Measuring paw preferences in dogs, cats and rats: Design requirements and innovations in methodology.

Studying behavioural lateralization in animals holds great potential for answering important questions in laterality research and clinical neuroscience. However, comparative research encounters challenges in reliability and validity, requiring new approaches and innovative designs to overcome. Although validated tests exist for some species, there is yet no standard test to compare lateralized manual behaviours between individuals, populations, and animal species. One of the main reasons is that different fine-motor abilities and postures must be considered for each species. Given that pawedness/handedness is a universal marker for behavioural lateralization across species, this article focuses on three commonly investigated species in laterality research: dogs, cats, and rats. We will present six apparatuses (two for dogs, three for cats, and one for rats) that enable an accurate assessment of paw preference. Design requirements and specifications such as zoometric fit for different body sizes and ages, reliability, robustness of the material, maintenance during and after testing, and animal welfare are extremely important when designing a new apparatus. Given that the study of behavioural lateralization yields crucial insights into animal welfare, laterality research, and clinical neuroscience, we aim to provide a solution to these challenges by presenting design requirements and innovations in methodology across species.

Rodent models for mood disorders - understanding molecular changes by investigating social behavior.

Mood disorders, including depressive and bipolar disorders, are the group of psychiatric disorders with the highest prevalence and disease burden. However, their pathophysiology remains poorly understood. Animal models are an extremely useful tool for the investigation of molecular mechanisms underlying these disorders. For psychiatric symptom assessment in animals, a meaningful behavioral phenotype is needed. Social behaviors constitute naturally occurring complex behaviors in rodents and can therefore serve as such a phenotype, contributing to insights into disorder related molecular changes. In this narrative review, we give a fundamental overview of social behaviors in laboratory rodents, as well as their underlying neuronal mechanisms and their assessment. Relevant behavioral and molecular changes in models for mood disorders are presented and an outlook on promising future directions is given.

Microglia and microbiome in schizophrenia: can immunomodulation improve symptoms?

In this overview, influences of microglia activation and disturbances of the microbiome in the devastating disorder schizophrenia are discussed. Despite previous assumptions of a primary neurodegenerative character of this disorder, current research underlines the important autoimmunological and inflammatory processes here. Early disturbances of microglial cells as well as cytokines could lead to weakness of the immunological system in the prodromal phase and then fully manifest in patients with schizophrenia. Measurements of microbiome features might allow identifying the prodromal phase. In conclusion, such thinking would imply several new therapeutic options regulating immune processes by old or new anti-inflammatory agents in patients.

Maternal separation and its developmental consequences on anxiety and parvalbumin interneurons in the amygdala.

The early postnatal period represents an exceptionally vulnerable phase for the development of neurobiological alterations, aberrant behavior, and psychiatric disorders. Altered GABAergic activity in the hippocampus and the amygdala have been identified in humans diagnosed with depression or anxiety disorders, as well as in respective animal models. Changes in GABAergic activity can be visualized by immunohistochemical staining of parvalbumin (PV) protein. Therewith, alterations in PV intensity as well as in the integrity of the perineural net surrounding PV positive (PV+) interneurons have been reported as consequences of early stress. In the current study, maternal separation (MS) was used to induce early life stress. Female and male Sprague-Dawley rats were subjected to MS over 4 h from postnatal days 2-20. Then, anxiety behavior and PV+ interneurons in the amygdala were analyzed using immunohistochemistry in adolescence or adulthood. MS induced increased anxiety behavior in the marble-burying test in adolescence as well as in the elevated plus maze in adulthood. No effect of sex was found. Concerning alterations of parvalbumin expression in the amygdala, a trend towards a lower number of parvalbumin-positive inhibitory interneurons was shown in the amygdala after MS in adolescence, with no differences in the total number of cells. The current study offers a developmental perspective, suggesting that the kind of anxiety behavior expressed by rats following MS changes over time from active to passive avoidance, indicating that effects of MS are highly dependent on developmental state. Moreover, a cell-type-specific effect of MS on the cellular composition of the amygdala is discussed. The presented study demonstrates the long-lasting consequences of early stress on behavior, offers a possible neurobiological correlate, and discusses possible mediators in the development of these alterations.

Maternal separation: Does it hold the potential to model consequences of postpartum depression?

The postpartum period is a sensitive time where women are especially vulnerable to develop postpartum depression (PPD), with 10%-15% of women affected. This review investigates whether the maternal separation (MS) paradigm in rodents holds the potential to help to understand mothers suffering from PPD. MS is a well-established stress model to investigate effects on infants, whereas effects on the dam are often overlooked. The database PubMed was searched for studies investigating effects of daily MS within the first weeks after parturition on dams in rats and mice and compared to findings in PPD mothers. MS was categorized as brief MS (5-45 min) with or without handling of pups and long MS (3-4 h and longer). MS alters maternal care, depressive-like behavior, anxiety, and aggression; leads to alterations in neuronal gene expression; and affects hormone and neurotransmitter levels similar to observations in PPD patients. Even though there are disparities between human and rodent mothers, with some results differing in directionality, as well as the reason for separation (self-induced in PPD, externally induced in MS), the overall effects found on neurobiological, hormonal, and behavioral levels mostly coincide. Thus, the MS paradigm can add relevant knowledge to existing PPD animal models, further advancing the study of PPD.

Poly I:C-induced maternal immune challenge reduces perineuronal net area and raises spontaneous network activity of hippocampal neurons in vitro.

Activation of the maternal immune system (MIA) during gestation is linked to neuropsychiatric diseases like schizophrenia. While many studies address behavioural aspects, less is known about underlying cellular mechanisms. In the following study, BALB/c mice received intraperitoneal injections of polyinosinic-polycytidylic acid (Poly I:C) (20 µg/ml) or saline (0.9%) at gestation day (GD) 9.5 before hippocampal neurons were isolated and cultured from embryonic mice for further analysis. Interestingly, strongest effects were observed when the perineuronal net (PNN) wearing subpopulation of neurons was analysed. Here, a significant reduction of aggrecan staining intensity, area and soma size could be detected. Alterations of PNNs are often linked to neuropsychiatric diseases, changes in synaptic plasticity and in electrophysiology. Utilizing multielectrode array analysis (MEA), we observed a remarkable increase of the spontaneous network activity in neuronal networks after 21 days in vitro (DIV) when mother mice suffered a prenatal immune challenge. As PNNs are associated with GABAergic interneurons, our data indicate that this neuronal subtype might be stronger affected by a prenatal MIA. Degradation or damage of this subtype might cause the hyperexcitability observed in the whole network. In addition, embryonic neurons of the Poly I:C condition developed significantly shorter axons after five days in culture, while dendritic parameters and apoptosis rate remained unchanged. Structural analysis of synapse numbers revealed an increase of postsynaptic density 95 (PSD-95) puncta after 14 DIV and an increase of presynaptic vesicular glutamate transporter (vGlut) puncta after 21 DIV, while inhibitory synaptic proteins were not altered.

Morc1 as a potential new target gene in mood regulation: when and where to find in the brain.

Recent animal and human studies connected the Morc family CW-type zinc finger 1 (Morc1) gene with early life stress and depression. Moreover, the Morc superfamily is related to epigenetic regulation in diverse nuclear processes. So far, the Morc1 gene was mainly studied in spermatogenesis, whereas its distribution and function in the brain are still unknown. In a first attempt to characterize Morc1 in the brain, we performed a Western Blot analysis as well as a real-time PCR analysis during different stages of development. Additionally, we detected Morc1 mRNA using real-time PCR in different mood-regulating brain areas in adult rats. We found that MORC1 protein as well as Morc1 mRNA is already expressed in the brain at embryonic day 14 and is stably expressed until adulthood. Furthermore, Morc1 mRNA is present in many important brain areas of mood regulation like the medial prefrontal cortex, the nucleus accumbens, the hippocampus, the hypothalamus, and the amygdala. The ample distribution in the brain and its molecular structure as a zinc finger protein indicate that Morc1 might act as a transcription factor. This function and its expression in mood-regulating areas already in the early brain development turn Morc1 into a possible candidate gene for mediating early life stress and depression.

The developmental inter-relationships between activity, novelty preferences, and delay discounting in male and female rats.

Increased locomotion, novelty-seeking, and impulsivity are risk factors associated with substance use. In this study, the inter-relationships between activity, novelty preferences, and delay discounting, a measure of impulsivity, were examined across three stages: juvenile/early adolescence (postnatal Day [P] 15, 19, and 42 for activity, novelty, and impulsivity, respectively), adolescent/late adolescent (P28, 32, 73), and adult (P90, 94, 137) in male and female rats. Our estimates of impulsive choice, where animals were trained to criterion, revealed an age × sex interaction where early adolescent females had the lowest levels of impulsivity. The relationships of activity and novelty to impulsivity significantly changed across age within each sex. Early adolescent males with high activity, but low novelty preferences, were more impulsive; however, low activity and high novelty preferences were related to high impulsivity in adult males. Female activity gradually increased across age, but did not show a strong relationship with impulsivity. Novelty preferences are moderately related to impulsivity into adulthood in females. These data show that males and females have different developmental trajectories for these behaviors. Males show greater sensation-seeking (e.g., activity) and risky behavior (e.g., novelty preferences) earlier in life, whereas these behaviors emerge during adolescence in females.

Bipolar Chronobiology in Men and Mice: A Narrative Review.

In patients with bipolar disorder, we do not only see a cycling of mood episodes, but also a shift in circadian rhythm. In the present overview, the circadian rhythm, the "internal clock", and their disruptions are briefly described. In addition, influences on circadian rhythms such as sleep, genetics, and environment are discussed. This description is conducted with a translational focus covering human patients as well as animal models. Concluding the current knowledge on chronobiology and bipolar disorder, implications for specificity and the course of bipolar disorder and treatment options are given at the end of this article. Taken together, circadian rhythm disruption and bipolar disorder are strongly correlated; the exact causation, however, is still unclear.

Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA.

Exposure to adversity during development is an identified risk factor for depression later in life. In humans, early adversity accelerates the onset of depressive symptoms, which manifest during adolescence. Animal studies have used maternal separation as a model of early adversity to produce adult depressive-like behaviors, but have yet to examine these behaviors during adolescence. Moreover, the nature of depressive-like behaviors has not been well characterized in this model. Here, we used the triadic model of learned helplessness to understand controllability, helplessness, and motivational factors following maternal separation in male and female adolescent rats. We found sex-dependent changes in the effects of separation, with males demonstrating loss of controllability in an escapable shock condition, whereas females demonstrated motivational impairment in a no-shock condition. The effect, however, did not endure as adult females were no longer helpless. Reductions in parvalbumin, a GABAergic marker, in the prefrontal cortex of separated subjects relative to age-matched controls were evident and paralleled depressive-like behavior. Understanding the risk factors for depression, the nature of depressive-like behaviors, and their unique sex dependency may ultimately provide insight into improved treatments.

Unraveling the mystery of white matter in depression: A translational perspective on recent advances.

BACKGROUND: Numerous cortical and subcortical structures have been studied extensively concerning alterations of their integrity as well as their neurotransmitters in depression. However, connections between these structures have received considerably less attention. OBJECTIVE: This systematic review presents results from recent neuroimaging as well as neuropathologic studies conducted on humans and other mammals. It aims to provide evidence for impaired white matter integrity in individuals expressing a depressive phenotype. METHODS: A systematic database search in accordance with the PRISMA guidelines was conducted to identify imaging and postmortem studies conducted on humans with a diagnosis of major depressive disorder, as well as on rodents and primates subjected to an animal model of depression. RESULTS: Alterations are especially apparent in frontal gyri, as well as in structures establishing interhemispheric connectivity between frontal regions. Translational neuropathological findings point to alterations in oligodendrocyte density and morphology, as well as to alterations in the expression of genes related to myelin synthesis. An important role of early life adversities in the development of depressive symptoms and white matter alterations across species is thereby revealed. Data indicating that stress can interfere with physiological myelination patterns is presented. Altered myelination is most notably present in regions that are subject to maturation during the developmental stage of exposure to adversities. CONCLUSION: Translational studies point to replicable alterations in white matter integrity in subjects suffering from depression across multiple species. Impaired white matter integrity is apparent in imaging as well as neuropathological studies. Future studies should focus on determining to what extent influencing white matter integrity is able to improve symptoms of depression in animals as well as humans.

Poly I:C Activated Microglia Disrupt Perineuronal Nets and Modulate Synaptic Balance in Primary Hippocampal Neurons in vitro.

Perineuronal nets (PNNs) are specialized, reticular structures of the extracellular matrix (ECM) that can be found covering the soma and proximal dendrites of a neuronal subpopulation. Recent studies have shown that PNNs can highly influence synaptic plasticity and are disrupted in different neuropsychiatric disorders like schizophrenia. Interestingly, there is a growing evidence that microglia can promote the loss of PNNs and contribute to neuropsychiatric disorders. Based on this knowledge, we analyzed the impact of activated microglia on hippocampal neuronal networks in vitro. Therefore, primary cortical microglia were cultured and stimulated via polyinosinic-polycytidylic acid (Poly I:C; 50 µg/ml) administration. The Poly I:C treatment induced the expression and secretion of different cytokines belonging to the CCL- and CXCL-motif chemokine family as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, the expression of matrix metalloproteinases (MMPs) could be verified via RT-PCR analysis. Embryonic hippocampal neurons were then cultured for 12 days in vitro (DIV) and treated for 24 h with microglial conditioned medium. Interestingly, immunocytochemical staining of the PNN component Aggrecan revealed a clear disruption of PNNs accompanied by a significant increase of glutamatergic and a decrease of γ-aminobutyric acid-(GABA)ergic synapse numbers on PNN wearing neurons. In contrast, PNN negative neurons showed a significant reduction in both, glutamatergic and GABAergic synapses. Electrophysiological recordings were performed via multielectrode array (MEA) technology and unraveled a significantly increased spontaneous network activity that sustained also 24 and 48 h after the administration of microglia conditioned medium. Taken together, we could observe a strong impact of microglial secreted factors on PNN integrity, synaptic plasticity and electrophysiological properties of cultured neurons. Our observations might enhance the understanding of neuron-microglia interactions considering the ECM.

Prefrontal dopamine D1 receptor manipulation influences anxiety behavior and induces neuroinflammation within the hippocampus.

BACKGROUND: Prefrontal dopamine D1 receptor (D1R) mediates behavior related to anxiety, reward and memory, and is involved in inflammatory processes, all of which are affected in bipolar disorder. Interleukin-6 (IL-6), a pro-inflammatory cytokine, is increased in patients with bipolar disorder in plasma samples, imaging studies and postmortem tissue and is an indicator for an inflammatory state. We could previously show that lentiviral overexpression of D1R in the medial prefrontal cortex (mPFC) of male adult rats and its termination induces bipolar disorder-like behavior. The purpose of this study was to investigate anxiety and the role of the immune system, specifically IL-6 positive neurons in this animal model. Due to its high density of inflammatory mediator receptors and therewith sensibility to immune activation, the hippocampus was investigated. METHODS: Expression of the gene for D1R in glutamatergic neurons within the mPFC of male, adult rats was manipulated through an inducible lentiviral vector. Animals over-expressing the gene (mania-like state), after termination of the expression (depressive-like) and their respective control groups were investigated. Anxiety behavior was studied in the elevated plus maze and marble burying test. Furthermore, IL-6-positive cells were counted within several subregions of the hippocampus. RESULTS: D1R manipulation in the mPFC had only mild effects on anxiety behavior in the elevated plus maze. However, subjects after termination buried more marbles compared to D1R over-expressing animals and their respective control animals indicating elevated anxiety behavior. In addition, animals in the depressive-like state showed higher numbers of IL-6 positive cells reflecting an elevated pro-inflammatory state in the hippocampus, in the CA3 and dentate gyrus. Consistently, inflammatory state in the whole hippocampus and anxiety behavior correlated positively, indicating a connection between anxiety and inflammatory state of the hippocampus. CONCLUSIONS: Behavioral and neurobiological findings support the association of manipulation of the D1R in the mPFC on anxiety and inflammation in the hippocampus. In addition, by confirming changes in the inflammatory state, the proposed animal model for bipolar disorder has been further validated.

Paw preferences in mice and rats: Meta-analysis.

Mice and rats are among the most common animal model species in both basic and clinical neuroscience. Despite their ubiquity as model species, many clinically relevant brain-behaviour relationships in rodents are not well understood. In particular, data on hemispheric asymmetries, an important organizational principle in the vertebrate brain, are conflicting as existing studies are often statistically underpowered due to small sample sizes. Paw preference is one of the most frequently investigated forms of hemispheric asymmetries on the behavioural level. Here, we used meta-analysis to statistically integrate findings on paw preferences in rats and mice. For both species, results indicate significant hemispheric asymmetries on the individual level. In mice, 81 % of animals showed a preference for either the left or the right paw, while 84 % of rats showed this preference. However, contrary to what has been reported in humans, population level asymmetries were not observed. These results are particularly significant as they point out that paying attention to potential individual hemispheric differences is important in both basic and clinical neuroscience.

Risky decision-making following prefrontal D1 receptor manipulation.

The prefrontal dopamine D1 receptor (D1R) is involved in cognitive processes. Viral overexpression of this receptor in rats further increases the reward-related behaviors and even its termination induces anhedonia and helplessness. In this study, we investigated the risky decision-making during D1R overexpression and its termination. Rats conducted the rodent version of the Iowa gambling task daily. In addition, the methyl CpG-binding protein-2 (MeCP2), one regulator connecting the dopaminergic system, cognitive processes, and mood-related behavior, was investigated after completion of the behavioral tasks. D1R overexpressing subjects exhibited maladaptive risky decision-making and risky decisions returned to control levels following termination of D1R overexpression; however, after termination, animals earned less reward compared to control subjects. In this phase, MeCP2-positive cells were elevated in the right amygdala. Our results extend the previously reported behavioral changes in the D1R-manipulated animal model to increased risk-taking and revealed differential MeCP2 expression adding further evidence for a bipolar disorder-like phenotype of this model.

Impact of Poly I:C induced maternal immune activation on offspring's gut microbiome diversity - Implications for schizophrenia.

Background Immunopathological concepts have been intensively discussed for schizophrenia. The polyriboinosinic-polyribocytidylic (PolyI:C) mouse model has been well validated to invasively study this disease. The intestinal microbiome exhibits broad immunological and neuronal activities. The relevance of microbiome alterations in the PolyI:C model to human schizophrenia should be explored. Methods Feces of offspring from mice mothers, who were administered to PolyI:C or NaCl (controls) at ED 9, were collected at PND 30 and 180 (PolyI:C and control mice (N = 32 each; half males and females). This was analyzed for bacterial 16S ribosomal DNA (rDNA) using a gut microbiome polymerase chain reaction (PCR) microarray tool. Results Differences were found in species richness of microbiome between animals of different ages (PND 30 and 180), but also between offspring from PolyI:C vs. NaCl treated mothers. In female mice at PND 30, the abundance of Prevotellaceae and Porphyromonadaceae was lower and that of Lactobacillales was higher, whereas in male mice at the same time point the abundance of four families of the Firmicutes phylum (Clostridia vadinBB60 group, Clostridiales Family XIII, Ruminococcaceae and Erysipelotrichaceae) was increased relative to the control group. Limitations No further analyses of cell types or cytokines involved in autoimmune gut and brain processes. Conclusions These finding seem to be similar to microbiome disturbances in patients with schizophrenia. The differential bacterial findings at day 30 (i.e., similar to the prodromal phase in patients with schizophrenia) correspond to the tremendous activation of the immune system with a strong increase in microglial cells which might be responsible for neuroplasticity reduction in cortical areas in patients with schizophrenia.

Maternal immune activation leads to atypical turning asymmetry and reduced DRD2 mRNA expression in a rat model of schizophrenia.

Atypical asymmetries have been reported in individuals diagnosed with schizophrenia, linking higher symptom severity to weaker lateralization. Furthermore, both lateralization and schizophrenia are influenced by the dopaminergic system. However, whether a direct link between the etiology of schizophrenia and atypical asymmetries exists is yet to be investigated. In this study, we examined whether maternal immune activation (MIA), a developmental animal model for schizophrenia and known to alter the dopaminergic system, induces atypical lateralization in adolescent and adult offspring. As the dopaminergic system is a key player in both, we analyzed neuronal dopamine D2 receptor (DRD2) mRNA expression. MIA was induced by injecting pregnant rats with 10 mg/kg polyinosinic:polycytidylic (PolyI:C) at gestational day 15. Controls were injected with 0.9 % NaCl. Offspring were tested at adolescence or early adulthood for asymmetry of turning behavior in the open field test. The total number of left and right turns per animal was assessed using DeepLabCut. Strength and preferred side of asymmetry were analyzed by calculating lateralization quotients. Additionally, DRD2 mRNA expression in the prefrontal cortex of offspring at both ages was analyzed using real-time PCR. MIA was associated with a rightward turning behavior in adolescents. In adults, MIA was associated with an absence of turning bias, indicating reduced asymmetry after MIA. The analysis of DRD2 mRNA expression revealed significantly lower mRNA levels after MIA compared to controls in adolescent, but not adult animals. Our results reinforce the association between atypical asymmetries, reduced DRD2 mRNA expression, and schizophrenia. However, more preclinical research is needed.

MORC1 methylation and BDI are associated with microstructural features of the hippocampus and medial prefrontal cortex.

BACKGROUND: Alterations in the hippocampus and prefrontal cortex (PFC) have frequently been reported in depressed patients. These parameters might prove to be a consistent finding in depression. In addition, peripheral DNA methylation of the MORC1 gene promoter showed stable associations with depression across independent samples. However, the question arises whether MORC1, supposedly acting as transcription factor, might also be involved in neurobiological alterations accompanying depression. This study further analyses the role of MORC1 in depression by investigating a potential correlation between peripheral MORC1 DNA methylation and neuronal structural properties previously associated with depression in humans. METHODS: Beck Depression Inventory (BDI) was assessed in 52 healthy participants. DNA was extracted from buccal cells and MORC1 methylation correlated with micro- and macrostructural properties derived from magnetic resonance imaging (MRI) and neurite orientation dispersion and density imaging (NODDI) in the hippocampus and medial prefrontal cortex (mPFC). RESULTS: MORC1 methylation was associated with volume reduction and neurite orientation dispersion and density markers in the hippocampus and mPFC. BDI was positively associated with neurite orientation dispersion and density markers in the hippocampus. LIMITATIONS: The study was conducted in a small sample of healthy participants with subclinical depressive symptoms. Peripheral tissue was analyzed. CONCLUSION: We found significant negative associations between peripheral MORC1 methylation and macro- and microstructural markers in the hippocampus and mPFC. Thus, MORC1 might be involved in neurobiological properties. Studies investigating neuronal methylation patterns of MORC1 are needed to support this hypothesis.

Maternal separation in rats induces neurobiological and behavioral changes on the maternal side.

The time after parturition is a sensitive period for mothers where they are prone to develop psychopathological symptoms. Studies investigating dams after separation from their pups (maternal separation, MS) showed that MS induces alterations similar to postpartum depression. This study aims to give further details on affected behavior and neurobiology of dams after MS. MS in rats from postnatal day 2-20 over four hours daily was performed. Upon reunion, maternal behavior, and ultrasonic vocalization (USV) of dams were measured. On the day of weaning, dams were tested for anxiety-like behavior in the elevated-plus-maze and marble burying test. Then Morc1 mRNA in the medial prefrontal cortex and Nr3c1 encoding the glucocorticoid receptor mRNA in the hippocampus were measured using real-time PCR to examine possible neurobiological correlates in psychopathology and social behavior. GABA and glutamate serum levels were analyzed by high-performance liquid chromatography as peripheral markers for stress-induced psychopathology. MS in dams increased maternal care towards pups even though both groups show high levels of maternal behavior even in late lactation. Furthermore, the emission of 50-kHz and 22-kHz USVs increased significantly. No differences in anxiety-like behavior were detected. MS further reduced Morc1 but not Nr3c1 expression. Serum GABA but not glutamate levels were significantly increased in separated dams. This study reinforces the benefit of investigating dams after MS for studying postpartum stress. Subclinical markers mainly connected to depression, namely Morc1 and GABA, proved to be useful allowing for earlier detection of symptoms of critical postpartum stress.