress (using both the CER and the conditioned freezing procedures) and characterized with typical (haloperidol) and atypical (clozapine and olanzapine) antipsychotic drugs administered either during the conditioning or the pre-exposure phases. An acute challenge with amphetamine, a dopamine releaser, was done to verify the enhancement of hyperactivity in C57BL/6 mice after the restraint stress sensitization. At all doses tested, diazepam decreased latent inhibition when administered during the pre-exposure phase (similarly to atypical antipsychotic drugs). Repeated restraint stress enhanced LI by blocking the CS-induced freezing in pre-exposed mice. In contrast, pre-treatment with diazepam before pre-exposure allowed the expression of CS-induced freezing in stressed mice pre-exposed to the tone. It is suggested that stress and anxiolytic drugs can have opposite effects on attention or perseveration processes during learning of conflicting contingency responses.

Neopterin is found at increased levels in biological fluids from individuals with inflammatory disorders. The biological role of this pteridine remains undefined; however, due to its capacity to increase hemeoxygenase-1 content, it has been proposed as a protective agent during cellular stress. Therefore, we investigated the effects of neopterin on motor, emotional and memory functions. To address this question, neopterin (0.4 and/or 4 pmol) was injected intracerebroventricularly before or after the training sessions of step-down inhibitory avoidance and fear conditioning tasks, respectively. Memory-related behaviors were assessed in Swiss and C57BL/6 mice, as well as in Wistar rats. Moreover, the putative effects of neopterin on motor and anxiety-related parameters were addressed in the open field and elevated plus-maze tasks. The effects of neopterin on cognitive performance were also investigated after intraperitoneal lipopolysaccharide (LPS) administration (0.33 mg/kg) in interleukin-10 knockout mice (IL-10-/-). It was consistently observed across rodent species that neopterin facilitated aversive memory acquisition by increasing the latency to step-down in the inhibitory avoidance task. This effect was related to a reduced threshold to generate the hippocampal long-term potentiation (LTP) process, and reduced IL-6 brain levels after the LPS challenge. However, neopterin administration after acquisition did not alter the consolidation of fear memories, neither motor nor anxiety-related parameters. Altogether, neopterin facilitated cognitive processes, probably by inducing an antioxidant/anti-inflammatory state, and by facilitating LTP generation. To our knowledge, this is the first evidence showing the cognitive enhancer property of neopterin. Fil: Ghisoni, Karina. Universidade Federal de Santa Catarina; Brasil Fil: Aguiar, Aderbal S.. Universidade Federal de Santa Catarina; Brasil Fil: Oliveira, Paulo Alexandre de. Universidade Federal de Santa Catarina; Brasil Fil: Matheus, Filipe Carvalho. Universidade Federal de Santa Catarina; Brasil Fil: Gabach, Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; Argentina Fil: Perez, Mariela Fernanda. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; Argentina Fil: Carlini, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Barbeito, Luis. Instituto Pasteur de Montevideo; Uruguay Fil: Mongeau, Raymond. Université Paris Descartes. INSERM UMR. Centre de Psychiatrie et Neurosciences; Francia Fil: Lanfumey, Laurence. Umr S894, Centre de Psychiatrie Et Neurosciences; Francia Fil: Prediger, Rui Daniel. Universidade Federal de Santa Catarina; Brasil Fil: Latini, Alexandra. Universidade Federal de Santa Catarina; Brasil

BACKGROUND: Desensitization and blockade of 5-HT2C receptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. METHODS: Mice lacking the 5-HT reuptake carrier (5-HTT(-/-)) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR-induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos-induced expression) levels. RESULTS: Although 5-HTT(-/-) mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR-mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR-mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT(-/-) mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT(-/-) mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR-like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT(-/-) mutants. CONCLUSIONS: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT(-/-) mice. This work was supported by grants from INSERM, UPMC, the European Community (FP7, “Devanx” consortium, F2-2007–201714), and ANR (Sercedit 2008–2010, contract ANR-06-Neuro-045-01). Dr CBP Martin was recipient of a fellowship from the French Ministère de la Recherche during performance of this work. Dr Trigo was supported by Fundacion Alicia Koplowitz. Dr Fink (DA07287, DA034488) and Dr Cunningham (DA024157, DA020087) were supported by the National Institute on Drug Abuse grants

Higher cognitive functions such as attention have been difficult to model in genetically tractable organisms. In humans, attention-distracting stimuli interfere with trace but not delay conditioning, two forms of associative learning. Attention has also been correlated with activation of anterior cingulate cortex (ACC), but its functional significance is unclear. Here we show that a visual distractor interferes selectively with trace but not delay auditory fear conditioning in mice. Trace conditioning is associated with increased neuronal activity in ACC, as assayed by relative levels of c-fos expression, and is selectively impaired by lesions of this structure. The effects of the ACC lesions are unlikely to be caused by indirect impairment of the hippocampus, which is required for mnemonic aspects of trace conditioning. These data suggest that trace conditioning may be useful for studying neural substrates of attention in mice, and implicate the ACC as one such substrate.

Small focal injections of manganese ion (Mn^(2+)) deep within the mouse central nervous system combined with in vivo high-resolution MRI delineate neuronal tracts originating from the site of injection. Previous work has shown that Mn^(2+) can be taken up through voltage-gated Ca^(2+) channels, transported along axons, and across synapses. Moreover, Mn^(2+) is a paramagnetic MRI contrast agent, causing positive contrast enhancement in tissues where it has accumulated. These combined properties allow for its use as an effective MRI detectable neuronal tract tracer. Injections of low concentrations of MnCl_2 into either the striatum or amygdala produced significant contrast enhancement along the known neuronal circuitry. The observed enhancement pattern is different at each injection site and enhancement of the homotopic areas was observed in both cases. Ten days postinjection, the Mn^(2+) had washed out, as evidenced by the absence of positive contrast enhancement within the brain. This methodology allows imaging of neuronal tracts long after the injection of the ion because Mn^(2+) concentrates in active neurons and resides for extended periods of time. With appropriate controls, differentiation of subsets of neuronal pathways associated with behavioral and pharmacological paradigms should be feasible.

Administration of cholecystokinin-tetrapeptide (CCK-4) triggers panic attacks in humans, but it is not known whether CCK-4 acts in the brain to produce this effect. Panic-like reactions (flight and tachycardia) induced in rats by injecting D, L-homocysteic acid (DLH) into the dorsal periaqueductal grey area (DPAG), were used as an animal model to investigate this issue. CCK-4 (2 micrograms) infused into the DPAG did not change these panic-like reactions. The DLH-induced tachycardia was prolonged by intracerebroventricular injection of CCK-4 (40 or 4 micrograms); however, the DLH-induced flight behavior was not changed by similar central injections of CCK-4 (40, 4, or 0.4 micrograms). Peripheral injection of t-butoxycarbonyl (BOC)-CCK-4 (40 micrograms) potentiated the flight behavior, but did not alter the tachycardia response. It was concluded that CCK tetrapeptide potentiates panic-like behaviors by acting on a peripheral target or on a circumventricular area of the brain. In contrast, increased brain CCK-4 prolongs tachycardia by acting in the brain at a level distinct from the DPAG.

Exercise improves the central nervous system (CNS) functions and is widely recommended for neurological patients with, e.g., Alzheimer’s and Parkinson’s disease (PD). However, exercise-induced neuroprotection is an open discussion. Here, the intranasal administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 65 mg/kg) caused death of dopaminergic neurons in the substantia nigra pars compacta and depletion of dopamine in the striatum of C57BL/6 mice. 1-Methyl-4-phenylpyridinium, the active metabolite of MPTP, also inhibited complex-I activity of mitochondria isolated from the CNS of mice. However, 6 weeks of exercise on voluntary running wheels did not protect against nigrostriatal neurodegeneration or mitochondrial inhibition, suggesting that benefits of exercise for PD may not be associated with neuroprotection. The literature presents other candidates, such as neurotrophins or increased antioxidant defenses.

Affective disorders such as major depression, bipolar disorders and seasonal affective disorders have been described as alterations of various neuronal systems. In addition to the classical monoaminergic hypotheses that have been long proposed to explain the pathophysiology of these disorders, a strong association between circadian rhythms and mood regulation has been suggested in the light of several clinical and preclinical findings. In this review, we summarize the different hypotheses on pathophysiology mechanisms underlying depressive disorders and put a special emphasis on the alterations of melatonin secretion and associated changes in biological rhythms that characterize mood disorders. Causal relationships between alterations in circadian rhythms and mood disorders are strongly supported by the antidepressant efficacy of innovative pharmacological treatments aimed at resynchronizing endogenous rhythms in depressed patients. Genetic, epigenetic and environmental factors generating desynchronization between endogenous biological rhythms and exogenous rhythms driven by environmental and societal constraints are very probably involved in the vulnerability to mood disorders. Further investigations of the molecular/cellular bases of the relationships between stress axis dysfunctions, endogenous biological rhythm dysregulations and associated functional and anatomical brain alterations should allow important progress in the knowledge of pathophysiological mechanisms of affective disorders and the downstream development of innovative, more effective and better tolerated, therapies.

Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

International audience; The vesicular monoamine transporter type 2 gene (VMAT2) plays a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of VMAT2 under the control of the serotonin transporter (slc6a4) promoter. VMAT2sert-cre mice showed a major (-95%) depletion of 5-HT levels in the brain with no major alterations of the other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2sert-cre mice but developed normal innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT1A autoreceptor coupling to G protein, as assessed with agonist stimulated [35S]GTP-γ-S binding, was observed in the raphe area, indicating an adaptive change to the reduced 5-HT transmission. Behavioral evaluation in adult VMAT2sert-cre mice showed an increase of escape-like reactions in response to tail suspension, and anxiolytic-like response in the novelty suppressed feeding test. In an aversive ultrasound-induced defense paradigm, VMAT2sert-cre mice displayed a major increase of escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, albeit in reduced amount, in synaptosomes from VMAT2sert-cre mice brain. These findings are coherent with the notion that 5-HT plays an important role in anxiety, and provide new insights on the role of endogenous 5-HT in defense behaviors.

There is evidence for altered 5-HT2C receptor function in anxiodepressive disorders and many antide-pressant drugs have a primary action at these receptors. Although the notion of 5-HT2C receptor desensitization following prolonged treatment with drugs that increase the synaptic availability of 5-HT is well anchored in the literature, this concept is based mainly on in vitro assays and/or behavioral assays that have little relevance to depression. Our objective herein is to provide a comprehensive overview of the studies that have assessed the effects of antidepressant treatments on 5-HT2C receptors. Relevant neurochemical (receptor binding and mRNA levels), physiological (5-HT2C-receptor-induced hyperthermia and hormone release) and behavioral (5-HT2C-receptor-inducedchanges in feeding, anxiety, defense and motor activity) data are summarized and discussed. Our extensive analysis of the literature clearly evidences a crucial lack of studies investigating changes in 5-HT2C receptor function in selected brain areas following antidepressant treatments, mainly because adequate tools are lacking. Although the notion of an overall desensitization of 5-HT2C receptors by antidepressant drugs has been challenged, the hypothesis appears nevertheless well supported by clear-cut data in selected brain areas. Setting the record straight about drug-induced changes in 5-HT2C receptor function should help in determining which pharmacotherapeutic strategy is the best in anxiety and depression.

Valproate may exert its effects by modulating signalling pathways controlling gene expression as they are known to alter both CREB and ERK pathways in the rat hippocampus and frontal cortex. The action of valproate on these signalling pathways has not been studied yet in limbic areas such as the nucleus accumbens and the amygdala which are central for the regulation of emotional behaviors. To this aim, the effect of valproate on phosphorylated CREB (p-CREB) and ERK (p-ERK) in the amygdala and nucleus accumbens, by using immunohistochemical and Western blot analysis, was investigated. The immunohistochemistry was followed by a stereological quantification of the number of immunoreactive cells. Acute valproate (80 mg/kg, i.p.) increased the density of p-CREB-positive cells and enhanced p-CREB, but not p-ERK, protein levels in the amygdala and the accumbens. In contrast, following chronic valproate (80 mg/kg/day for 4 weeks) p-CREB and p-ERK protein levels were markedly attenuated in the amygdala, while the number of p-CREB immunoreactive cells was increased in the accumbens. These data suggest that valproate exert differential effects depending on the brain region examined, the duration and the dose of treatment. The increasing effect of chronic valproate on p-CREB levels in the accumbens is consistent with previous studies in the cortex and the hippocampus, while the decrease of amygdalar p-CREB levels might be specific to mood stabilizers compared to antidepressant drugs, and might be linked to the anti-manic action of valproate.

The effect of two alpha-adrenergic receptor antagonists widely employed in the therapy of benign prostatic hyperplasia, tamsulosin [(-)-(R)-5-[2-[[2-(0-ethoxyphenoxy) ethyl]amino]propyl]-2-methoxybenzenesulfonamide] and alfuzosin [(+/-)-N-[3-[(4-amino-6,7-dimethoxy-2-quinazolinyl) methylamino]propyl] tetrahydro-2-furancarboxamide], was investigated in the rat vas deferens. Because several clinical studies have shown that tamsulosin causes ejaculatory disorders, this study also evaluated the possible mechanisms implicated in these disorders by comparing the effect of tamsulosin with that of alfuzosin. Tamsulosin competitively antagonized the contractions induced by noradrenaline in vitro in the epididymal portion of the vas deferens with a potency pA(2) value of 9.2 +/- 0.8. In the prostatic portion, tamsulosin increased the amplitude of intermittent spikes induced by exogenous noradrenaline (100-1000 microM). In both portions of the vas deferens, alfuzosin behaved as an alpha-adrenergic antagonist blocking the contractions induced by exogenous noradrenaline without altering spikes. The administration of tamsulosin (3 microg/kg i.v.) significantly reduced the contractions evoked by electrical pulses in the epididymal portion, whereas it increased those produced in the prostatic portion. Intravenous tamsulosin antagonized the contraction produced by exogenous noradrenaline, whereas alfuzosin administration (10 microg/kg i.v.) did not change the electrically induced contractions in both portions of the rat vas deferens and did not antagonize the contractions produced by exogenous noradrenaline. The fact that tamsulosin unusually enhances noradrenaline-induced intermittent spike contractions and nerve stimulation-induced twitches in the prostatic portions might be linked to its greater propensity to cause sexual dysfunctions.

Several converging lines of evidence indicate that drugs of abuse may exert their long-term effects on the central nervous system by modulating signaling pathways controlling gene expression. Cannabinoids produce, beside locomotor effects, cognitive impairment through central CB1 cannabinoid receptors. Data clearly indicate that the cerebellum, an area enriched with CB1 receptors, has a role not only in motor function but also in cognition. This immunohistochemical study examines the effect of delta9-tetrahydrocannabinol (delta9-THC), the principal psychoactive component of marijuana, on the levels of phosphorylated CREB (p-CREB) in the rat cerebellum. Acute treatments with delta9-THC at doses of 5 or 10 mg/kg induced a significant increase of p-CREB in the granule cell layer of the cerebellum, an effect blocked by the CB1 receptor antagonist SR 141716A. Following chronic delta9-THC administration (10 mg/kg/day for 4 weeks), the density of p-CREB was markedly attenuated compared to controls, and this attenuation persisted 3 weeks after withdrawal from delta9-THC. These data provide evidence for the involvement of cerebellar granule cells in the adaptive changes occurring during acute and chronic delta9-THC exposure. This might be a mechanism by which delta9-THC interferes with motor and cognitive functions.

Quetiapine is a novel atypical antipsychotic drug with multi-receptorial affinity. Using in vivo microdialysis, we investigated if quetiapine modulates extracellular noradrenaline and dopamine in brain areas generally believed to be involved in the pathophysiology of schizophrenia and in the action of antipsychotic drugs. Quetiapine (5, 10 and 20 mg/kg, i.p.) increased levels of noradrenaline in both the prefrontal cortex and the caudate nucleus, while it increased dopamine levels mainly in the prefrontal cortex. It is argued that the marked increase of dopaminergic transmission in the prefrontal cortex induced by quetiapine might be relevant to its therapeutical action.

International audience; Stress-induced alterations in neuronal plasticity and in hippocampal functions have been suggested to be involved in the development of mood disorders. In this context, we investigated in the hippocampus the activation of intracellular signaling cascades, the expression of epigenetic markers and plasticity-related genes in a mouse model of stress-induced hyperactivity and of mixed affective disorders. We also determined whether the antidepressant drug agomelatine, a MT1/MT2 melatonergic receptor agonist/5-HT2C receptor antagonist, could prevent some neurobiological and behavioral alterations produced by stress. C57BL/6J mice, exposed for 3 weeks to daily unpredictable socio-environmental stressors of mild intensity, were treated during the whole procedure with agomelatine (50 mg kg−1 per day, intraperitoneal). Stressed mice displayed robust increases in emotional arousal, vigilance and motor activity, together with a reward deficit and a reduction in anxiety-like behavior. Neurobiological investigations showed an increased phosphorylation of intracellular signaling proteins, including Atf1, Creb and p38, in the hippocampus of stressed mice. Decreased hippocampal level of the repressive epigenetic marks HDAC2 and H3K9me2, as well as increased level of the permissive mark H3K9/14ac suggested that chronic mild stress was associated with increased gene transcription, and clear-cut evidence was further indicated by changes in neuroplasticity-related genes, including Arc, Bcl2, Bdnf, Gdnf, Igf1 and Neurod1. Together with other findings, the present data suggest that chronic ultra-mild stress can model the hyperactivity or psychomotor agitation, as well as the mixed affective behaviors often observed during the manic state of bipolar disorder patients. Interestingly, agomelatine could normalize both the behavioral and the molecular alterations induced by stress, providing further insights into the mechanism of action of this new generation antidepressant drug.

Purpose of the study: Traumatic brain injury (TBI) results in white matter injury (WMI), evidenced by demyelination. WMI may originate from neuroinflammation and is associated with late emotional and cognitive disorders in patients. Experimental research focuses on brain lesions and neurological deficits occurring days after injury, occulting long-term consequences of TBI. The present study aims to evaluate the time-course of neuroinflammation, WMI and behavioral disorders related to anxiety, compulsiveness, depression, behavioral flexibility and spatial memory after TBI. Methods used: Three groups of male Swiss mice were studied: non-operated, sham-operated and traumatic brain injured mice. TBI was induced by a controlled cortical impact. Neuroinflammation was determined by microglia activation using CD11b immunostaining. The expression of myelin basic protein (MBP), a marker of myelination, was measured in the corpus callosum using Western blot (for all biomarkers evaluations n = 5−6/group). At 15 days, 1, 2 and 3 months, sensorimotor performances were determined with pole test and spontaneous locomotion with actimeter. Spatial learning and memory, and behavioral flexibility were assessed using Barnes maze: two weeks after TBI, mice were trained to find a target hole 3 trials/day during 4 days, followed by a probe test (i.e. in absence of target box) on the last day. At 1, 2 and 3 months, mice were trained to find a new target hole location as previously described. Compulsiveand anxiety-like behaviors using the marble-burying, the open field and the elevated plus maze tests and immobility/escape behavior in the forced swim test, more relevant to depression, were assessed 3 months postTBI (for all behavioral measurements: n = 9−12/group). Data were analyzed with one-way ANOVA. Results: Number of CD11b+ cells was increased from 24 h (P< 0.001) up to 7 days post-TBI (P< 0.001), showing neuroinflammation. MBP expression was decreased at 7 days and 3 months (P< 0.01) in ipsiand contralateral corpus callosum demonstrating acute and long-term bilateral WMI. Spontaneous locomotor activity, motor coordination performances, anxiety-like and despair-like behavioral measurements were undisturbed by TBI. Although, spatial learning performances were not disturbed at 2 weeks post-TBI, deficit was apparent when moving the target hole location 1, 2 and 3 months after TBI. While all mice discriminated the target zone correctly during the probe tests, latencies to find the new target hole, distances travelled and number of errors, were all significantly higher in injured mice during the first training day (P< 0.05), suggesting that TBI induces an early deficit in learning processes, behavioral adaptability and flexibility disorder. Conclusions: These data clearly demonstrate that TBI induces acute microglial activation that precedes acute and long term demyelination, suggesting the involvement of neuroinflammation in WMI. Moreover, the latter is associated with a reduction of flexibility and a spatial learning deficit, in the absence of sensorimotor and emotional states impairments, suggesting that changes in the escape behavior to the target hole, were related to behavioral flexibility rather than defense behavior. Behavioral evaluation is still ongoing until 12 months after TBI. This model will provide an overview of various late behavioral disorders and demyelination that could help to validate pharmacological strategies.

Objectives5-HT2C receptors are well known to be involved in anxiety, but their implication in stress-induced changes of 5-HT transmission remained to be investigated. We thus assess the behavioral and neurochemical effects of 5-HT2C receptor activation in naïve and stressed mice, and after chronic paroxetine known to exert anxiolytic effects in humans.Methods and resultsThe effects of the preferential 5-HT2C agonists m-chlorophenylpiperazine (mCPP) and RO60-0175, the selective 5-HT2C receptor antagonist SB242,084 and restraint-stress on anxiety-like behavior in mice were assessed using the social interaction test, while the neurochemical effects of these treatments on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT were determined using HPLC and microdialysis. Both mCPP and restraint-stress increased anxiety-like behavior in the social interaction test, and these effects were blocked by pretreatment with SB242,084. Restraint-stress increased 5-HT turnover in various brain areas, and this effect could be prevented by the 5-HT2C receptor agonist RO60-0175. Acute administration of SB242,084 potentiated the stress-induced increase in 5-HT turnover and blocked the inhibitory effect of RO60-0175. Microdialysis studies in frontal cortex revealed that RO60-0175 has an inhibitory effect on the stress-induced increase in extracellular 5-HT levels, but not on basal 5-HT levels. Chronic paroxetine prevented the anxiogenic effect of mCPP and prevented the inhibitory effect of RO60-0175 on restraint-stress-induced increase in 5-HT turnover.ConclusionsThese data strongly suggest that 5-HT2C receptor activation mediates the anxiogenic effect of stress. In addition, the anxiolytic action of long term treatment with SSRIs might be causally related to a clear-cut 5-HT2C receptor desensitization.

[3H]U69,593 and [3H]ethylketazocine (μ + δ suppressed) binding was measured in homogenates of guinea-pig brain. Both ligands bind with high affinity to a single class of opioid sites. The relative equilibrium dissociation constant (KD) for [3H]U69,593 is 1.15 nM, while [3H]ethylketazocine has a KD value of 0.33 nM. Their respective maximum binding capacities are 4.49 and 4.48 pmol/g of wet tissue. Various μ-selective, δ-selective, κ-selective, and nonselective opioids were tested in competition studies against the binding of [3H]U69,593 or [3H]ethylketazocine (in the presence of μ- and δ-blockers) to measure their relative affinity. [D-Ala2, MePhe4,Gly5-ol]enkephalin (μ-selective) has low affinity (600–3000 nM) and [D-Pen2,D-Pen5]enkephalin and [D-Ser2, Leu5, Thr6]enkephalin (δ-selective) have very low affinities (> 20 000 nM) at the sites labelled with [3H]U69,593 or [3H]ethylketazocine. On the other hand, unlabelled U69,593, U50,488H, and tifluadom (all three κ-selective substances) display high affinity (1–5 nM) at those sites. Nonselective opioids, such as bremazocine, levorphanol, and ethylketazocine show similar affinities at the sites labelled with [3H]U69,593 and at the sites labelled with [3H]ethylketazocine. These data indicate that [3H]U69,593 is a selective high-affinity ligand for the same sites that are labelled with [3H]ethylketazocine (in the presence of μ- and δ-blockers) and that these are κ-sites.