Orexin A injection into the ventral medial prefrontal cortex induces antidepressant-like effects: Possible involvement of local Orexin-1 and Trk receptors.

RATIONALE: Reduced levels of orexin-A (OXA) in the central nervous system (CNS) have been associated with the pathophysiology of depression and its exogenous administration promotes antidepressant-like effect. The mechanisms associated with these effects are, however, not yet known. Herein, we investigated the hypothesis that OXA effects could be associated with orexin 1 receptor (OX1R) and tyrosine receptor kinase B (TrkB) activation, in the ventromedial prefrontal cortex (vmPFC), a brain region that is central to depression neurobiology. OBJECTIVES: 1. To Investigate the effects induced by OXA administration into the vmPFC; 2. Evaluate the participation of OX1R and TrkB in behavioral responses induced by OXA. METHODS: Male Wistar rats received intra-vmPFC injections of OXA (10, 50 and 100 pmol/0.2 µL) and were exposed to the forced swimming test (FST) or the open field test (OFT). Independent groups received an intra-vmPFC injection of SB334867 (OX1R antagonist, 10 nmol/0.2 µL) or K252a (non-selective Trk antagonist, 10 pmol/0.2 µL), before local injection of OXA, and were exposed to the same tests. RESULTS: OXA injection (100 pmol/0.2 µL) into the vmPFC induced antidepressant-like effect, which was prevented by SB334867 and K252a pretreatments. CONCLUSION: OXA signaling in the vmPFC induces antidepressant-like effect in the FST which is dependent on OX1R and Trk receptors.

New evidence for refinement of anesthetic choice in procedures preceding the forced swimming test and the elevated plus-maze.

Previous studies indicated that some general anesthetics induce long-term antidepressant and/or anxiolytic-like effects. This raises the concern about the use of anesthesia in surgeries that precede psychopharmacological tests, since it may be a potential bias on results depending on the experimental design used. Thus, we evaluated whether general anesthetics used in surgeries preceding psychopharmacological tests would affect rats behavior in tests predictive of antidepressant or anxiolytic-like effects. We tested if a single exposure to sub-anesthetic or anesthetic doses of tribromoethanol, chloral hydrate, thiopental or isoflurane would change rats behavior in the forced swimming test (FST) or in the elevated plus-maze (EPM) test, at 2 h or 7 days after their administration. We also evaluated whether prior anesthesia would interfere in the detection of the antidepressant-like effect of imipramine or the anxiolytic-like effect of diazepam. Previous anesthesia with the aforementioned anesthetics did not change rats behaviors in FST per se nor it changed the antidepressant-like effect induced by imipramine treatment. Rats previously anesthetized with tribromoethanol or chloral hydrate exhibited, respectively, anxiogenic-like and anxiolytic-like behaviors in the EPM. Prior anesthesia with thiopental or isoflurane did not produce any per se effect in rats behaviors in the EPM nor disturbed the anxiolytic-like effect of diazepam. Our results suggest that, in our experimental conditions, tribromoethanol and chloral hydrate are improper anesthetics for surgeries that precede behavioral analysis in the EPM. Isoflurane or thiopental may be suitable for anesthesia before evaluation in the EPM or in the FST.

Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype: Putative Role for BDNF Metabolism in Prefrontal Cortex.

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

Mice lacking interleukin-18 gene display behavioral changes in animal models of psychiatric disorders: Possible involvement of immunological mechanisms.

Preclinical and clinical evidence suggests pro-inflammatory cytokines might play an important role in the neurobiology of schizophrenia and stress-related psychiatric disorders. Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines and it is widely expressed in brain regions involved in emotional regulation. Since IL-18 involvement in the neurobiology of mental illnesses, including schizophrenia, remains unknown, this work aimed at investigating the behavior of IL-18 null mice (KO) in different preclinical models: 1. the prepulse inhibition test (PPI), which provides an operational measure of sensorimotor gating and schizophrenic-like phenotypes; 2. amphetamine-induced hyperlocomotion, a model predictive of antipsychotic activity; 3. resident-intruder test, a model predictive of aggressive behavior. Furthermore, the animals were submitted to models used to assess depressive- and anxiety-like behavior. IL-18KO mice showed impaired baseline PPI response, which was attenuated by d-amphetamine at a dose that did not modify PPI response in wild-type (WT) mice, suggesting a hypodopaminergic prefrontal cortex function in those mice. d-Amphetamine, however, induced hyperlocomotion in IL-18KO mice compared to their WT counterparts, suggesting hyperdopaminergic activity in the midbrain. Moreover, IL-18KO mice presented increased basal levels of IL-1ß levels in the hippocampus and TNF-α in the prefrontal cortex, suggesting an overcompensation of IL-18 absence by increased levels of other proinflammatory cytokines. Although no alteration was observed in the forced swimming or in the elevated plus maze tests in naïve IL-18KO mice, these mice presented anxiogenic-like behavior after exposure to repeated forced swimming stress. In conclusion, deletion of the IL-18 gene resembled features similar to symptoms observed in schizophrenia (positive and cognitive symptoms, aggressive behavior), in addition to increased susceptibility to stress. The IL-18KO model, therefore, could provide new insights into how changes in brain immunological homeostasis induce behavioral changes related to psychiatric disorders, such as schizophrenia.

Involvement of CB1 and TRPV1 receptors located in the ventral medial prefrontal cortex in the modulation of stress coping behavior.

Cannabinoid type-1 (CB1) and transient receptor potential vanilloid type-1 (TRPV1) receptors may have opposite roles in modulating neural activity and, consequently, in regulating the stress response. These receptors are widely expressed in several brain structures, including the ventral medial prefrontal cortex (vmPFC). The functional consequences of the interaction between CB1 and TRPV1, however, have scarcely been explored. Therefore, we investigated if CB1 and TRPV1 receptors located in the vmPFC would be involved in the behavioral changes induced by the stress of the forced swim test (FST). Rats with cannulae implanted into the vmPFC were given the dual blocker of TRPV1 receptors and fatty acid amide hydrolase (FAAH), Arachidonyl serotonin (AA-5HT, 0.125/0.25/0.5nmol), TRPV1 antagonist, SB366791 (0.5/1/10nmol), FAAH inhibitor, URB597 (0.001/0.01/0.1/1nmol), or vehicle and were submitted to the FST, or to the open-field test. Another group received intra-vmPFC injection of SB366791 or vehicle, followed by a second injection of URB597 or vehicle, and was submitted to the FST. Lastly, a group received intra-vmPFC injection of a CB1 antagonist, in sub-effective dose or vehicle, followed by AA-5HT, SB366791 or vehicle. The results showed that AA-5HT, SB366791 and URB597 significantly reduced the immobility time without changing the locomotor activity. Furthermore, the co-administration of URB597 and SB366791 in sub-effective doses induced an antidepressant-like effect in the FST. Additionally, the antidepressant-like effect of AA-5HT was prevented by the CB1 antagonist. Together, these results suggest that both, CB1 and TRPV1 receptors located in the vmPFC are involved in the behavioral responses to stress, although in opposite ways.

Antidepressant-like effect of cannabidiol injection into the ventral medial prefrontal cortex-Possible involvement of 5-HT1A and CB1 receptors.

RATIONALE: Systemic administration of cannabidiol (CBD), the main non-psychotomimetic constituent of Cannabis sativa, induces antidepressant-like effects. The mechanism of action of CBD is thought to involve the activation of 5-HT1A receptors and the modulation of endocannabinoid levels with subsequent CB1 activation. The brain regions involved in CBD-induced antidepressant-like effects remain unknown. The ventral medial prefrontal cortex (vmPFC), which includes the infralimbic (IL) and prelimbic (PL) subregions, receives dense serotonergic innervation and plays a significant role in stress responses. OBJECTIVE: To test the hypothesis that the administration of CBD into the IL or PL would induce an antidepressant-like effect through 5-HT1A and CB1 activation. METHODS: Rats received intra-IL or -PL microinjections of CBD (10-60 nmol/side), 8-OH-DPAT (5-HT1A agonist, 5-10 nmol/side), anandamide (AEA, 0.5 pmol/side) or vehicle (0.2 µl/side) and were submitted to the forced swimming (FST) or to the open field (OFT) tests. Independent CBD-treated groups were pre-treated with WAY100635 (10, 30 nmol/side, 5-HT1A antagonist) or AM251 (10 pmol/side, CB1 antagonist) and submitted to the same tests. An additional group was treated with WAY100635 followed by anandamide. RESULTS: CBD (PL: 10-60 nmol; IL:45-60 nmol) and 8-OH-DPAT (10 nmol) administration significantly reduced the immobility time in the FST, without changing locomotor activity in the OFT. WAY100635 (30 nmol) did not induce effect per se but blocked CBD, 8-OH-DPAT and AEA effects. Additionally, AM251 blocked CBD-effects. CONCLUSION: administration of CBD into the vmPFC induces antidepressant-like effects possibly through indirect activation of CB1 and 5-HT1A receptors.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats.

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats' brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery.

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D2) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for 'in-situ' hybridisation study using (35)S-labeled oligonucleotide probe complementary to D2 receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D2 receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D2 receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Involvement of medial prefrontal cortex neurons in behavioral and cardiovascular responses to contextual fear conditioning.

To explore the ventral medial prefrontal cortex (vMPFC) involvement in behavioral and autonomic fear-conditioned responses to context, vMPFC synaptic transmission was temporarily inhibited by bilateral microinjections of 200 nL of the nonselective synapse blocker CoCl(2) (1 mM). Behavioral activity (freezing, motor activity and rearing) as well as evoked cardiovascular responses (arterial pressure and heart rate) was analyzed. Rats were pre-exposed to the footshock chamber (context) and shock stimulus was used unconditioned stimulus. During re-exposure to context, conditioned rats spent 80% of the session in freezing while non-conditioned rats (no shock group) spent less than 15% of the session time in freezing. Conditioned rats had significantly lower activity scores than non-conditioned animals. Exposure to context increased mean arterial pressure (MAP) and heart rate (HR) of both groups. MAP and HR of the conditioned animals were markedly increased and remained at a high and stable level, whereas MAP and HR increases in non-conditioned animals were less pronounced and declined during the session. CoCl(2) microinjected in the vMPFC significantly reduced freezing and attenuated MAP and HR increase of the conditioned group. Cobalt-induced vMPFC inhibition also significantly reduced MAP and HR increase observed in non-conditioned animals, without any behavioral changes. The effect of vMPFC acute ablation on MAP and HR did not seem to be specific to the fear response because they were also evident in non-conditioned animals. The results indicate that vMPFC integrity is crucial for expression of fear-conditioned responses to context, such as freezing and cardiovascular changes, suggesting that fear-conditioned responses to context involve cortical processing prior to amygdalar output. They also indicate a cardiovascular response observed during re-exposure of non-conditioned rats to the context is completely dependent on vMPFC integrity.