Sex-specific modulation of safety learning in Shank2-deficient mice.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impaired perceptual processing and social communication, intellectual disabilities, and repetitive behaviors. Interestingly, while not a core symptom, anxiety disorders frequently co-occur in individuals with ASD and deficits in safety learning have been described in patients with anxiety-related disorders. Because genetic factors, such as SHANK deficiency (loss-of-function mutations), have been linked to ASD, the aim of the present study was to investigate whether Shank2 deficiency interferes with associative fear and safety signal learning. To first investigate trait anxiety, male and female Shank2-deficient mice were exposed to a light-dark box test. Mice were then submitted to a combination of contextual fear conditioning and single-cue safety conditioning. The results show that Shank2 deficiency increases trait anxiety but reduces contextual fear learning. In male but not female Shank2-deficient mice, reduced single-cued safety learning was observed. This safety learning deficit was not caused by altered anxiety levels, increased locomotor activity, or reduced contextual fear since these changes were also observed in female Shank2-deficient mice. Concluding, our data indicate that the observed safety learning deficits in Shank2-deficient male mice could contribute to the emotional symptoms observed in ASD and the high comorbidity with anxiety-related disorders.

An appeasing pheromone ameliorates fear responses in the brown rat (Rattus norvegicus).

The brown rat (Rattus norvegicus) is one of the major animals both in the laboratory and in urban centers. Brown rats communicate various types of information using pheromones, the chemicals that mediate intra-species communication in minute amounts. Therefore, analyses of pheromones would further our understanding of the mode of life of rats. We show that a minute amount of 2-methylbutyric acid (2-MB) released from the neck region can ameliorate fear responses both in laboratory rats and in wild brown rats. Based on these findings, we conclude that 2-MB is an appeasing pheromone in the brown rat. A better understanding of rats themselves would allow us to perform more effective ecologically based research on social skills and pest management campaigns with low animal welfare impacts, which might contribute to furthering the advancement of science and improving public health.

Nasal administration of orexin A partially rescues dizocilpine-induced cognitive impairments in female C57BL/6 J mice.

Sex difference has been reported in several behavioral endophenotypes of neuropsychiatric disorder in both rodents and humans. However, sex difference in cognitive symptoms associated with neuropsychiatric disorders has not been studied in detail. In this study, we induced cognitive impairment using the NMDA receptor antagonist, dizocilpine (MK-801), in male and female C57BL/6 J mice and performed a visual discrimination task in an automated touchscreen system. We found that discrimination performance decreased with increased doses of MK-801 in both sexes. However, female mice showed stronger deficit in discrimination performance than the male mice especially after administration of low (0.01 mg/kg) and high (0.15 mg/kg) doses of MK-801. Furthermore, we tested if administration of orexin A, orexin-1 receptor antagonist SB-334867 or orexin-2 receptor antagonist EMPA rescued MK-801 (0.15 mg/kg) induced cognitive impairment in visual discrimination. We found that nasal administration of orexin A partially rescued the cognitive impairment induced by MK-801 in females but not in males. Taken together, our data show that female C57BL/6 J mice are more sensitive compared to males to some doses of MK-801 in discrimination learning task and that orexin A partially rescues this cognitive impairment in females.

Anxiolytic-like Effects of the Positive GABAB Receptor Modulator GS39783 Correlate with Mice's Individual Basal Anxiety and Stress Reactivity.

Positive gamma-aminobutyric acid type B (GABAB) receptor modulators such as GS39783 have showed anxiolytic-like effects in several studies while such effects were absent in other studies. These conflicting findings led us hypothesize that the anxiolytic-like effects of such compounds depend on the individual basal anxiety and/or the anxiogenic properties of the used tests. The present study addresses this hypothesis by testing GS39783 effects on mice's anxiety-like behavior in a light-dark box. We found that GS39783 had no effects on a whole-group level. However, after grouping the mice for their basal anxiety, GS39783 reduced anxiety-like behavior in the subgroup with highest basal anxiety. Moreover, GS39783 effects correlated with individual basal anxiety. Next, the anxiogenic properties of the light-dark box test were increased by prior stress exposure. Again, GS39783 was not effective on a whole-group level. However, GS39783 had an anxiolytic-like effect in the most stress-responsive subgroup. Moreover, GS39783 effects correlated with individual stress responsiveness. Finally, we show that GS39783 brain levels were within a behaviorally relevant range. Overall, our study demonstrates that GS39783 effects depend on individual basal anxiety and stress responsiveness. This suggests that anxiety tests should generally be designed to capture individual basal anxiety and/or stress responsiveness as well as individual compound effects.

Orexin deficiency affects sensorimotor gating and its amphetamine-induced impairment.

The orexin neuropeptides have an important role in the regulation of the sleep/wake cycle and foraging, as well as in reward processing and emotions. Furthermore, recent research implicates the orexin system in different behavioral endophenotypes of neuropsychiatric diseases such as social avoidance and cognitive flexibility. Utilizing orexin-deficient mice, the present study tested the hypothesis that orexin is involved in two further mouse behavioral endophenotypes of neuropsychiatric disorders, i.e., sensorimotor gating and amphetamine sensitivity. The data revealed that orexin-deficient mice expressed a deficit in sensorimotor gating, measured by prepulse inhibition of the startle response. Amphetamine treatment impaired prepulse inhibition in wildtype and heterozygous orexin-deficient mice, but had no effects in homozygous orexin-deficient mice. Furthermore, locomotor activity and center time in the open field was not affected by orexin deficiency but was similarly increased or decreased, respectively, by amphetamine treatment in all genotypes. These data indicate that the orexin system modulates prepulse inhibition and is involved in mediating amphetamine's effect on prepulse inhibition. Future studies should investigate whether pharmacological manipulations of the orexin system can be used to treat neuropsychiatric diseases associated with deficits in sensorimotor gating, such as schizophrenia or attention deficit hyperactivity disorder.

Dissociative Effects of Neuropeptide S Receptor Deficiency and Nasal Neuropeptide S Administration on T-Maze Discrimination and Reversal Learning.

Cognitive flexibility refers to the ability to modify learned behavior in response to changes in the environment. In laboratory rodents, cognitive flexibility can be assessed in reversal learning, i.e., the change of contingencies, for example in T-maze discrimination learning. The present study investigated the role of the neuropeptide S (NPS) system in cognitive flexibility. In the first experiment, mice deficient of NPS receptors (NPSR) were tested in T-maze discrimination and reversal learning. In the second experiment, C57BL/6J mice were tested in the T-maze after nasal administration of NPS. Finally, the effect of nasal NPS on locomotor activity was evaluated. NPSR deficiency positively affected the acquisition of T-maze discrimination but had no effects on reversal learning. Nasal NPS administration facilitated reversal learning and supported an allocentric learning strategy without affecting acquisition of the task or locomotor activity. Taken together, the present data show that the NPS system is able to modulate both acquisition of T-maze discrimination and its reversal learning. However, NPSR deficiency only improved discrimination learning, while nasal NPS administration only improved reversal learning, i.e., cognitive flexibility. These effects, which at first glance appear to be contradictory, could be due to the different roles of the NPS system in the brain regions that are important for learning and cognitive flexibility.

Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization.

Rats can acquire fear by observing conspecifics that express fear in the presence of conditioned fear stimuli. This process is called observational fear learning and is based on the social transmission of the demonstrator rat's emotion and the induction of an empathy-like or anxiety state in the observer. The aim of the present study was to investigate the role of trait anxiety and ultrasonic vocalization in observational fear learning. Two experiments with male Wistar rats were performed. In the first experiment, trait anxiety was assessed in a light-dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure, and 22 kHz and 50 kHz calls were analyzed. The results of our study show that trait anxiety differently affects direct fear learning and observational fear learning. Direct fear learning was more pronounced with higher trait anxiety, while observational fear learning was the best with a medium-level of trait anxiety. There were no indications in the present study that ultrasonic vocalization, especially emission of 22 kHz calls, but also 50 kHz calls, are critical for observational fear learning.

Deficiency of the immunoproteasome subunit ß5i/LMP7 supports the anxiogenic effects of mild stress and facilitates cued fear memory in mice.

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.

Rhodiola rosea root extract has antipsychotic-like effects in rodent models of sensorimotor gating.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant arctic root (Rhodiola rosea, L.) is growing in northern regions of Europe, Asia and North America. Extracts of R. rosea are used in traditional medicine for various conditions related to nervous system function. According to scientific studies from the last decades, the plant might have potential for use in the treatment of memory impairments, stress and depression, but reports concerning other neuropsychiatric disorders are scarce. AIM OF THE STUDY: In this context, our study aimed to examine potential antipsychotic-like effects of R. rosea root extract. MATERIALS AND METHODS: We tested the effects of R. rosea root extract on prepulse inhibition in rats and mice. Prepulse inhibition is an established operational measure of sensorimotor gating, which is impaired in schizophrenia and other psychotic disorders. RESULTS: R. rosea root extract increased prepulse inhibition in rats and mice. Interestingly, the R. rosea extract had stronger effects in those individual animals that had low baseline levels of prepulse inhibition. Therefore, we performed further experiments in which we pharmacologically induced a prepulse inhibition deficit by two different psychostimulants, either the dopamine D2 receptor agonist apomorphine or the NMDA receptor antagonist dizocilpine (MK-801). Pre-treatment with the R. rosea extract significantly restored both, apomorphine- and dizocilpine-induced prepulse inhibition deficits. CONCLUSIONS: The present study demonstrates that R. rosea extract robustly reverses prepulse inhibition deficits in rodents. This suggests antipsychotic-like effects of R. rosea extract. Future studies should focus on the pharmacological mechanisms underlying these effects.

Memory generalization after one-trial contextual fear conditioning: Effects of sex and neuropeptide S receptor deficiency.

One-trial contextual fear conditioning in laboratory mice results in a fear memory which is relatively specific to the original conditioning context shortly after conditioning but becomes more unspecific after an incubation time of one month. This process is called generalization of fear memory and is used to investigate processes which might be involved in the pathogenesis of post-traumatic stress disorder. In the present study, we investigated the effects of sex and neuropeptide S receptor (NPSR) deficiency in one-trial contextual fear conditioning. In addition to contextual fear, we also measured startle reactivity, anxiety and corticosterone plasma levels of the mice. Our data show main effects of sex and NPSR-deficiency on freezing behavior, startle magnitude, and anxiety levels. However, generalization of contextual fear memory after incubation time was not affected by sex. Notably, NPSR-deficient mice had a more specific fear memory shortly after conditioning than their wildtype littermates but after incubation time, all genotypes had a generalized fear memory. The present data further show that plasma corticosterone levels are increased after incubation time. This increase was significantly more pronounced in NPSR-deficient mice. Taken together, our study confirms the suitability of one-trial contextual fear conditioning to study the effects of incubation time on fear memory generalization but also indicates the need for control groups without incubation. We further demonstrate that the increase of plasma corticosterone levels after incubation time is exaggerated in NPSR-deficient mice. The latter finding suggests an important role of the NPS system in the regulation of corticosterone release.

The role of trait anxiety in associative learning during and after an aversive event.

Trait anxiety is considered to be a risk factor for anxiety disorders. The aim of the present study was to investigate how trait anxiety affects associative learning during and after an aversive event in laboratory rats. For this, rats were first submitted to a light-dark box test, followed by relief, safety, and fear learning. Our data demonstrate that all types of learning were affected by trait anxiety, both on a group and on an individual level. Whereas high levels of anxiety impaired relief and safety learning, fear learning was more pronounced. These findings help to show how trait anxiety might be involved in the etiology of anxiety disorders and pathological sensation- and risk-seeking.

Role of the mesolimbic dopamine system in relief learning.

The relief from an aversive event is rewarding. Since organisms are able to learn which environmental cues can cease an aversive event, relief learning helps to better cope with future aversive events. Literature data suggest that relief learning is affected in various psychopathological conditions, such as anxiety disorders. Here, we investigated the role of the mesolimbic dopamine system in relief learning. Using a relief learning procedure in Sprague Dawley rats, we applied a combination of behavioral experiments with anatomical tracing, c-Fos immunohistochemistry, and local chemogenetic and pharmacological interventions to broadly characterize the role of the mesolimbic dopamine system. The present study shows that a specific part of the mesolimbic dopamine system, the projection from the posterior medial ventral tegmental area (pmVTA) to the nucleus accumbens shell (AcbSh), is activated by aversive electric stimuli. 6-OHDA lesions of the pmVTA blocked relief learning but fear learning and safety learning were not affected. Chemogenetic silencing of the pmVTA-AcbSh projection using the DREADD approach, as well as intra-AcbSh injections of the dopamine D2/3 receptor antagonist raclopride inhibited relief learning. Taken together, the present data demonstrate that the dopaminergic pmVTA-AcbSh projection is critical for relief learning but not for similar learning phenomena. This novel finding may have clinical implications since the processing of signals predicting relief and safety is often impaired in patients suffering from anxiety disorders. Furthermore, it may help to better understand psychological conditions like non-suicidal self-injury, which are associated with pain offset relief.

Relief learning requires a coincident activation of dopamine D1 and NMDA receptors within the nucleus accumbens.

Relief learning is the association of a stimulus with the offset of an aversive event. Later, the now conditioned relief stimulus induces appetitive-like behavioral changes. We previously demonstrated that the NMDA receptors within the nucleus accumbens (NAC) are involved in relief learning. The NAC is also important for reward learning and it has been shown that reward learning is mediated by an interaction of accumbal dopamine and NMDA glutamate receptors. Since conditioned relief has reward-like properties, we hypothesized that (a) acquisition of relief learning requires the activation of dopamine D1 receptors in the NAC, and (b) if D1 receptors are involved in this process as expected, a concurrent dopamine D1 and NMDA receptor activation may mediate this learning. The present study tested these hypotheses. Therefore, rats received intra-NAC injections of the dopamine D1 receptor antagonist SCH23390 and the NMDA antagonist AP5, either separately or together, at different time points of a relief conditioning procedure. First, we showed that SCH23390 dose-dependently blocked acquisition and the expression of conditioned relief. Next, we demonstrated that co-injections of SCH23390 and AP5 into the NAC, at doses that were ineffective when applied separately, blocked acquisition but not consolidation or expression of relief learning. Notably, neither of the injections affected the locomotor response of the animals to the aversive stimuli suggesting that their perception is not changed. This data indicates that a co-activation of dopamine D1 and NMDA receptors in the NAC is required for acquisition of relief learning.

Metabotropic Glutamate Receptors 7 within the Nucleus Accumbens are Involved in Relief Learning in Rats.

Relief learning is an appetitive association of a formally neutral cue with relief induced by the offset of an aversive stimulus. Since the nucleus accumbens mediates relief learning and accumbal metabotropic glutamate receptors 7 (mGluR7) modulate appetitive-like processes, we hypothesized that accumbal mGluR7 may be involved in the modulation of relief learning. Therefore, we injected the allosteric mGluR7 agonist AMN082 into the nucleus accumbens and tested the effects of these injections on acquisition and expression of relief memory, as well as on the reactivity to electric stimuli. AMN082 injections blocked acquisition but not expression of relief memory. In addition, accumbal AMN082 injections strongly reduced the locomotor reactivity to electric stimuli indicating antinociceptive effects. These antinociceptive effects might be causal for the blockade of relief learning after AMN082 injections. Taken together, the present study indicates that functional activation of accumbal mGluR7 has antinociceptive effects that interfere with relief learning.

Relief memory consolidation requires protein synthesis within the nucleus accumbens.

Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias.

Standardized extract of Ficus platyphylla reverses apomorphine-induced changes in prepulse inhibition and locomotor activity in rats.

Preparations of Ficus platyphylla are used in Nigeria's folk medicine to manage a plethora of diseases including, insomnia, psychoses, depression, epilepsy, pain and inflammation. In this study, we examined the effects of the standardized methanol extract of F. platyphylla stem bark (FP) on apomorphine-induced changes in prepulse inhibition and locomotor activity in rats, as well as on the retrieval of a conditioned reaction in one-way active avoidance in mice. FP did not affect basal prepulse inhibition, but significantly reduced locomotor activity. The apomorphine-induced prepulse inhibition deficit and hyperactivity were significantly reversed by co-administration of clozapine or FP. Furthermore, FP inhibited the retrieval of a conditioned avoidance reaction. Our results revealed that FP contains psychoactive ingredients with neuroleptic-like properties, thus supporting the isolation and development of the biologically active components of this medicinal plant as antipsychotic agents.

Injections of the somatostatin receptor type 2 agonist L-054,264 into the amygdala block expression but not acquisition of conditioned fear in rats.

The amygdala is a crucial brain site for acquisition and expression of conditioned fear. Neuropeptides such as somatostatin play a critical role in regulating the activity of the amygdala. In the present study, the specific somatostatin receptor type 2 (SSTR2) agonist L-054,264 was injected into the amygdala of rats either before fear acquisition or before the expression test on conditioned fear measured by fear-potentiated startle. L-054,264 injections strongly attenuated fear expression but did not affect fear acquisition. This suggests that amygdaloid SSTR2 plays an important role in the modulation of fear memory expression.

Gene expression profile after intense second messenger activation in cortical primary neurones.

Numerous stimuli induce immediate early gene (IEG) expression in neurones, but a comprehensive overview of the late-response genes is lacking. Therefore we aimed to identify changes in the neuronal gene expression profile following intense stimulation. Forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA), direct activators of intracellular second messengers, were applied to primary cultured cortical neurones. The gene expression profiles were analyzed on Affymetrix DNA chips which cover around 8000 rat genes. Out of these, 95 genes (1.2%) were increased at least three-fold, and 43 genes (0.5%) were at least three-fold decreased. The gene chip results were verified by testing 15 of the altered genes by quantitative real-time PCR. The majority of the up-regulated genes were transcription factors, neurotrophic factors or (putative) neuropeptides. Furthermore, there were marked changes in intracellular signal processing enzymes and in postsynaptic structural proteins (e.g. vesl, arc, narp), which have been implicated in synaptic plasticity. Notably, classical players in neurotransmission or plasticity such as glutamate and GABA receptors or voltage-gated ion channels were not increased. It is likely that the increased production of components of intracellular signalling and of postsynaptic proteins is involved in neuronal plasticity.