CACNA1C gene regulates behavioral strategies in operant rule learning.

Behavioral experiments are usually designed to tap into a specific cognitive function, but animals may solve a given task through a variety of different and individual behavioral strategies, some of them not foreseen by the experimenter. Animal learning may therefore be seen more as the process of selecting among, and adapting, potential behavioral policies, rather than mere strengthening of associative links. Calcium influx through high-voltage-gated Ca2+ channels is central to synaptic plasticity, and altered expression of Cav1.2 channels and the CACNA1C gene have been associated with severe learning deficits and psychiatric disorders. Given this, we were interested in how specifically a selective functional ablation of the Cacna1c gene would modulate the learning process. Using a detailed, individual-level analysis of learning on an operant cue discrimination task in terms of behavioral strategies, combined with Bayesian selection among computational models estimated from the empirical data, we show that a Cacna1c knockout does not impair learning in general but has a much more specific effect: the majority of Cacna1c knockout mice still managed to increase reward feedback across trials but did so by adapting an outcome-based strategy, while the majority of matched controls adopted the experimentally intended cue-association rule. Our results thus point to a quite specific role of a single gene in learning and highlight that much more mechanistic insight could be gained by examining response patterns in terms of a larger repertoire of potential behavioral strategies. The results may also have clinical implications for treating psychiatric disorders.

Influence of regional cerebral blood volume on voxel-based morphometry.

The investigation of structural brain alterations is one focus in research of brain diseases like depression. Voxel-based morphometry (VBM) based on high-resolution 3D MRI images is a widely used non-invasive tool for such investigations. However, the result of VBM might be sensitive to local physiological parameters such as regional cerebral blood volume (rCBV) changes. In order to investigate whether rCBV changes may contribute to variation in VBM, we performed analyses in a study with the congenital learned helplessness (cLH) model for long-term findings. The 3D structural and rCBV data were acquired with T2 -weighted rapid acquisition with relaxation enhancement (RARE) pulse sequences. The group effects were determined by standard statistical parametric mapping (SPM) and biological parametric mapping (BPM) and examined further using atlas-based regions. In our genetic animal model of depression, we found co-occurrence of differences in gray matter volume and rCBV, while there was no evidence of significant interaction between both. However, the multimodal analysis showed similar gray matter differences compared with the standard VBM approach. Our data corroborate the idea that two group VBM differences might not be influenced by rCBV differences in genetically different strains. Copyright © 2016 John Wiley & Sons, Ltd.

Daily exposure to a touchscreen-paradigm and associated food restriction evokes an increase in adrenocortical and neural activity in mice.

The translational assessment of mechanisms underlying cognitive functions using touchscreen-based approaches for rodents is growing in popularity. In these paradigms, daily training is usually accompanied by extended food restriction to maintain animals' motivation to respond for rewards. Here, we show a transient elevation in stress hormone levels due to food restriction and touchscreen training, with subsequent adaptation effects, in fecal corticosterone metabolite concentrations, indicating effective coping in response to physical and psychological stressors. Corticosterone concentrations of experienced but training-deprived mice revealed a potential anticipation of task exposure, indicating a possible temporary environmental enrichment-like effect caused by cognitive challenge. Furthermore, the analyses of immediate early gene (IEG) immunoreactivity in the hippocampus revealed alterations in Arc, c-Fos and zif268 expression immediately following training. In addition, BDNF expression was altered as a function of satiation state during food restriction. These findings suggest that standard protocols for touchscreen-based training induce changes in hippocampal neuronal activity related to satiation and learning that should be considered when using this paradigm.

A matter of timing: harm reduction in learned helplessness.

BACKGROUND: Learned helplessness has excellent validity as an animal model for depression, but problems in reproducibility limit its use and the high degree of stress involved in the paradigm raises ethical concerns. We therefore aimed to identify which and how many trials of the learned helplessness paradigm are necessary to distinguish between helpless and non-helpless rats. FINDINGS: A trial-by-trial reanalysis of tests from 163 rats with congenital learned helplessness or congenital non-learned helplessness and comparison of 82 rats exposed to inescapable shock with 38 shock-controls revealed that neither the first test trials, when rats showed unspecific hyperlocomotion, nor trials of the last third of the test, when almost all animals responded quickly to the stressor, contributed to sensitivity and specificity of the test. Considering only trials 3-10 improved the classification of helpless and non-helpless rats. CONCLUSIONS: The refined analysis allows abbreviation of the test for learned helplessness from 15 trials to 10 trials thereby reducing pain and stress of the experimental animals without losing statistical power.

Learned helplessness: unique features and translational value of a cognitive depression model.

The concept of learned helplessness defines an escape or avoidance deficit after uncontrollable stress and is regarded as a depression-like coping deficit in aversive but avoidable situations. Based on a psychological construct, it ideally complements other stress-induced or genetic animal models for major depression. Because of excellent face, construct, and predictive validity, it has contributed to the elaboration of several pathophysiological concepts and has brought forward new treatment targets. Whereas learned helplessness can be modeled not only in a broad variety of mammals, but also in fish and Drosophila, we will focus here on the use of this model in rats and mice, which are today the most common species for preclinical in vivo research in psychiatry.

A glass full of optimism: enrichment effects on cognitive bias in a rat model of depression.

Investigations of cognitive biases in animals are conceptually and translationally valuable because they contribute to animal welfare research and help to extend and refine our understanding of human emotional disorders, where biased information processing is a critical causal and maintenance factor. We employed the "learned helplessness" genetic rat model of depression in studying cognitive bias and its modification by environmental manipulations. Using a spatial judgment task, responses to ambiguous spatial cues were assessed before and after environmental enrichment to test whether this manipulation would cause an optimistic shift in emotional state. Twenty-four congenitally helpless and nonhelpless male rats were trained to discriminate two different locations, "rewarded" versus "aversive." After successful acquisition of this spatial discrimination, cognitive bias was probed by measuring responses to three ambiguous locations. Latencies to "reach" and to actively "choose" a goal pot were recorded alongside exploratory behaviors. An overall strain difference was observed, with helpless rats displaying longer "reach" latencies than nonhelpless rats. This implies a "pessimistic" response bias in helpless rats, underscoring their depressive-like phenotype. No strain differences were observed regarding other behavioral measures. Half of the animals were then transferred to enriched cages and retested. Environmental enrichment resulted in reduced "choose" latencies in both rat strains, associating enrichment with more optimistic interpretations of ambiguous cues. Our results emphasize the suitability of cognitive bias measurement for animal emotion assessment. They extend the methodological repertoire for characterizing complex phenotypes and bear implications for animal welfare research and for the use of animal models in preclinical research.

Persistent inhibition of hippocampal long-term potentiation in vivo by learned helplessness stress.

The persistent cognitive disruptive effects of stress have been strongly implicated in the pathophysiology of depression and post-traumatic stress disorder. Here we examined factors influencing the time course of recovery from the inhibitory effect of acute inescapable stressors on the ability to induce long-term potentiation (LTP) in the dorsal hippocampus in vivo. We tested different forms of LTP, different stressors and different inbred strains of rats. Acute elevated platform stress completely, but transiently (<3 h), inhibited induction of both NMDA receptor-dependent LTP induced by a standard high frequency (200 Hz) conditioning stimulus and an additional LTP that required voltage-dependent Ca(2+) channel activation triggered by strong (400 Hz) conditioning stimulation. In contrast, acute inescapable footshock stress, used to study learned helplessness, inhibited LTP for at least 4 weeks. Contrary to expectations, there was no clear relationship between the ability of the footshock to trigger helpless behavior, a model of stress-induced depression, and the magnitude of LTP inhibition. Moreover, LTP did not appear to be affected by genetic susceptibility to learned helplessness, a model of genetic vulnerability to depression. This long-lasting synaptic plasticity disruption may underlie persistent impairment of hippocampus-dependent cognition by excessive acute inescapable stress.

Aging and depression vulnerability interaction results in decreased serotonin innervation associated with reduced BDNF levels in hippocampus of rats bred for learned helplessness.

Epidemiological studies have revealed a strong genetic contribution to the risk for depression. Both reduced hippocampal serotonin neurotransmission and brain-derived neurotrophic factor (BDNF) levels have been associated with increased depression vulnerability and are also regulated during aging. Brains from young (5 months old) and old (13 months old) congenital Learned Helplessness rats (cLH), and congenital Non Learned Helplessness rats (cNLH) were immunohistochemically stained for the serotonin transporter and subsequently stereologically quantified for estimating hippocampal serotonin fiber density. Hippocampal BDNF protein levels were measured by ELISA. An exacerbated age-related loss of serotonin fiber density specific for the CA1 area was observed in the cLH animals, whereas reduced hippocampal BDNF levels were seen in young and old cLH when compared with age-matched cNLH controls. These observations indicate that aging should be taken into account when studying the neurobiological factors behind the vulnerability for depression and that understanding the effect of aging on genetically predisposed individuals may contribute to a better understanding of the pathophysiology behind depression, particularly in the elderly.

Imaging new neurons in vivo: a pioneering tool to study the cellular biology of depression?

Hippocampal neurogenesis has been implicated in the pathogenesis of and recovery from depression. However, most of the underlying studies were endpoint investigations in experimental animals yielding conflicting results, and it has been under debate to which extent these results could be transferred to human patients. Now, researchers have developed a powerful new tool to address these questions by a non-invasive method in humans and animals in vivo, using magnetic resonance spectroscopy to detect a biomarker for proliferating progenitor cells that give rise to new neurons. This makes it possible to study the role of neural progenitor cells in a wide variety of human brain disorders.

The influence of ketamine's repeated treatment on brain topology does not suggest an antidepressant efficacy.

As ketamine is increasingly used as an effective antidepressant with rapid action, sustaining its short-lived efficacy over a longer period of time using a schedule of repeated injections appears as an option. An open question is whether repeated and single administrations would affect convergent neurocircuits. We used a combination of one of the most robust animal models of depression with high-field neuroimaging to perform a whole-brain delineation of functional mechanisms underlying ketamine's effects. Rats from two genetic strains, depressive-like and resilient, received seven treatments of 10 mg/kg S-ketamine (N = 14 depressive-like, N = 11 resilient) or placebo (N = 12 depressive-like, N = 10 resilient) and underwent resting-state functional magnetic resonance imaging. Using graph theoretical models of brain networks, we compared effects of repeated ketamine with those of single administration from a separate dataset of our previous study. Compared to single treatment, repeated ketamine evoked strain-specific brain network randomization, resembling characteristics of the depressive-like strain and patients. Several affected regions belonged to the auditory, visual, and motor circuitry, hinting at possible cumulative side effects. Finally, when compared to saline, repeated ketamine affected only a few local topological properties and had no effects on global properties. In combination with the lack of clear differences compared to placebo, our findings point toward an inefficacy of ketamine's long-term administration on brain topology, making questionable the postulated effect of repeated administration and being consistent with the recently reported absence of repeated ketamine's antidepressant efficacy in several placebo-controlled studies.

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.

Differences between ketamine's short-term and long-term effects on brain circuitry in depression.

Ketamine acts as a rapid clinical antidepressant at 25 min after injection with effects sustained for 7 days. As dissociative effects emerging acutely after injection are not entirely discernible from therapeutic action, we aimed to dissect the differences between short-term and long-term response to ketamine to elucidate potential imaging biomarkers of ketamine's antidepressant effect. We used a genetical model of depression, in which we bred depressed negative cognitive state (NC) and non-depressed positive cognitive state (PC) rat strains. Four parallel rat groups underwent stress-escape testing and a week later received either S-ketamine (12 NC, 13 PC) or saline (12 NC, 12 PC). We acquired resting-state functional magnetic resonance imaging time series before injection and at 30 min and 48 h after injection. Graph analysis was used to calculate brain network properties. We identified ketamine's distinct action over time in a qualitative manner. The rapid response entailed robust and strain-independent topological modifications in cognitive, sensory, emotion, and reward-related circuitry, including regions that exhibited correlation of connectivity metrics with depressive behavior, and which could explain ketamine's dissociative and antidepressant properties. At 48 h ketamine had mainly strain-specific action normalizing habenula, midline thalamus, and hippocampal connectivity measures in depressed rats. As these nodes mediate cognitive flexibility impaired in depression, action within this circuitry presumably reflects ketamine's procognitive effects induced only in depressed patients. This finding is especially valid, as our model represents cognitive aspects of depression. These empirically defined circuits explain ketamine's distinct action over time and might serve as translational imaging correlates of antidepressant response in preclinical testing.

Recurrent stress across life may improve cognitive performance in individual rats, suggesting the induction of resilience.

Depressive symptoms are often accompanied by cognitive impairments and recurrent depressive episodes are discussed as a potential risk for dementia. Especially, stressful life events are considered a potent risk factor for depression. Here, we induced recurrent stress-induced depressive episodes over the life span of rats, followed by cognitive assessment in the symptom-free period. Rats exposed to stress-induced depressive episodes learned faster than control rats. A high degree of stress-induced depressive-like behavior early in the paradigm was a predictor of improved cognitive performance, suggesting induction of resilience. Subsequently, exposure to lorazepam prior to stress-induced depressive episodes and cognitive testing in a nonaversive environment prevented the positive effect. This indicates a beneficial effect of the stress-associated situation, with the existence of individual coping abilities. Altogether, stress may in some have a beneficial effect, yet for those individuals unable to tackle these aversive events, consecutive unpleasant episodes may lead to worse cognitive performance later in life.

Proton magnetic resonance spectroscopic creatine correlates with creatine transporter protein density in rat brain.

Creatine (Cr) is an amino acid, which upon phosphorylation is utilized as an energy reservoir in cells with high-energy demand. The ongoing catabolism of creatine to creatinine requires a permanent creatine replenishment into the cells. Because neurons themselves cannot synthesize creatine, they have to take it up via the creatine transporter (CrT). Thus, the concentration of intracellular Cr available for the Cr/PCr shuttle system depends on the expression level of CrT protein. The proton magnetic resonance spectroscopy (MRS) creatine peak (total creatine=tCr) constitutes of two metabolites, namely Cr and phosphocreatine (PCr). We have quantified the level of CrT protein expression with western blotting and compared it to tCr content as estimated by in vitro MRS in Sprague-Dawley rats. Under the assumption of hemispheric symmetry, we took identical samples from left and right hemisphere, which were used for in vitro MRS (tCr) and for western blotting (CrT), respectively. Altogether, it was possible to take 90 corresponding brain samples from 31 animals. A Pearson linear regression analysis for CrT and tCr revealed p<0.0001, explaining 14% of the variance. Since MR-detectable alterations of tCr in the human brain are widespread (e.g. in most major psychiatric disorders proton MRS detectable tCr alterations have been described as regionally and usually state dependent) it is stringent to elucidate their meaning. An influence of tCr on the brain's energy regulating system seems plausible.

Cross-species evidence from human and rat brain transcriptome for growth factor signaling pathway dysregulation in major depression.

An enhanced understanding of the pathophysiology of depression would facilitate the discovery of new efficacious medications. To this end, we examined hippocampal transcriptional changes in rat models of disease and in humans to identify common disease signatures by using a new algorithm for signature-based clustering of expression profiles. The tool identified a transcriptomic signature comprising 70 probesets able to discriminate depression models from controls in both Flinders Sensitive Line and Learned Helplessness animals. To identify disease-relevant pathways, we constructed an expanded protein network based on signature gene products and performed functional annotation analysis. We applied the same workflow to transcriptomic profiles of depressed patients. Remarkably, a 171-probesets transcriptional signature which discriminated depressed from healthy subjects was identified. Rat and human signatures shared the SCARA5 gene, while the respective networks derived from protein-based significant interactions with signature genes contained 25 overlapping genes. The comparison between the most enriched pathways in the rat and human signature networks identified a highly significant overlap (p-value: 3.85 × 10-6) of 67 terms including ErbB, neurotrophin, FGF, IGF, and VEGF signaling, immune responses and insulin and leptin signaling. In conclusion, this study allowed the identification of a hippocampal transcriptional signature of resilient or susceptible responses in rat MDD models which overlapped with gene expression alterations observed in depressed patients. These findings are consistent with a loss of hippocampal neural plasticity mediated by altered levels of growth factors and increased inflammatory responses causing metabolic impairments as crucial factors in the pathophysiology of MDD.

Lateral habenula perturbation reduces default-mode network connectivity in a rat model of depression.

Hyperconnectivity of the default-mode network (DMN) is one of the most widely replicated neuroimaging findings in major depressive disorder (MDD). Further, there is growing evidence for a central role of the lateral habenula (LHb) in the pathophysiology of MDD. There is preliminary neuroimaging evidence linking LHb and the DMN, but no causal relationship has been shown to date. We combined optogenetics and functional magnetic resonance imaging (fMRI), to establish a causal relationship, using an animal model of treatment-resistant depression, namely Negative Cognitive State rats. First, an inhibitory light-sensitive ion channel was introduced into the LHb by viral transduction. Subsequently, laser stimulation was performed during fMRI acquisition on a 9.4 Tesla animal scanner. Neural activity and connectivity were assessed, before, during and after laser stimulation. We observed a connectivity decrease in the DMN following laser-induced LHb perturbation. Our data indicate a causal link between LHb downregulation and reduction in DMN connectivity. These findings may advance our mechanistic understanding of LHb inhibition, which had previously been identified as a promising therapeutic principle, especially for treatment-resistant depression.

Elevated spectroscopic glutamate/gamma-amino butyric acid in rats bred for learned helplessness.

The theory of depression is dominated by the monoamine hypothesis but there is increasing evidence that beyond monoamines, glutamate (Glu) and gamma-aminobutyric acid (GABA) play an essential role in the pathogenesis of depression. In this study, the effect of alterations of GABA and Glu were investigated in the congenital learned helplessness paradigm. Proton magnetic resonance spectroscopy is an important monitoring tool to bridge the findings in clinical and preclinical studies. We found increased Glu/GABA ratios in the hippocampus and prefrontal cortex of placebo-treated (saline intraperitoneally) congenital learned helplessness rats versus wild-type rats, and a treatment-induced (desipramine 10 mg/kg intraperitoneally or electroconvulsive shock) decrease of this monoamine ratio in both brain regions. Our results corroborate previous findings of an amino-acid influence on the pathomechanisms of mood disorders.

Reduced expression of complexins I and II in rats bred for learned helplessness.

Disturbed synaptic transmission contributes to the pathophysiology of mood disorders. Post mortem studies reported reduced expression of the synaptic vesicle protein (SVP) complexins I and II in depression. Antidepressants were found to induce the expression of these genes. Since animals with congenital susceptibility to learned helplessness provide a valid animal model of depression, we investigated the expression of different SVPs in this system by semiquantitative in situ hybridization. Rats bred for congenital learned helpless behavior (cLH, N=6) failed to interrupt foot shock currents by lever pressing (mean 12.3 failures out of 15 trials). These animals showed significantly lower expression of complexins I and II mRNA in hippocampal, limbic and cortical brain areas compared to not helpless animals (cNLH, N=6) with a mean failure rate of 0.83 out of 15 trials. Expression levels of complexins I and II significantly correlated with the failure rate in the test paradigm. In contrast, the expressions of synaptotagmin I and synaptophysin were found unchanged. This investigation provides a further validation of the LH model of depression. The experimental data fit well into current pathogenetic concepts of mood disorders and support the hypothesis, that complexins are pivotal players in the pathophysiology of depression and tentative targets of antidepressants.

Correlation between MR-spectroscopic rat hippocampal choline levels and phospholipase A2.

Hippocampal choline-containing compounds (Cho) determined with 1H MR spectroscopy (MRS) are decreased in major depression episodes and return to baseline levels after antidepressive electroconvulsive therapy (ECT). A rise in hippocampal Cho has been observed in rats upon electroconvulsive shocks (ECS), an analogue of human ECT. Choline production involves the activity of various phospholipases. In order to investigate whether the increase of Cho correlates with an enhanced expression of phosphoslipase A2 (PLA2) we took rectangular tissue samples from the region of the MRS voxel for immunoblotting. Our data show a significant inverse correlation (p = 0.018) between PLA2 protein levels and MRS Cho/NAA levels suggesting a possible downregulation of PLA2 in compensation for an upregulation of other phospholipases.

MicroRNA Profiling in the Medial and Lateral Habenula of Rats Exposed to the Learned Helplessness Paradigm: Candidate Biomarkers for Susceptibility and Resilience to Inescapable Shock.

Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miRNAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.