Probing cognitive flexibility in Shank2-deficient mice: Effects of D-cycloserine and NMDAR signaling hub dynamics.

Neurodevelopmental disorders such as autism spectrum disorder (ASD) have a heterogeneous etiology but are largely associated with genetic factors. Robust evidence from recent human genetic studies has linked mutations in the Shank2 gene to idiopathic ASD. Modeling these Shank2 mutations in animal models recapitulates behavioral changes, e.g. impaired social interaction and repetitive behavior of ASD patients. Shank2-deficient mice exhibit NMDA receptor (NMDAR) hypofunction and associated behavioral deficits. Of note, NMDARs are strongly implicated in cognitive flexibility. Their hypofunction, e.g. observed in schizophrenia, or their pharmacological inhibition leads to impaired cognitive flexibility. However, the association between Shank2 mutations and cognitive flexibility is poorly understood. Using Shank2-deficient mice, we explored the role of Shank2 in cognitive flexibility measured by the attentional set shifting task (ASST) and whether ASST performance in Shank2-deficient mice can be modulated by treatment with the partial NMDAR agonist D-cycloserine (DCS). Furthermore, we investigated the effects of Shank2 deficiency, ASST training, and DCS treatment on the expression level of NMDAR signaling hub components in the orbitofrontal cortex (OFC), including NMDAR subunits (GluN2A, GluN2B, GluN2C), phosphoglycerate dehydrogenase and serine racemase. Surprisingly, Shank2 deficiency did not affect ASST performance or alter the expression of the investigated NMDAR signaling hub components. Importantly, however, DCS significantly improved ASST performance, demonstrating that positive NMDAR modulation facilitates cognitive flexibility. Furthermore, DCS increased the expression of GluN2A in the OFC, but not that of other NMDAR signaling hub components. Our findings highlight the potential of DCS as a pharmacological intervention to improve cognitive flexibility impairments downstream of NMDAR modulation and substantiate the key role of NMDAR in cognitive flexibility.

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.

Orexin deficiency modulates the dipsogenic effects of angiotensin II in a sex-dependent manner.

The orexin (hypocretin) neuropeptide system is an important regulator of ingestive behaviors, i.e., it promotes food and water intake. Here, we investigated the role of orexin in drinking induced by the potent dipsogen angiotensin II (ANG II). Specifically, male and female orexin-deficient mice received intracerebroventricular (ICV) injections of ANG II, followed by measuring their water intake within 15 min. We found that lower doses of ANG II (100 ng) significantly stimulated drinking in males but not in females, indicating a general sex-dependent effect that was not affected by orexin deficiency. However, higher doses of ANG II (500 ng) were sufficient to induce drinking in female wild-type mice, while female orexin-deficient mice still did not respond to the dipsogenic properties of ANG II. In conclusion, these results suggest sex-dependent effects in ANG II-induced drinking and further support the sexual dimorphism of orexin system functions.

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.

Cognitive Flexibility in Mice: Effects of Puberty and Role of NMDA Receptor Subunits.

Cognitive flexibility refers to the ability to adapt flexibly to changing circumstances. In laboratory mice, we investigated whether cognitive flexibility is higher in pubertal mice than in adult mice, and whether this difference is related to the expression of distinct NMDA receptor subunits. Using the attentional set shifting task as a measure of cognitive flexibility, we found that cognitive flexibility was increased during puberty. This difference was more pronounced in female pubertal mice. Further, the GluN2A subunit of the NMDA receptor was more expressed during puberty than after puberty. Pharmacological blockade of GluN2A reduced the cognitive flexibility of pubertal mice to adult levels. In adult mice, the expression of GluN2A, GluN2B, and GluN2C in the orbitofrontal cortex correlated positively with performance in the attentional set shifting task, whereas in pubertal mice this was only the case for GluN2C. In conclusion, the present study confirms the observation in humans that cognitive flexibility is higher during puberty than in adulthood. Future studies should investigate whether NMDA receptor subunit-specific agonists are able to rescue deficient cognitive flexibility, and whether they have the potential to be used in human diseases with deficits in cognitive flexibility.

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.

Chronic unilateral inhibition of GABA synthesis in the amygdala increases specificity of conditioned fear in a discriminative fear conditioning paradigm in rats.

Neural activity in the amygdala is critical for fear learning. In anxiety disorder patients, bilateral hyperactivity of the amygdala can be observed. This hyperactivation is often associated with the facilitation of fear learning and/or over-generalization of conditioned fear. In contrast, hypoactivity of the amygdala, e.g. by pharmacological interventions, attenuates or blocks fear learning. To date, little is known about how neural excitability of the amygdala affects specificity or generalization of fear. Therefore, the present study utilized chronic inhibition of GABA synthesis in the amygdala to increase excitability and investigated the effect on the specificity of fear learning. In rats, unilateral cannulas aiming at the amygdala were implanted. The cannulas were connected to subcutaneously implanted osmotic mini pumps that delivered either the GABA synthesis inhibitor L-allylglycine or its inactive enantiomer D-allylglycine. Following one week of chronic GABA synthesis manipulation, the rats were submitted to a discriminative fear conditioning protocol. In addition, anxiety-like behavior in the light-dark box was measured. Our data show that chronic unilateral L-AG infusions into the amygdala improve the specificity of learned fear, support safety learning, and reduce fear generalization and anxiety. This data demonstrates that moderately increased amygdala excitability can be beneficial for the specificity of fear learning and highlights the potential application for therapeutic interventions.

Sex-dependent role of orexin deficiency in feeding behavior and affective state of mice following intermittent access to a Western diet - Implications for binge-like eating behavior.

Binge eating disorder is a debilitating disease characterized by recurrent episodes of excessive food consumption and associated with psychiatric comorbidities. Despite a growing body of research investigating the neurobiological underpinnings of eating disorders, specific treatments are lacking. Given its fundamental role in feeding behaviors, we investigated the role of the orexin (hypocretin) neuropeptide system in binge-like eating and associated phenotypes. Specifically, we submitted female and male orexin-deficient mice to a paradigm of intermittent access (once weekly for 24 h) to a Western diet (WD) to induce binge-like eating. Additionally, we measured their anxiety-like behavior and plasma corticosterone levels. All mice showed binge-like eating in response to the intermittent WD access, but females did so to a greater extent than males. While orexin deficiency did not affect binge-like eating in this paradigm, we found that female orexin-deficient mice generally weighed more, and they expressed increased hypophagia and stress levels compared to wild-type mice following binge-like eating episodes. These detrimental effects of orexin deficiency were marginal or absent in males. Moreover, male wild-type mice expressed post-binge anxiety, but orexin-deficient mice did not. In conclusion, these results extend our knowledge of orexin's role in dysregulated eating and associated negative affective states, and contribute to the growing body of evidence indicating a sexual dimorphism of the orexin system. Considering that many human disorders, and especially eating disorders, have a strong sex bias, our findings further emphasize the importance of testing both female and male subjects.

D-cycloserine rescues scopolamine-induced deficits in cognitive flexibility in rats measured by the attentional set-shifting task.

Cognitive flexibility facilitates adaptions to a changing environment in humans and animals and can be assessed with the attentional set shifting task (ASST). In various learning paradigms for laboratory rodents, the partial NMDA receptor agonist D-cycloserine has been found to have pro-cognitive effects. However, D-cycloserine has not yet been investigated for its effects on cognitive flexibility. The aim of the present study was to determine whether D-cycloserine is able to improve cognitive flexibility measured by the ASST in rats. Rats were first pre-treated with the muscarinic antagonist scopolamine (0.5 mg/kg) before the D-cycloserine administrations (20 mg/kg) to induce deficits in ASST performance. Our findings showed impaired ASST performance after scopolamine administration with significant effects on reversal phases and extra-dimensional shift. D-cycloserine treatment selectively improved the performance in the extra-dimensional shift and the last reversal phase, where scopolamine effects were most pronounced. These findings suggest that D-cycloserine can rescue deficits in cognitive flexibility.

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.

Intracerebroventricular infusion of the selective orexin 1 receptor antagonist SB-334867 impairs cognitive flexibility in a sex-dependent manner.

Orexin neuropeptides are well known for their role in sleep/wake cycle, feeding behavior and motivation-related behaviors. However, their role in cognition is not clearly understood. We recently published that orexin deficiency impairs intra-dimensional set shifting in female homozygous orexin-deficient mice but improves the first reversal phase in male homozygous orexin-deficient mice in the attentional set shifting task (ASST), a well-established rodent test for cognitive flexibility. In the present study, we tested if intracerebroventricular injections of the selective orexin 1 receptor antagonist SB-334867 (4 µg/2 µL) affects cognitive flexibility in the different phases of ASST. We found that SB-334867 injections impaired the first and second reversal phases in female C57BL/6J mice but not in males. In addition, we also showed that at this particular dose of SB-334867, the consumption of the reward that was used in the ASST was not affected in both males and females. Our findings indicate that cognitive flexibility is impaired by orexin 1 receptor antagonism in a sex-dependent manner and reiterates the sexually dimorphic role of orexin in cognitive flexibility.

Let's get wild: A review of free-ranging rat assays as context-enriched supplements to traditional laboratory models.

More than 24,000 rodent studies are published annually, with the vast majority of these studies focused on genetically undiverse animals in highly-controlled laboratory settings. However, findings from the laboratory have become increasingly unreliable for predicting outcomes in field and clinical settings, leading to a perceived crisis in translational research. One cause of this disparity might be that most human societies, in contrast to laboratory rodents, are genetically diverse and live in super-enriched environments. Methods for importing wild rats into the laboratory, and also exporting laboratory-style chambers into natural environments are not well-known outside their respective disciplines. Therefore, we have reviewed the current status of supplements to the laboratory rodent assay. We progress logically from highly-controlled experiments with natural breeding colonies to purely naturalistic approaches with free-ranging rats. We then highlight a number of approaches that allow genetically-diverse wild rats to be utilized in context-enriched paradigms. While considering the benefits and shortcomings of each available approach, we detail protocols for random sampling, remote-sensing, and deployment of laboratory chambers in the field. As supplements to standardized laboratory trials, some of these assays could offer key insights to help unify outcomes between laboratory and field studies. However, we note several outstanding questions that must be addressed such as: the trade-off between control and context, possible reductions in sample size, ramifications for the 'standardization fallacy', and ethical dilemmas of working with wild animals. Given these challenges, further innovation will be required before supplemental assays can be made broadly-accessible and thus, transferrable across disciplines.

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.

BDNF haploinsufficiency induces behavioral endophenotypes of schizophrenia in male mice that are rescued by enriched environment.

Brain-derived neurotrophic factor (BDNF) is implicated in a number of processes that are crucial for healthy functioning of the brain. Schizophrenia is associated with low BDNF levels in the brain and blood, however, not much is known about BDNF's role in the different symptoms of schizophrenia. Here, we used BDNF-haploinsufficient (BDNF+/-) mice to investigate the role of BDNF in different mouse behavioral endophenotypes of schizophrenia. Furthermore, we assessed if an enriched environment can prevent the observed changes. In this study, male mature adult wild-type and BDNF+/- mice were tested in mouse paradigms for cognitive flexibility (attentional set shifting), sensorimotor gating (prepulse inhibition), and associative emotional learning (safety and fear conditioning). Before these tests, half of the mice had a 2-month exposure to an enriched environment, including running wheels. After the tests, BDNF brain levels were quantified. BDNF+/- mice had general deficits in the attentional set-shifting task, increased startle magnitudes, and prepulse inhibition deficits. Contextual fear learning was not affected but safety learning was absent. Enriched environment housing completely prevented the observed behavioral deficits in BDNF+/- mice. Notably, the behavioral performance of the mice was negatively correlated with BDNF protein levels. These novel findings strongly suggest that decreased BDNF levels are associated with several behavioral endophenotypes of schizophrenia. Furthermore, an enriched environment increases BDNF protein to wild-type levels and is thereby able to rescue these behavioral endophenotypes.

Intranasal oxytocin compensates for estrus cycle-specific reduction of conditioned safety memory in rats: Implications for psychiatric disorders.

Stress and anxiety disorder patients frequently fail to benefit from psychotherapies which often consist of inhibitory fear learning paradigms. One option to improve the therapy outcome is medication-enhanced psychotherapy. Research in humans and laboratory rodents has demonstrated that oxytocin (OT) reduces fear and facilitates fear extinction. However, the role of OT in conditioned safety learning, an understudied but highly suitable type of inhibitory fear learning, remains to be investigated. The present study aimed at investigating the effect of intranasal OT on conditioned safety. To test this, Sprague Dawley rats (♂n = 57; ♀n = 72) were safety conditioned. The effects of pre-training or pre-testing intranasal OT on conditioned safety and contextual fear, both measured by the acoustic startle response, and on corticosterone plasma levels were assessed. Furthermore, the involvement of the estrous cycle was analyzed. The present data show that intranasal OT administration before the acquisition or recall sessions enhanced conditioned safety memory in female rats while OT had no effects in male rats. Further analysis of the estrus cycle revealed that vehicle-treated female rats in the metestrus showed reduced safety memory which was compensated by OT-treatment. Moreover, all vehicle-treated rats, regardless of sex, expressed robust contextual fear following conditioning. Intranasal OT-treated rats showed a decrease in contextual fear, along with reduced plasma corticosterone levels. The present data demonstrate that intranasal OT has the capacity to compensate deficits in safety learning, along with a reduction in contextual fear and corticosterone levels. Therefore, add-on treatment with intranasal OT could optimize the therapy of anxiety disorders.

Regulation of CREB Phosphorylation in Nucleus Accumbens after Relief Conditioning.

Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKIIα, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning.