Psilocybin does not induce the vulnerability marker HSP70 in neurons susceptible to Olney's lesions.

S-ketamine, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and psilocybin, a 5-hydroxy-tryptamine (serotonin) 2A receptor (5-HT2AR) agonist, are reported as effective rapid-acting antidepressants. Both compounds increase glutamate signalling and evoke cortical hyperexcitation. S-ketamine induces neurotoxicity especially in the retrosplenial cortex (Olney's lesions). Whether psilocybin produces similar neurotoxic effects has so far not been investigated. We performed an immunohistochemical whole-brain mapping for heat shock protein 70 (HSP70) in rats treated with psilocybin, S-ketamine, and MK-801. In contrast to S-ketamine- and MK-801-treated animals, we did not detect any HSP70-positive neurons in retrosplenial cortex of rats treated with psilocybin. Our results suggest that psilocybin might be safer for clinical use compared to S-ketamine regarding neuronal damage.

Evidence-based severity assessment of the forced swim test in the rat.

The forced swim test (FST) is a traditional assay, which has been used for more than 40 years to assess antidepressant effects of novel drug candidates. In recent years, a debate about the test has focused on the assumption that the FST is highly aversive and burdening for the animals because of the earlier anthropomorphic interpretation and designation as a "behavioral despair test". The Directive 2010/63/EU and the German Animal Welfare law require a prospective severity classification of the planned experimental procedures. Still, an objective examination of the animals' burden in this test has not been performed yet. To fill this gap, we conducted an evidence-based severity assessment of the forced swim test in rats according to a 'standard protocol' with a water temperature of 25°C. We examined parameters representing the physiological and the affective state, and natural as well as locomotion-associated behaviors in three separate experiments to reflect as many dimensions as possible of the animal's condition in the test. Hypothermia was the only effect observed in all animals exposed to the FST when using this standard protocol. Additional adverse effects on body weight, food consumption, and fecal corticosterone metabolite concentrations occurred in response to administration of the antidepressant imipramine, which is frequently used as positive control when testing for antidepressant effects of new substances. We conclude that this version of the FST itself is less severe for the animals than assumed, and we suggest a severity classification of 'moderate' because of the acute and short-lasting effects of hypothermia. To refine the FST according to the 3Rs, we encourage confirming the predictive validity in warmer water temperatures to allow the rats to maintain physiological body temperature.

A systematic mapping review of the evolution of the rat Forced Swim Test: Protocols and outcome parameters.

As depression is projected to become the leading mental disease burden globally by 2030, understanding the underlying pathology, as well as screening potential anti-depressants with a higher efficacy, faster onset of action, and/or fewer side-effects is essential. A commonly used test for screening novel antidepressants and studying depression-linked aspects in rodents is the Porsolt Forced Swim Test. The present systematic mappping review gives a comprehensive overview of the evolution and of the most prevalently used set-ups of this test in rats, including the choice of animals (strain, sex, and age), technical aspects of protocol and environment, as well as reported outcome measures. Additionally, we provide an accessible list of all existing publications, to support informed decision-making for procedural and technical aspects of the test, to thereby enhance reproducibility and comparability. This should further contribute to reducing the number of unnecessarily replicated experiments, and consequently, reduce the number of animals used in future.

Region-Specific Enhancement of c-fos Expression by Combined Treatment With NMDA Receptor Agonists and Antagonists With Antidepressant Potential.

Rapastinel, formerly Glyx-13, is a novel positive allosteric modulator of the N-methyl-D-aspartate-receptor (NMDAR) that counteracts psychotomimetic actions of NMDAR antagonists. We set out to evaluate the effect of rapastinel alone or in combination with the global and GluN2B subunit-specific NMDAR antagonists MK-801 and Ro25-6981, respectively, on neuronal activation in relevant regions using c-fos brain mapping. Whereas rapastinel alone did not trigger significant c-fos expression beyond the prelimbic cortex, it strongly increased the c-fos expression induced by MK-801 in hippocampal, cingulate, and retrosplenial areas. Similar results were obtained when rapastinel was replaced by D-cycloserine. Our results reveal new interactions at network level between NMDAR modulators with possible implications regarding their therapeutic effects.

Dopamine Transporter Knockout Rats Show Impaired Wellbeing in a Multimodal Severity Assessment Approach.

In preclinical psychiatry research, animals are central to modeling and understanding biological mechanisms of behavior and psychiatric disorders. We here present the first multimodal severity assessment of a genetically modified rat strain used in psychiatric research, lacking the dopamine transporter (DAT) gene and showing endophenotypes of several dopamine-associated disorders. Absence of the DAT leads to high extracellular dopamine (DA) levels and has been associated with locomotor hyperactivity, compulsive behaviors and stereotypies in the past. The German Animal Welfare Law, which is based on the EU Directive (2010/63/EU), requires a prospective severity assessment for every animal experiment, depending on the extent of the expected degree of pain, suffering, distress or lasting harm that the animals will experience. This should consider all procedures but also the impact of the genotype on the phenotype. Therefore, we examined multiple parameters indicating animal welfare, like burrowing behavior, social interaction, saccharin preference, baseline stress hormone levels and nesting behavior. Additionally, a footprint analysis was performed and home cage activity was analyzed for a more detailed characterization of locomotion. DAT KO rats demonstrated reduced burrowing, social interaction and saccharin preference. We also found pronounced stereotypies and alterations in the gait analysis in DAT KO rats. Moreover, we confirmed the hyperactivity and the impaired sensorimotor gating mechanisms to assure that our rats are exhibiting the correct phenotype. In conclusion, we provide evidence that DAT KO rats show alterations in natural behavior patterns and deduce that the marked stereotypies are a sign for coping difficulties, both indicating a negative influence of the genotype on wellbeing. We suggest to assess further rat models in an objectified severity assessment as previously done in mice to create a relative severity assessment based on scientific evidence. Until then, we propose the classification of homozygous DAT KO rats as "moderate" in accordance with the criteria of the EU directive 2010/63.

Comparative Severity Assessment of Genetic, Stress-Based, and Pharmacological Mouse Models of Depression.

The use of animals in neurosciences is pivotal to gaining insights into complex functions and dysfunctions of behavior. For example, various forms of physical and/or psychological stress are inherent to various animal models for psychiatric disorders, e.g., depression. Regarding animal welfare, it would be mandatory to use models that inflict the least amount of stress necessary to address the underlying scientific question. This study compared the severity of different approaches to induce depression in mice: mutagenesis in GluA1 knockout, immobilization stress, and stress-induction via stress hormone treatment. While genetic alterations potentially represent a lifelong burden, the temporary intervention only affects the animals for a limited time. Therefore, we used home cage-based behavioral and physiological parameters, including nest building, burrowing, body weight, and fecal corticosterone metabolites, to determine the well-being of male and female mice. In addition, we performed an evidence-based estimate of severity using a composite score for relative severity assessment (RELSA) with this data. We found that even though restraint stress and supplementation of corticosterone in the diet both aimed at depression-related precipitating stress effects, the latter affected the well-being much stronger, especially in females. Restraint leads to less noticeable well-being impairments but causes depression-associated anhedonic behavior. Mice of both sexes recovered well from the stress treatment. GluA1 KO and their littermates showed diminished well-being, comparable to the immobilization experiments. However, since this is a lifelong condition, this burden is not reversible and potentially accumulative. In line with the 3Rs (Replacement, Reduction, and Refinement), the process of choosing the most suitable model should ideally include an evidence-based severity assessment to be able to opt for the least severe alternative, which still induces the desired effect. Promoting refinement, in our study, this would be the restraint stress.

Dopamine transporter silencing in the rat: systems-level alterations in striato-cerebellar and prefrontal-midbrain circuits.

Silencing of dopamine transporter (DAT), a main controlling factor of dopaminergic signaling, results in biochemical and behavioral features characteristic for neuropsychiatric diseases with presumed hyperdopaminergia including schizophrenia, attention deficit hyperactivity disorder (ADHD), bipolar disorder, and obsessive-compulsive disorder (OCD). Investigation of DAT silencing thus provides a transdiagnostic approach towards a systems-level understanding of common underlying pathways. Using a high-field multimodal imaging approach and a highly sensitive cryogenic coil, we integrated structural, functional and metabolic investigations in tandem with behavioral assessments on a newly developed preclinical rat model, comparing DAT homozygous knockout (DAT-KO, N = 14), heterozygous knockout (N = 8) and wild-type male rats (N = 14). We identified spatially distributed structural and functional brain alterations encompassing motor, limbic and associative loops that demonstrated strong behavioral relevance and were highly consistent across imaging modalities. DAT-KO rats manifested pronounced volume loss in the dorsal striatum, negatively correlating with cerebellar volume increase. These alterations were associated with hyperlocomotion, repetitive behavior and loss of efficient functional small-world organization. Further, prefrontal and midbrain regions manifested opposite changes in functional connectivity and local network topology. These prefrontal disturbances were corroborated by elevated myo-inositol levels and increased volume. To conclude, our imaging genetics approach provides multimodal evidence for prefrontal-midbrain decoupling and striato-cerebellar neuroplastic compensation as two key features of constitutive DAT blockade, proposing them as transdiagnostic mechanisms of hyperdopaminergia. Thus, our study connects developmental DAT blockade to systems-level brain changes, underlying impaired action inhibition control and resulting in motor hyperactivity and compulsive-like features relevant for ADHD, schizophrenia and OCD.

Cre-Activation in ErbB4-Positive Neurons of Floxed Grin1/NMDA Receptor Mice Is Not Associated With Major Behavioral Impairment.

Extensive evidence suggests a dysfunction of the glutamate NMDA receptor (NMDAR) in schizophrenia, a severe psychiatric disorder with putative early neurodevelopmental origins, but clinical onset mainly during late adolescence. On the other hand, pharmacological models using NMDAR antagonists and the clinical manifestation of anti-NMDAR encephalitis indicate that NMDAR blockade/hypofunction can trigger psychosis also at adult stages, without any early developmental dysfunction. Previous genetic models of NMDAR hypofunction restricted to parvalbumin-positive interneurons indicate the necessity of an early postnatal impairment to trigger schizophrenia-like abnormalities, whereas the cellular substrates of NMDAR-mediated psychosis at adolescent/adult stages are unknown. Neuregulin 1 (NRG1) and its receptor ErbB4 represent schizophrenia-associated susceptibility factors that closely interact with NMDAR. To determine the neuronal populations implicated in "late" NMDAR-driven psychosis, we analyzed the effect of the inducible ablation of NMDARs in ErbB4-expressing cells in mice during late adolescence using a pharmacogenetic approach. Interestingly, the tamoxifen-inducible NMDAR deletion during this late developmental stage did not induce behavioral alterations resembling depression, schizophrenia or anxiety. Our data indicate that post-adolescent NMDAR deletion, even in a wider cell population than parvalbumin-positive interneurons, is also not sufficient to generate behavioral abnormalities resembling psychiatric disorders. Other neuronal substrates that have to be revealed by future studies, may underlie post-adolescent NMDAR-driven psychosis.

Correction: Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

[This corrects the article DOI: 10.1371/journal.pone.0240439.].

Rapastinel alleviates the neurotoxic effect induced by NMDA receptor blockade in the early postnatal mouse brain.

Rapastinel is a novel psychoactive substance that acts as an N-methyl-D-aspartate-receptor (NMDAR) agonist and triggers antidepressant- and antipsychotic-like effects in animal models. However, it is unknown if rapastinel possesses a better side-effect profile than fast-acting glutamatergic antidepressants, like ketamine, which trigger neurotoxicity in the perinatal rodent cortex and protracted schizophrenia-like alterations. Here we found a remarkable neuroprotective effect of rapastinel against apoptosis induced by the NMDAR antagonist MK-801 in comparison to that elicited by clozapine and the mGlu2/3 agonist LY354740. These results suggest the potential therapeutic/prophylactic effect of rapastinel in ameliorating deleterious effects induced by NMDAR blockade during neurodevelopment.

Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

Early life stress compromises brain development and can contribute to the development of mental illnesses. A common animal model used to study different facets of psychiatric disorders is social isolation from early life on. In rats, this isolation can induce long-lasting alterations in molecular expression and in behavior. Since social isolation models severe psychiatric symptoms, it is to be expected that it affects the overall wellbeing of the animals. As also promoted by the 3Rs principle, though, it is pivotal to decrease the burden of laboratory animals by limiting the number of subjects (reduce, replace) and by improving the animals' wellbeing (refine). The aim of this study was therefore to test possible refinement strategies such as resocialization and mere adult social isolation. We examined whether the alternatives still triggered the necessary phenotype while minimizing the stress load on the animals. Interestingly, we did not find reduced wellbeing-associated burrowing performance in isolated rats. The hyperactive phenotype seen in socially isolated animals was observed for rats undergoing the adult-only isolation, but resocializing ameliorated the locomotor abnormality. Isolation strongly affected markers of neuroplasticity in the prefrontal cortex independent of timing: mRNA levels of Arc, Bdnf and the pool of Bdnf transcripts with the 3' long UTR were reduced in all groups. Bdnf splice variant IV expression was reduced in lifelong-isolated animals. Some of these deficits normalized after resocialization; likewise, exon VI Bdnf mRNA levels were reduced only in animals persistently isolated. Conversely, social deprivation did not affect the expression of Gad67 and Pvb, two GABAergic markers, whereas changes occurred in the expression of dopamine d1 and d2 receptors. As adult isolation was sufficient to trigger the hyperactive phenotype and impaired neuroplasticity in the prefrontal cortex, it could be a candidate for a refinement strategy for certain research questions. To fully grade the severity of post-weaning social isolation and the alternatives, adult isolation and resocialization, a more profound and multimodal assessment approach is necessary.

The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific.

Handling is a well-known source of stress to laboratory animals and can affect variability of results and even compromise animal welfare. The conventional tail handling in mice has been shown to induce aversion and anxiety-like behaviour. Recent findings demonstrate that the use of alternative handling techniques, e.g. tunnel handling, can mitigate negative handling-induced effects. Here, we show that technique and frequency of handling influence affective behaviour and stress hormone release of subjects in a sex-dependent manner. While frequent tail handling led to a reduction of wellbeing-associated burrowing and increased despair-like behaviour in male mice, females seemed unaffected. Instead, they displayed a stress response to a low handling frequency, which was not detectable in males. This could suggest that in terms of refinement, the impact in handling could differ between the sexes. Independently from this observation, both sexes preferred to interact with the tunnel. Mice generally explored the tunnel more often than the tail-handling hands of the experimenter and showed more positively rated approaches, e.g. touching or climbing, and at the same time, less defensive burrowing, indicating a strong preference for the tunnel.

Systematic analysis of severity in a widely used cognitive depression model for mice.

Animal models in psychiatric research are indispensable for insights into mechanisms of behaviour and mental disorders. Distress is an important aetiological factor in psychiatric diseases, especially depression, and is often used to mimic the human condition. Modern bioethics requires balancing scientific progress with animal welfare concerns. Therefore, scientifically based severity assessment of procedures is a prerequisite for choosing the least compromising paradigm according to the 3Rs principle. Evidence-based severity assessment in psychiatric animal models is scarce, particularly in depression research. Here, we assessed severity in a cognitive depression model by analysing indicators of stress and well-being, including physiological (body weight and corticosterone metabolite concentrations) and behavioural (nesting and burrowing behaviour) parameters. Additionally, a novel approach for objective individualised severity grading was employed using clustering of voluntary wheel running (VWR) behaviour. Exposure to the paradigm evoked a transient elevation of corticosterone, but neither affected body weight, nesting or burrowing behaviour. However, the performance in VWR was impaired after recurrent stress exposure, and the individual severity level increased, indicating that this method is more sensitive in detecting compromised welfare. Interestingly, the direct comparison to a somatic, chemically induced colitis model indicates less distress in the depression model. Further objective severity assessment studies are needed to classify the severity of psychiatric animal models in order to balance validity and welfare, reduce the stress load and thus promote refinement.

Effects of Soy in Laboratory Rodent Diets on the Basal, Affective, and Cognitive Behavior of C57BL/6 Mice.

Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Neural Mechanisms of Early-Life Social Stress as a Developmental Risk Factor for Severe Psychiatric Disorders.

BACKGROUND: To explore the domain-general risk factor of early-life social stress in mental illness, rearing rodents in persistent postweaning social isolation has been established as a widely used animal model with translational relevance for neurodevelopmental psychiatric disorders such as schizophrenia. Although changes in resting-state brain connectivity are a transdiagnostic key finding in neurodevelopmental diseases, a characterization of imaging correlates elicited by early-life social stress is lacking. METHODS: We performed resting-state functional magnetic resonance imaging of postweaning social isolation rats (N = 23) 9 weeks after isolation. Addressing well-established transdiagnostic connectivity changes of psychiatric disorders, we focused on altered frontal and posterior connectivity using a seed-based approach. Then, we examined changes in regional network architecture and global topology using graph theoretical analysis. RESULTS: Seed-based analyses demonstrated reduced functional connectivity in frontal brain regions and increased functional connectivity in posterior brain regions of postweaning social isolation rats. Graph analyses revealed a shift of the regional architecture, characterized by loss of dominance of frontal regions and emergence of nonfrontal regions, correlating to our behavioral results, and a reduced modularity in isolation-reared rats. CONCLUSIONS: Our result of functional connectivity alterations in the frontal brain supports previous investigations postulating social neural circuits, including prefrontal brain regions, as key pathways for risk for mental disorders arising through social stressors. We extend this knowledge by demonstrating more widespread changes of brain network organization elicited by early-life social stress, namely a shift of hubness and dysmodularity. Our results highly resemble core alterations in neurodevelopmental psychiatric disorders such as schizophrenia, autism, and attention-deficit/hyperactivity disorder in humans.

May the use of different background strains 'strain' the stress-related phenotype of GR+/- mice?

Genetically altered mice are available on different background strains. While respective backcrosses are often performed for pragmatic reasons, e.g. references, comparability, or existing protocols, the interaction between the mutations per se and the background strain often remains a neglected factor. The heterozygous mutation of the glucocorticoid receptor gene (GR) represents a well-examined model for depressive-like behavior in mice. To address the question in how far a robust depressive-like phenotype on a distinct background strain may allow a generalized conclusion, we analyzed respective phenotypes in two commonly used inbred strains: i.) C57BL/6N and ii.) BALB/c. Beside the use of different genetic models, we also extended our approach by applying two alternative paradigms to induce a depressive-like phenotype. Our study therefore comprised the model of 'unpredictable chronic mild stress' (UCMS) for four weeks and 'learned helplessness' (LH), which were used to study the role of GR, a key player in the development of depression. In the course of the experiment two cohorts of male GR+/- mice on either C57BL/6N or BALB/c background strain underwent a behavioral test battery to assess basal and depressive-like features. While both stress paradigms were functional in inducing depressive-like changes, the results were strictly strain-dependent. The genetic consequences became even more obvious under non-stress conditions with significant effects detected in BALB/c mice, which indicates a different basal stress predisposition due to differences in the genetic background.

Puberty marks major changes in the hippocampal and cortical c-Fos activation pattern induced by NMDA receptor antagonists.

Non-selective and subunit (GluN2B)-specific N-methyl-d-aspartate receptor (NMDAR) antagonists represent promising alternative antidepressant drugs with fast onset of the therapeutic action. The neuronal activation pattern induced by NMDAR antagonists is well characterized by c-Fos expression analysis only in the adult rodent brain. In contrast, there is little information available regarding their effects during postnatal development. Here we performed a systematic c-Fos brain mapping of the non-selective NMDAR antagonist MK-801 and the GluN2B-specific antagonist Ro 25-6981 from postnatal day 16 (P16) to P40. We found significant regional differences with gender-specificity in the activation pattern compared to the adult. Surprisingly, in the hippocampus, MK-801 triggered at pre-pubertal stages (especially at P24) very strong c-Fos expression, followed by low levels after P30, the approximate time point of puberty onset in mice. The cortical distribution of MK-801-triggered c-Fos expression before puberty differed also substantially from the adult brain, showing high levels only in deep cortical layers at pre-pubertal stages. In comparison, the cortical activation induced by Ro 25-6981 diminished from high pre-pubertal levels and was in comparison with that triggered by MK-801 low in the hippocampus. These results reveal highly dynamic changes in the c-Fos activation pattern induced by NMDAR antagonists during puberty. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Role of the nitric oxide donor sodium nitroprusside in the antidepressant effect of ketamine in mice.

Ketamine may represent an efficient alternative antidepressant with rapid therapeutic onset; however, the clinical use of ketamine is hampered by psychosis-like side-effects. Recent studies suggest that the nitric oxide (NO) donor sodium nitroprusside (SNP) prevents psychosis-like abnormalities triggered by ketamine or another NMDA receptor (NMDAR) antagonist, phencyclidine (PCP) in rats. SNP was shown to elicit antipsychotic effects also in humans. Considering the tight interrelation between NMDAR activation and neuronal NO synthesis, we evaluated the effect of pre-treatment with SNP on the antidepressant action of ketamine. We found that SNP (0.5-1mg/kg, i.p.) did not alter the antidepressant effect of ketamine (30 mg/kg) in the Porsolt Forced Swim Test (FST) in mice. Additionally, SNP by itself produced no effect in the FST or in the openfield. This suggests indirectly a differential involvement of the nitrinergic system in the antidepressant vs. psychotomimetic effect of ketamine, although an influence of species-specific differences cannot be excluded in this interpretation.

Inducible forebrain-specific ablation of the transcription factor Creb during adulthood induces anxiety but no spatial/contextual learning deficits.

The cyclic AMP (cAMP)-response element binding protein (CREB) is an activity-dependent transcription factor playing a role in synaptic plasticity, learning and memory, and emotional behavior. However, the impact of Creb ablation on rodent behavior is vague as e.g., memory performance of different Creb mutant mice depends on the specific type of mutation per se but additionally on the background and learning protocol differences. Here we present the first targeted ablation of CREB induced during adulthood selectively in principal forebrain neurons in a pure background strain of C57BL/6 mice. All hippocampal principal neurons exhibited lack of CREB expression. Mutant mice showed a severe anxiety phenotype in the openfield and novel object exploration test as well as in the Dark-Light Box Test, but unaltered hippocampus-dependent long-term memory in the Morris water maze and in context dependent fear conditioning. On the molecular level, CREB ablation led to CREM up regulation in the hippocampus and frontal cortex which may at least in part compensate for the loss of CREB. BDNF, a postulated CREB target gene, was down regulated in the frontal lobe but not in the hippocampus; neurogenesis remained unaltered. Our data indicate that in the adult mouse forebrain the late onset of CREB ablation can, in case of memory functionality, be compensated for and is not essential for memory consolidation and retrieval during adulthood. In contrast, the presence of CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior.

Impact of adolescent GluA1 AMPA receptor ablation in forebrain excitatory neurons on behavioural correlates of mood disorders.

Glutamatergic dysfunctions have recently been postulated to play a considerable role in mood disorders. However, molecular mechanisms underlying these effects have been poorly deciphered. Previous work demonstrated the contribution of GluA1-containing AMPA receptors (AMPAR) to a depression-like and anxiety-like phenotype. Here we investigated the effect of temporally and spatially restricted gene manipulation of GluA1 on behavioural correlates of mood disorders in mice. Here we show that tamoxifen-induced GluA1 deletion restricted to forebrain glutamatergic neurons of post-adolescent mice does not induce depression- and anxiety-like changes. This differs from the phenotype of mice with global AMPAR deletion suggesting that for mood regulation AMPAR may be particularly important on inhibitory interneurons or already early in development.