Cannabidiol induces systemic analgesia through activation of the PI3Kγ/nNOS/NO/KATP signaling pathway in neuropathic mice. A KATP channel S-nitrosylation-dependent mechanism.

BACKGROUND: Cannabidiol (CBD) is the second most abundant pharmacologically active component present in Cannabis sp. Unlike Δ-9-tetrahydrocannabinol (THC), it has no psychotomimetic effects and has recently received significant interest from the scientific community due to its potential to treat anxiety and epilepsy. CBD has excellent anti-inflammatory potential and can be used to treat some types of inflammatory and neuropathic pain. In this context, the present study aimed to evaluate the analgesic mechanism of cannabidiol administered systemically for the treatment of neuropathic pain and determine the endogenous mechanisms involved with this analgesia. METHODS: Neuropathic pain was induced by sciatic nerve constriction surgery, and the nociceptive threshold was measured using the paw compression test in mice. RESULTS: CBD produced dose-dependent antinociception after intraperitoneal injection. Selective inhibition of PI3Kγ dose-dependently reversed CBD-induced antinociception. Selective inhibition of nNOS enzymes reversed the antinociception induced by CBD, while selective inhibition of iNOS and eNOS did not alter this antinociception. However, the inhibition of cGMP production by guanylyl cyclase did not alter CBD-mediated antinociception, but selective blockade of ATP-sensitive K+ channels dose-dependently reversed CBD-induced antinociception. Inhibition of S-nitrosylation dose-dependently and completely reversed CBD-mediated antinociception. CONCLUSION: Cannabidiol has an antinociceptive effect when administered systemically and this effect is mediated by the activation of PI3Kγ as well as by nitric oxide and subsequent direct S-nitrosylation of KATP channels on peripheral nociceptors.

The Reversal of Empathy-Induced Hypernociception in Male Mice by Intra-Amygdala Administration of Midazolam and Cannabidiol Depends on 5-HT3 Receptors.

Introduction: Empathy is a fundamental prosocial behavior. It has been defined as perception, awareness, and understanding of others' emotional states, including painful processes. Mice living in pairs with conspecific chronic suffering from constriction injury exhibit pain hypersensitivity mediated by the amygdaloid complex. Nevertheless, the underlying mechanisms in the amygdala responsible for this response remain to be determined. This study investigated if the anxiolytic benzodiazepine midazolam (MDZ) and cannabidiol (CBD), a phytocannabinoid with multiple molecular targets, would attenuate this behavioral change. We also investigated if serotonergic and γ-aminobutyric acid (GABA)ergic mechanisms in the amygdala are involved in this effect. Materials and Methods: Male Swiss mice were housed in pairs for 28 days. The pairs were divided into two groups on the 14th day: cagemate nerve constriction and cagemate sham. On the 24th day, cagemates underwent a stereotaxic surgery and, on the 28th day, were evaluated on the writhing test. Results: The results showed that living with chronic pain leads to hypernociception in the cagemate and increases the expression of 5-HT3 receptor (5-HT3R) and glutamic acid decarboxylase 67 within the amygdala. MDZ (3.0 and 30 nmol) and CBD (30 and 60 nmol) attenuated the hypernociceptive behavior. The 5-HT3R antagonist ondansetron (0.3 nmol) prevented the antinociceptive effects of MDZ and CBD. Conclusion: These findings indicate that 5-HT3R and GABAergic mechanisms within the amygdala are involved in the pain hypersensitivity induced by the empathy for pain model. They also suggest that MDZ and CBD could be a new potential therapy to alleviate emotional pain disorders.

Oficina para elaboração de testes de múltipla escolha de ciências básicas aplicadas: relato de experiência / Workshop on item writing to assess basic sciences in health professional education: an experience report

Resumo: Introdução: A avaliação do estudante é componente essencial de todo programa educacional. O aprendizado das ciências básicas é fundamental para dar sentido ao que se aprende na fase clínica da formação de um profissional em saúde. Entretanto, a maioria dos treinamentos de elaboradores de testes de múltipla escolha (TME) é voltada à formulação de questões clínicas e não inclui abordagem específica para questões das ciências básicas. Relato de experiência: Foi realizada uma oficina para a capacitação docente na elaboração de TME de aplicação dos conhecimentos de ciências básicas, visando à elaboração de uma prova a ser aplicada no final do ciclo básico de seis cursos da saúde. O material instrucional foi elaborado pelos autores, que ofereceram uma oficina no formato on-line. Um diferencial dessa capacitação foi a aplicação de modelos de elaboração de enunciados com contextos definidos, utilizando momentos de preparo assíncronos e encontro síncrono. Após a oficina, aplicaram-se questionários sobre a satisfação e aprendizagem dos participantes. A maioria avaliou a oficina como boa ou muito boa e referiu aumento da percepção de capacidade para elaborar TME, e, ao final, somente 7% se sentiram pouco preparados para elaborar um TME seguindo as boas práticas. Houve melhora na qualidade dos TME elaborados, tendo como referencial os índices de dificuldade e discriminação. Discussão: Existem evidências do valor do desenvolvimento do corpo docente na melhoria da qualidade das questões produzidas. O formato de oficina proposto foi bem avaliado pelos participantes e contribuiu para a qualidade das questões de provas aplicadas ao final do ciclo básico. Conclusão: Estratégias como a descrita qualificam as avaliações dentro da escola e contribuem para a organização de provas externas.

Abstract: Introduction: Student assessment is an essential component of all educational programs. Basic science learning is essential for making clinical knowledge meaningful to healthcare students. However, most item writer training is focused on the formulation of clinical questions and does not include a specific approach to basic science questions. Experience Report: Workshops on item writing for knowledge application on basic sciences were carried out with the aim of planning a test to be applied at the end of the basic cycle of six health courses. The instructional material was prepared by the authors, who offered online workshops. A differential of this training was the application of models of item lead-in elaboration with defined contexts, using moments of asynchronous preparation and synchronous encounter. After each workshop, surveys were applied to assess participants' satisfaction and learning. Most participants rated the workshop as good or very good and reported an increase in their perceived ability to prepare single best answer multiple-choice questions. At the end, only 7% reported they were not prepared to write an item following good practices. There was an improvement in the quality of the items prepared, using the difficulty and discrimination indexes as a reference. Discussion: There is evidence of the value of faculty development in improving the quality of the questions produced. The proposed workshop format was well evaluated by the participants and contributed to the quality of tests applied to students at the end of the basic science cycle. Conclusion: Strategies such this qualify assessments within the school and contribute to the organization of external exams.

Increased blood neutrophil extracellular traps (NETs) associated with early life stress: translational findings in recent-onset schizophrenia and rodent model.

Higher levels of interleukin (IL)-6 and elevated neutrophil counts are consistently reported in the blood of patients with schizophrenia. Stressors during childhood and/or adolescence are major socioenvironmental risk factors for schizophrenia and may contribute to immune dysregulation. Previous studies using blood cytokines to stratify patients with schizophrenia suggest that only a subset presents a low-grade inflammatory state. However, these studies have not addressed whether environmental factors such as childhood maltreatment contributed to identifying inflammatory clusters. Moreover, a neutrophil-related mechanism (Neutrophil Extracellular Traps; NETs) central to both the initiation and chronicity of autoimmune and inflammatory diseases has never been investigated in psychiatry. Elevated NETs in schizophrenia may predispose patients to inflammatory and autoimmune diseases resulting in reduced life expectancy. We, therefore, investigated NETs as a novel mechanism and biological target in early schizophrenia and their role together with IL-6 and childhood maltreatment in identifying cluster subgroups. We found increased NETs in the plasma of patients with early schizophrenia (n = 78) compared to both their unaffected siblings (n = 25) and community controls (n = 78), irrespective of sex, body mass index, psychoactive drug use, or tobacco smoking. Increased NETs in patients were unrelated to antipsychotic treatment, which was further tested in vitro using fresh neutrophils. By applying unsupervised two-step clustering analysis, we integrated values of NETs, IL-6, and childhood maltreatment scores. We identified two main clusters; childhood maltreatment scores and NETs were the most important variables contributing to cluster separation (high-CL1 and low-CL2), while IL-6 was the least contributor. Patients allocated in the high-CL1 (61.5%) had significantly higher childhood maltreatment scores, NETs, and IL-6 levels than the remaining groups (patients low-CL2, siblings, and controls high-CL1 and low-CL2). We complemented these findings with a rat model based on stress exposure during adolescence that results in several schizophrenia-like changes in adulthood. We found that adolescent stressed rats had higher NETs and IL-6 levels in serum compared to non-stressed rats with a tendency to produce more NETs from the bone marrow. Altogether, this study brings a novel cellular-based mechanism in schizophrenia that, combined with early-stress, could be useful to identify subgroups for more personalised treatments.

The anxiolytic effect of cannabidiol depends on the nature of the trauma when patients with post-traumatic stress disorder recall their trigger event

Objectives: We assessed whether administering cannabidiol (CBD) before recalling the traumatic event that triggered their disorder attenuates anxiety in patients with post-traumatic stress disorder (PTSD). As an exploratory pilot analysis, we also investigated whether this effect depends on the nature of the event (sexual vs. nonsexual trauma). Methods: Thirty-three patients of both sexes with PTSD were recruited and randomized 1:1 into two groups. One group received oral CBD (300 mg), and the other received a placebo before listening to a digital audio playback of their previously recorded report of the trigger event. Subjective and physiological measurements were taken before and after recall. We analyzed the data in two subsamples: trigger events involving sexual and nonsexual trauma. Results: In the nonsexual trauma group, the differences between measurements before and after recall were significantly smaller with CBD than placebo; this held true for anxiety and cognitive impairment. However, in the sexual trauma group, the differences were non-significant for both measurements. Conclusion: A single dose of CBD (300mg) attenuated the increased anxiety and cognitive impairment induced by recalling a traumatic event in patients with PTSD when the event involved nonsexual trauma.

The Cannabidiol Analog PECS-101 Prevents Chemotherapy-Induced Neuropathic Pain via PPARγ Receptors.

Chemotherapy-induced peripheral neuropathy (CIPN) is the main dose-limiting adverse effect of chemotherapy drugs such as paclitaxel (PTX). PTX causes marked molecular and cellular damage, mainly in the peripheral nervous system, including sensory neurons in the dorsal root ganglia (DRG). Several studies have shown the therapeutic potential of cannabinoids, including cannabidiol (CBD), the major non-psychotomimetic compound found in the Cannabis plant, to treat peripheral neuropathies. Here, we investigated the efficacy of PECS-101 (former HUF-101), a CBD fluorinated analog, on PTX-induced neuropathic pain in mice. PECS-101, administered after the end of treatment with PTX, did not reverse mechanical allodynia. However, PECS-101 (1 mg/kg) administered along with PTX treatment caused a long-lasting relief of the mechanical and cold allodynia. These effects were blocked by a PPARγ, but not CB1 and CB2 receptor antagonists. Notably, the effects of PECS-101 on the relief of PTX-induced mechanical and cold allodynia were not found in macrophage-specific PPARγ-deficient mice. PECS-101 also decreased PTX-induced increase in Tnf, Il6, and Aif1 (Iba-1) gene expression in the DRGs and the loss of intra-epidermal nerve fibers. PECS-101 did not alter motor coordination, produce tolerance, or show abuse potential. In addition, PECS-101 did not interfere with the chemotherapeutic effects of PTX. Thus, PECS-101, a new fluorinated CBD analog, could represent a novel therapeutic alternative to prevent mechanical and cold allodynia induced by PTX potentially through the activation of PPARγ in macrophages.

Cannabidiol Confers Neuroprotection in Rats in a Model of Transient Global Cerebral Ischemia: Impact of Hippocampal Synaptic Neuroplasticity.

Evidence for the clinical use of neuroprotective drugs for the treatment of cerebral ischemia (CI) is still greatly limited. Spatial/temporal disorientation and cognitive dysfunction are among the most prominent long-term sequelae of CI. Cannabidiol (CBD) is a non-psychotomimetic constituent of Cannabis sativa that exerts neuroprotective effects against experimental CI. The present study investigated possible neuroprotective mechanisms of action of CBD on spatial memory impairments that are caused by transient global cerebral ischemia (TGCI) in rats. Hippocampal synaptic plasticity is a fundamental mechanism of learning and memory. Thus, we also evaluated the impact of CBD on neuroplastic changes in the hippocampus after TGCI. Wistar rats were trained to learn an eight-arm aversive radial maze (AvRM) task and underwent either sham or TGCI surgery. The animals received vehicle or 10 mg/kg CBD (i.p.) 30 min before surgery, 3 h after surgery, and then once daily for 14 days. On days 7 and 14, we performed a retention memory test. Another group of rats that received the same pharmacological treatment was tested in the object location test (OLT). Brains were removed and processed to assess neuronal degeneration, synaptic protein levels, and dendritic remodeling in the hippocampus. Cannabidiol treatment attenuated ischemia-induced memory deficits. In rats that were subjected to TGCI, CBD attenuated hippocampal CA1 neurodegeneration and increased brain-derived neurotrophic factor levels. Additionally, CBD protected neurons against the deleterious effects of TGCI on dendritic spine number and the length of dendritic arborization. These results suggest that the neuroprotective effects of CBD against TGCI-induced memory impairments involve changes in synaptic plasticity in the hippocampus.

Um curso de história da música para estudantes de medicina? / "A course on music history for medical students?"

RESUMO: A participação em atividades lúdicas e reflexivas poderia aumentar a resiliência dos estudantes ao estresse associado ao curso de medicina. Nesse sentido, a música poderia facilitar o desenvolvimento de sentimentos e atitudes, como inclusão social, equilíbrio, atenção, imaginação e reflexão essenciais à prática clínica. Neste artigo relatamos nossa experiência com a disciplina optativa "Introdução à história da música erudita ocidental". Ela é oferecida anualmente aos estudantes de medicina desde 2015. As avaliações anônimas recebidas por 62,5% dos 64 estudantes que frequentaram a disciplina até o momento evidenciaram uma elevada satisfação (100% de avaliações caracterizadas como boa ou ótima) e o reconhecimento de sua importância para a formação médica (95% de avaliações caracterizadas como boa ou ótima). Estes resultados são encorajadores para a continuidade da disciplina e a introdução de atividades optativas similares no currículo da nossa Faculdade. (AU)

ABSTRACT: Participation in playful and reflective activities could increase the students' resilience to the stress associated with the medical course. In this sense, music could facilitate the development of feelings and attitudes, such as social inclusion, balance, attention, imagination, and reflection, essential for clinical practice. In this article, I report my experience with the optional discipline "Introduction to the history of Western classical music." This course has been offered annually to medical students since 2015. The anonymous evaluations received by 62.5% of the 64 students who have attended the discipline so far showed a high satisfaction rate (100% of the assessments marked as good or excellent) and the recognition of its importance for medical training (95% of evaluations marked as good or excellent). These results encourage the continuation of the discipline and the introduction of similar activities into our curriculum.

Cannabidiol on 5-FU-induced oral mucositis in mice.

PURPOSE: The aim of this study was to evaluate the clinical, histological, hematological, and oxidative stress effects of cannabidiol (CBD) in mice with induced oral mucositis. METHODS: We used 90 mice of the CF-1 strain in which oral mucositis was induced using a protocol with 5-fluorouracil (5-FU) chemotherapy. The animals were divided randomly into 10 study groups. Three groups were treated with different doses of CBD (3, 10, and 30 mg/kg), while 2 were control groups (positive control: 5-FU + mechanical trauma + placebo; and negative control: mechanical trauma + placebo), and 2 experimental times were studied (4 and 7 days). All treatments were by intraperitoneal administration. RESULTS: In the clinical evaluation, the groups treated with CBD showed less severity of oral lesions compared with the positive control at both experimental times. The intensity of the inflammatory response was also lower in the groups treated with this drug, but there was no statistically significant difference when compared with the positive control. With regard to erythrocyte, leukocyte, and platelet counts and anti-oxidant enzyme activity, the groups treated with CBD showed better results, but only some of these variables showed statistically significant differences. CONCLUSIONS: CBD seems to exert an anti-inflammatory and anti-oxidant activity favoring a faster resolution of oral mucositis in this animal model.

Cannabidiol attenuates aggressive behavior induced by social isolation in mice: Involvement of 5-HT1A and CB1 receptors.

Long-term single housing increases aggressive behavior in mice, a condition named isolation-induced aggression or territorial aggression, which can be attenuated by anxiolytic, antidepressant, and antipsychotic drugs. Preclinical and clinical findings indicate that cannabidiol (CBD), a non-psychotomimetic compound from Cannabis sativa, has anxiolytic, antidepressant, and antipsychotic properties. Few studies, however, have investigated the effects of CBD on aggressive behaviors. Here, we investigated whether CBD (5, 15, 30, and 60 mg/kg; i.p.) could attenuate social isolation-induced aggressive behavior in the resident-intruder test. Male Swiss mice (7-8 weeks) were single-housed for 10 days (resident mice) to induce aggressive behaviors, while conspecific mice of same sex and age (intruder mice) were group-housed. During the test, the intruder was placed into the resident's home-cage and aggressive behaviors initiated by the resident, including the latency for the first attack, number of attacks, and total duration of aggressive encounters, were recorded. The involvement of 5-HT1A and CB1 receptors (CB1R) in the effects of CBD was also investigated. All tested CBD doses induced anti-aggressive effects, indicated by a decrease in the number of attacks. CBD, at intermediary doses (15 and 30 mg/kg), also increased latency to attack the intruder and decreased the duration of aggressive encounters. No CBD dose interfered with locomotor behavior. CBD anti-aggressive effects were attenuated by the 5-HT1A receptor antagonist WAY100635 (0.3 mg/kg) and the CB1 antagonist AM251 (1 mg/kg), suggesting that CBD decreases social isolation-induced aggressive behaviors through a mechanism associated with the activation of 5-HT1A and CB1 receptors. Also, CBD decreased c-Fos protein expression, a neuronal activity marker, in the lateral periaqueductal gray (lPAG) in social-isolated mice exposed to the resident-intruder test, indicating a potential involvement of this brain region in the drug effects. Taken together, our findings suggest that CBD may be therapeutically useful to treat aggressive behaviors that are usually associated with psychiatric disorders.

Neuronal preservation and reactive gliosis attenuation following neonatal sciatic nerve axotomy by a fluorinated cannabidiol derivative.

Immature peripheral nervous system damage, such as the transection of a peripheral nerve, results in the extensive degeneration of motoneurons and dorsal root ganglia (DRG) sensory neurons, mostly due to apoptotic events. We have previously shown that cannabidiol (CBD), the most abundant non-psychotropic molecule present in the Cannabis sativa plant, exhibits neuroprotective action when administered daily at a dose of 15 mg/kg. This study shows that use of the fluorinated synthetic version of CBD (4'-fluoro-cannabidiol, HUF-101) significantly improves neuronal survival by 2-fold compared to that achieved with traditional CBD at one-third the dose. Furthermore, we show that HUF-101 administration significantly upregulates anti-apoptotic genes and blocks the expression of pro-apoptotic nuclear factors. Two-day-old Wistar rats were subjected to unilateral sectioning of the sciatic nerve and treated daily with HUF-101 (1, 2.5, 5 mg/kg/day, i.p.) or a vehicle solution for five days. The results were evaluated by Nissl staining, immunohistochemistry, and qRT-PCR. Neuronal counting revealed a 47% rescue of spinal motoneurons and a 79% rescue of DRG neurons (HUF-101, 5 mg/kg). Survival was associated with complete depletion of p53 and a 60-fold elevation in BCL2-like 1 gene expression. Additionally, peroxisome proliferator-activated receptor gamma (PPAR-gamma) gene expression was downregulated by 80%. Neuronal preservation was coupled with a high preservation of synaptic coverage and a reduction in astroglial and microglial reactions that were evaluated in nearby spinal motoneurons present in the ventral horn of the lumbar intumescence. Overall, these data strongly indicate that HUF-101 exerts potent neuroprotective effects that are related to anti-apoptotic protection and the reduction of glial reactivity.

Effects of cannabidiol, a Cannabis sativa constituent, on oral wound healing process in rats: Clinical and histological evaluation.

The effects of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa, were assessed on oral wound healing in an in vivo model. Standardized ulcers were induced in 60 Wistar rats using a 5-mm biopsy punch on the midline of the ventral tongue. Animals received intraperitoneal injections of CBD at doses of 0 (control), 5, and 10 mg/kg daily. Animals were weighed daily, and wound healing was clinically and histologically evaluated after 3 and 7 days of treatment. CBD treatment did not influence the wound area of ulcerative lesions at either observation time. Conversely, microscopic findings revealed that at Day 3 postwounding, CBD-treated lesions exhibited significantly lower inflammatory scores than those in the control group. However, this difference was not observed at Day 7. Collectively, these findings indicate that CBD exert an antiinflammatory effect in early phase of wound healing process although it was not sufficient promote clinical improvement of oral traumatic ulcerative lesions.

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders.

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ(9)-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca(2+)) increase, etc.), on CBD behavioural effects.

Cannabidiol-treated rats exhibited higher motor score after cryogenic spinal cord injury.

Cannabidiol (CBD), a non-psychoactive constituent of cannabis, has been reported to induce neuroprotective effects in several experimental models of brain injury. We aimed at investigating whether this drug could also improve locomotor recovery of rats submitted to spinal cord cryoinjury. Rats were distributed into five experimental groups. Animals were submitted to laminectomy in vertebral segment T10 followed or not by application of liquid nitrogen for 5 s into the spinal cord at the same level to cause cryoinjury. The animals received injections of vehicle or CBD (20 mg/kg) immediately before, 3 h after and daily for 6 days after surgery. The Basso, Beattie, and Bresnahan motor evaluation test was used to assess motor function post-lesion one day before surgery and on the first, third, and seventh postoperative days. The extent of injury was evaluated by hematoxylin-eosin histology and FosB expression. Cryogenic lesion of the spinal cord resulted in a significant motor deficit. Cannabidiol-treated rats exhibited a higher Basso, Beattie, and Bresnahan locomotor score at the end of the first week after spinal cord injury: lesion + vehicle, day 1: zero, day 7: four, and lesion + Cannabidiol 20 mg/kg, day 1: zero, day 7: seven. Moreover, at this moment there was a significant reduction in the extent of tissue injury and FosB expression in the ventral horn of the spinal cord. The present study confirmed that application of liquid nitrogen to the spinal cord induces reproducible and quantifiable spinal cord injury associated with locomotor function impairments. Cannabidiol improved locomotor functional recovery and reduced injury extent, suggesting that it could be useful in the treatment of spinal cord lesions.

Neuronal NOS inhibitor and conventional antidepressant drugs attenuate stress-induced fos expression in overlapping brain regions.

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naïve). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats.

RATIONALE: Systemic administration of cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is able to attenuate cardiovascular and behavioral (freezing) changes induced by re-exposure to a context that had been previously paired with footshocks. The brain sites mediating this effect, however, remain unknown. The medial prefrontal cortex (mPFC) has been related to contextual fear conditioning. OBJECTIVES: (1) To verify, using c-Fos immunocytochemistry, if the mPFC is involved in the attenuation of contextual fear induced by systemic administration of CBD; (2) to investigate if direct microinjections of CBD into mPFC regions would also attenuate contextual fear. RESULTS: Confirming previous results systemic administration of CBD (10mg/kg) decreased contextual fear and associated c-Fos expression in the prefrontal cortex (prelimbic and infralimbic regions). The drug also attenuated c-Fos expression in the bed nucleus of the stria terminalis (BNST). Direct CBD (30 nmol) microinjection into the PL prefrontal cortex reduced freezing induced by re-exposure to the aversively conditioned context. In the infralimbic (IL) prefrontal cortex, however, CBD (30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning. This result was confirmed by an additional experiment where the conditioning session was performed under a less aversive protocol. CONCLUSION: These results suggest that the PL prefrontal cortex may be involved in the attenuation of contextual fear induced by systemic injection of CBD. They also support the proposition that the IL and PL play opposite roles in fear conditioning. A possible involvement of the BNST in CBD effects needs to be further investigated.

Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator.

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive-related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra-dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic-like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra-dlPAG injections of the selective nNOS inhibitor, N-propyl-L-arginine (NP), or the glutamate antagonist, AP7 (2-amino-7-phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos-like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat-induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double-labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double-labeled cells). These results suggest that glutamate/NO-mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses.

Antidepressant treatment reduces Fos-like immunoreactivity induced by swim stress in different columns of the periaqueductal gray matter.

Antidepressant treatment attenuates behavioral changes induced by uncontrollable stress. The periaqueductal gray matter (PAG) is proposed to be a brain site involved in the behavioral responses to uncontrollable stress and antidepressant effects. The main goal of the present study was to investigate the effect of antidepressant treatment on the pattern of neural activation of the PAG along its mediolateral and rostrocaudal subregions after a forced swim stress episode. Male Wistar rats were sub-acutely treated with desipramine (a selective noradrenaline re-uptake blocker, three injections of 10 mg/kg in 24 h) or clomipramine (a non-selective serotonin and noradrenaline re-uptake blocker, three injections of 10 mg/kg in 24 h) and submitted to the forced swimming test (FST). Two hours after the test their brain were removed for Fos immunohistochemistry. Fos-like immunoreactivity (FLI) in rostral, intermediate and caudal portions of dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG) PAG were quantified by a computerized system. The FST session increased FLI in most parts of the PAG. Previous treatment with desipramine or clomipramine reduced FLI in all columns of the PAG. FLI in the PAG correlated positively with to the immobility time and negatively with to climbing behavior scored during the test. These results indicate that neurons in the PAG are activated by uncontrollable stress. Moreover, inhibitory action of antidepressants on this activity may be associated with the anti-immobility effects of these drugs in the FST.

Further evidence that anxiety and memory are regionally dissociated within the hippocampus.

The hippocampus has been implicated in the regulation of anxiety and memory processes. Nevertheless, the precise contribution of its ventral (VH) and dorsal (DH) division in these issues still remains a matter of debate. The Trial 1/2 protocol in the elevated plus-maze (EPM) is a suitable approach to assess features associated with anxiety and memory. Information about the spatial environment on initial (Trial 1) exploration leads to a subsequent increase in open-arm avoidance during retesting (Trial 2). The objective of the present study was to investigate whether transient VH or DH deactivation by lidocaine microinfusion would differently interfere with the performance of EPM-naive and EPM-experienced rats. Male Wistar rats were bilaterally-implanted with guide cannulas aimed at the VH or the DH. One-week after surgery, they received vehicle or lidocaine 2.0% in 1.0 microL (0.5 microL per side) at pre-Trial 1, post-Trial 1 or pre-Trial 2. There was an increase in open-arm exploration after the intra-VH lidocaine injection on Trial 1. Intra-DH pre-Trial 2 administration of lidocaine also reduced the open-arm avoidance. No significant changes were observed in enclosed-arm entries, an EPM index of general exploratory activity. The cautious exploration of potentially dangerous environment requires VH functional integrity, suggesting a specific role for this region in modulating anxiety-related behaviors. With regard to the DH, it may be preferentially involved in learning and memory since the acquired response of inhibitory avoidance was no longer observed when lidocaine was injected pre-Trial 2.

Activation of neurons containing the enzyme nitric oxide synthase following exposure to an elevated plus maze.

The elevated plus-maze (EPM) is one of the most used animal models of anxiety. Exposure to the EPM activates brain regions related to anxiety/fear. Systemic or intra-dorsolateral periaqueductal gray (dlPAG) inhibition of nitric oxide synthase (NOS) induces anxiolytic effect in animals submitted to an EPM. Additionally, exposure to an innate fear stimulus, such as a live predator, activates neurons containing NOS in regions related to defensive behavior. Considering these pieces of evidence, the present study investigated if neurons containing NOS localized in regions related to anxiety/fear are also activated after exposure to an EPM. Male Wistar rats were exposed to the EPM for 15 min and 2 h later their brains were removed and processed for c-Fos immunohistochemistry (a marker of neuronal functional activation) and NADPH-diaphorase histochemistry (NADPH-d; used to detect the presence of NOS neurons). Exposure to the EPM significantly increased double-stained cells (c-Fos + NADPHd positive neurons) in the parvocellular paraventricular (pPVN) and lateral (LH) hypothalamic nuclei, dlPAG and dorsal raphe nucleus (DRN), but not in the amygdaloid complex, bed nucleus of stria terminallis, dorsal premammillary nucleus of hypothalamus and inferior colicullus. These results suggest that exposure to an EPM activates NOS containing neurons in brain areas related to fear/anxiety.