Background Aging is associated with cognitive decline and behavioral alterations linked to neurobiological changes. Rodent models are crucial for investigating age-related variations in cognitive flexibility, anxiety, and circadian rhythms. Comparing two inbred strains, C57BL/6J and 129S2/SvPasCrl, helps clarify how genetic background influences these aging-related outcomes. New method Advanced RFID-based automated home-cage platforms (Mouse Matrix and IntelliCage) were utilized to quantify circadian locomotor activity, core body temperature dynamics, and cognitive performance in group-housed mice. These systems enable continuous, high-resolution monitoring of behavioral and physiological data within an automated, ethologically relevant environment. Results Significant age- and strain-dependent differences were observed in temperature rhythms, locomotor activity, anxiety-like behavior, and adaptability to environmental changes. Aged mice showed reduced activity and impaired anticipatory behavior, indicating disrupted circadian regulation. The 129S2/SvPasCrl strain displayed higher anxiety and slower adaptation, whereas C57BL/6J mice exhibited greater cognitive flexibility and more rapid learning. Comparison with existing methods Conventional behavioral tests performed outside the home-cage, such as open field or maze-based tasks, rely on brief, experimenter-dependent sessions that may introduce handling stress and novelty effects. In contrast, MM and IC provide continuous, unbiased assessment within the familiar home-cage environment, reducing stress and improving ecological validity. Conclusions Advanced home-cage monitoring systems offer reliable platforms for detecting strain- and age-specific behavioral and physiological differences. These findings highlight the importance of automated, naturalistic approaches for improving reproducibility and translational relevance in aging and neuropsychiatric research.

The involvement of non-scientific staff in discussions about animal welfare and scientific quality is essential for biomedical research progress. In this study, we developed a survey to collect the self-perception of animal care staff (ACS) and laboratory technicians about their involvement in scientific planning and conduct. Participants were contacted to complete an anonymous online questionnaire. We obtained 850 responses, mainly from Europe: 564 from ACS and 286 from laboratory technicians. Job satisfaction was assessed as positive by ACS and laboratory technicians despite the low frequency of culture of care activities and mental health meetings. Both groups expressed their desire to be trained in research planning and conduct; however, regular training was not reported. In addition, the inability to act on animal welfare concerns owing to experimental reasons was reported by both groups. Over half of the participants felt valued and appreciated by the lead scientists or animal facility manager; however, it is not clear how they are acknowledged, as their names on the authors list or in the manuscript acknowledgments are barely included. Our results indicated that involvement of ACS and laboratory technicians in planning and conducting studies would improve their understanding of how experiments are done, and therefore communication processes, work satisfaction, animal welfare, and scientific quality. Finally, we provided recommendations to improve the engagement of ACS and laboratory technicians in discussions about animal research planning and conduct.

Training in laboratory animals is important to ensure that scientific studies are reliable, reproducible, and ethically acceptable. Well-trained animals experience less stress and exhibit fewer unwanted behaviors, improving both welfare and research outcomes. Clicker training is widely used in animal training as a positive reinforcement method to reduce distress. The present study was designed as a pilot study to examine the effects of a four-day clicker training protocol applied to both dams and their offspring, with behavioral outcomes assessed exclusively in the offspring. The results indicated that clicker training potentially increased voluntary interaction with the experimenter and promoted body weight gain during the training period. No significant effects of offspring training were found for classical anxiety-related measures (EPM open arm time, OF center time, Nest Building Test, Sucrose Preference Test, Forced Swim Test) or plasma corticosterone. A potential sex effect was observed across locomotor, center-zone, and corticosterone measures. Maternal training effects on offspring outcomes should be interpreted as preliminary exploratory observations, as only two dams per maternal group were available. Given the exploratory nature and limited sample size of this pilot study, the findings should be interpreted with caution. Under the present condition, clicker training produced a context-specific improvement in human-animal interaction without evidence of harm, but did not produce generalized reductions in anxiety-like behaviors as assessed by standard paradigms. Further studies with larger sample sizes are needed to confirm these findings.

Implant-mediated electrical stimulation (ES) is a promising approach in the treatment of intestinal motility disorders. The preclinical application of a prototype implant was technically successful. A clear contraction in the ascending colon was detected in the video analysis synchronized with the electromyography after ES with 5 mA.

INTRODUCTION:Gastric electrical stimulation (GES) is an effective treatment for gastroparesis. However, the available devices are equipped with bulky batteries that need to be replaced regularly by surgery. METHODS:Our new implantable system consists of only 6 passive components in addition to a diode and does not require a battery. Two acute porcine experiments were carried out with a robotic surgical system for placement of the prototypes. The stimulation parameters were set with an extracorporeal unit and GES was performed. The recorded electromyography (EMG) signal was subjected to a multiresolution analysis. RESULTS:The robot-assisted placement of the prototypes was successful. The inductive energy transfer was confirmed to be functional and EMG analysis revealed changes in gastric electrical activity. CONCLUSIONS:Further technological and rapid regulatory solutions are being sought in order to start a clinical trial with the next generation devices in the near future.

Improving laboratory animal welfare by minimizing stress and promoting species-appropriate handling is a central goal of contemporary biomedical research worldwide. Clicker training, a widely recognized form of positive reinforcement training, uses a click sound as a conditioned reinforcer to bridge the desired behavior and the reward, enabling animals to learn more quickly and with reduced stress. Our research group has previously demonstrated that clicker training functions as a form of cognitive enrichment in mice and rats. In the present study, we successfully adapted this training approach for Mongolian gerbils (Meriones unguiculatus), tailoring the protocol to the species-specific behavioral characteristics of gerbils. A cohort of 43 inbred gerbils (27 females and 16 males) underwent a standardized 10-day clicker training protocol, during which they learned to voluntarily approach and interact with the experimenter's hand in exchange for a food reward. Following the training period, animals were subjected to behavioral assessments, including an Open Field Test and a standardized human interaction test, to evaluate the effects of training on exploratory behavior and human-animal interaction. Our findings demonstrate that the implementation of clicker training in gerbils is fast, efficient, and well-tolerated. Trained animals, particularly females, showed increased voluntary interaction with the experimenter's hand and reduced anxiety-like behaviors. These results suggest that species-adapted clicker training protocols can facilitate the development of trust between experimenter and animal, ultimately decreasing stress and improving both animal welfare and the reliability of experimental outcomes.

Facilities involved in laboratory animal research often face ethical challenges such as: what should I do with the animals that are no longer suitable for experimental purposes? One of the common answers to this question is to kill them. And while numerous scientifically justifiable reasons exist for killing laboratory animals, we must not overlook our ethical responsibility towards these sentient beings. Animal facility managers and scientists frequently find themselves in a moral dilemma, torn between furthering their research and addressing the well-being of experimental animals required for their studies. We elaborated a concept consisting of six decision trees and recommendations for making informed decisions about the need to kill laboratory animals in research facilities, considering legal and ethical considerations. The concept is based on the German regulatory perspective. However, the measures and decisions for animal welfare can be implemented in all laboratory animal facilities. These recommendations suggest several courses of action, including implementing consistent breeding plans, exploring alternative uses, reassigning surplus animals and their organs, and establishing appropriate housing capacity limits that ensure species-appropriate care. We encourage scientists and animal facility managers to develop and implement decision-making frameworks and procedures tailored to their specific facilities, in the hope that this work will promote a thoughtful and responsible approach to the complex challenges associated with the killing of laboratory animals, advancing scientific progress and the humane treatment of these animals.

Excitation/inhibition (E/I) balance plays important roles in mental disorders. Bioactive phospholipids like lysophosphatidic acid (LPA) are synthesized by the enzyme autotaxin (ATX) at cortical synapses and modulate glutamatergic transmission, and eventually alter E/I balance of cortical networks. Here, we analyzed functional consequences of altered E/I balance in 25 human subjects induced by genetic disruption of the synaptic lipid signaling modifier PRG-1, which were compared to 25 age and sex matched control subjects. Furthermore, we tested therapeutic options targeting ATX in a related mouse line. Using EEG combined with TMS in an instructed fear paradigm, neuropsychological analysis and an fMRI based episodic memory task, we found intermediate phenotypes of mental disorders in human carriers of a loss-of-function single nucleotide polymorphism of PRG-1 (PRG-1R345T/WT). Prg-1R346T/WT animals phenocopied human carriers showing increased anxiety, a depressive phenotype and lower stress resilience. Network analysis revealed that coherence and phase-amplitude coupling were altered by PRG-1 deficiency in memory related circuits in humans and mice alike. Brain oscillation phenotypes were restored by inhibtion of ATX in Prg-1 deficient mice indicating an interventional potential for mental disorders.

Aim: Multisegmental gastrointestinal (GI) dysmotilities, such as chronic intestinal pseudo-obstruction, may lead to life-threatening events. The interdisciplinary INTAKT project is developing wirelessly communicating GI devices for intelligent sensor technology and electrostimulation-based motility modulation. However, a concrete vision of clinical implementation has not been previously described. Methods: An interdisciplinary team of surgeons, gastroenterologists, biologists, engineers, veterinarians and product designers developed a clinical scenario based on pre-clinical experiments, contextual analysis and usage scenarios. Results: The surgical experiences of our previous studies with conventional open, laparoscopic and robotic setups were analyzed. Patient selection, a surgical strategy and perioperative management are described in detail. Conclusion: Further technological solutions may enable first-in-human trials according to our protocol.

Emerging from the development of single-energy Computed Tomography (CT) and Dual-Energy Computed Tomography, Multi-Energy Computed Tomography (MECT) is a promising tool allowing advanced material and tissue decomposition and thereby enabling the use of multiple contrast materials in preclinical research.The scope of this work was to evaluate whether a usual preclinical micro-CT system is applicable for the decomposition of different materials using MECT together with a matrix-inversion method and how different changes of the measurement-environment affect the results.A matrix-inversion based algorithm to differentiate up to five materials (iodine, iron, barium, gadolinium, residual material) by applying four different acceleration voltages/energy levels was established. We carried out simulations using different ratios and concentrations (given in fractions of volume units, VU) of the four different materials (plus residual material) at different noise-levels for 30 keV, 40 keV, 50 keV, 60 keV, 80 keV and 100 keV (monochromatic). Our simulation results were then confirmed by using region of interest-based measurements in a phantom-study at corresponding acceleration voltages. Therefore, different mixtures of contrast materials were scanned using a micro-CT. Voxel wise evaluation of the phantom imaging data was conducted to confirm its usability for future imaging applications and to estimate the influence of varying noise-levels, scattering, artifacts and concentrations.The analysis of our simulations showed the smallest deviation of 0.01 (0.003-0.15) VU between given and calculated concentrations of the different contrast materials when using an energy-combination of 30 keV, 40 keV, 50 keV and 100 keV for MECT. Subsequent MECT phantom measurements, however, revealed a combination of acceleration voltages of 30 kV, 40 kV, 60 kV and 100 kV as most effective for performing material decomposition with a deviation of 0.28 (0-1.07) mg/ml. The feasibility of our voxelwise analyses using the proposed algorithm was then confirmed by the generation of phantom parameter-maps that matched the known contrast material concentrations. The results were mostly influenced by the noise-level and the concentrations used in the phantoms.

The development of realistic dummies for training the distal phalanx amputation (DPA) technique in mouse pups is a promising alternative to reduce and replace animals in training for research and teaching. To test this, we obtained micro-CT data from postnatal day-five mouse pups, meticulously segmented them, and converted them into a 3D mesh format suitable for 3D printing. Once the dummy was printed, it was evaluated during actual training courses in two different groups: in the first group, users received no dummies to train the DPA, and in the second group, users were trained with three dummies. To assess the effectiveness of the dummy, we conducted a survey followed by an expert veterinarian evaluation. Our results showed that DPA is a complex procedure, and it is commonly poorly performed. When implementing the dummies, users who were not provided with dummies to practice only had an 8.3% success rate in DPA, while users provided with three dummies had a 45.5% success rate, respectively. Despite additional research being needed, our dummy offered improved practical training by providing a safe and effective alternative in line with ethical considerations while demonstrating the feasibility of using 3D printing technology to promote the 3Rs in experimental research.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

The Phospholipid Phosphatase Related 4 gene (PLPPR4,  *607813) encodes the Plasticity-Related-Gene-1 (PRG-1) protein. This cerebral synaptic transmembrane-protein modulates cortical excitatory transmission on glutamatergic neurons. In mice, homozygous Prg-1 deficiency causes juvenile epilepsy. Its epileptogenic potential in humans was unknown. Thus, we screened 18 patients with infantile epileptic spasms syndrome (IESS) and 98 patients with benign familial neonatal/infantile seizures (BFNS/BFIS) for the presence of PLPPR4 variants. A girl with IESS had inherited a PLPPR4-mutation (c.896C > G, NM_014839; p.T299S) from her father and an SCN1A-mutation from her mother (c.1622A > G, NM_006920; p.N541S). The PLPPR4-mutation was located in the third extracellular lysophosphatidic acid-interacting domain and in-utero electroporation (IUE) of the Prg-1p.T300S construct into neurons of Prg-1 knockout embryos demonstrated its inability to rescue the electrophysiological knockout phenotype. Electrophysiology on the recombinant SCN1Ap.N541S channel revealed partial loss-of-function. Another PLPPR4 variant (c.1034C > G, NM_014839; p.R345T) that was shown to result in a loss-of-function aggravated a BFNS/BFIS phenotype and also failed to suppress glutamatergic neurotransmission after IUE. The aggravating effect of Plppr4-haploinsufficiency on epileptogenesis was further verified using the kainate-model of epilepsy: double heterozygous Plppr4-/+|Scn1awt|p.R1648H mice exhibited higher seizure susceptibility than either wild-type, Plppr4-/+, or Scn1awt|p.R1648H littermates. Our study shows that a heterozygous PLPPR4 loss-of-function mutation may have a modifying effect on BFNS/BFIS and on SCN1A-related epilepsy in mice and humans.

Das INTAKT-Projekt hat innerhalb der letzten 5 Jahre die präklinische Erforschung und interdisziplinäre technische Entwicklung von vernetzten Implantaten zur Behandlung gastrointestinaler Motilitätsstörungen vorangetrieben. Unter der Rubrik „Videobeitrag“ werden die Meilensteine der Entwicklung verständlich erläutert und Anwendungsszenarios in einer graphischen 3D-Animation veranschaulicht.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Phospholipid levels are influenced by peripheral metabolism. Within the central nervous system, synaptic phospholipids regulate glutamatergic transmission and cortical excitability. Whether changes in peripheral metabolism affect brain lipid levels and cortical excitability remains unknown. Here, we show that levels of lysophosphatidic acid (LPA) species in the blood and cerebrospinal fluid are elevated after overnight fasting and lead to higher cortical excitability. LPA-related cortical excitability increases fasting-induced hyperphagia, and is decreased following inhibition of LPA synthesis. Mice expressing a human mutation (Prg-1R346T) leading to higher synaptic lipid-mediated cortical excitability display increased fasting-induced hyperphagia. Accordingly, human subjects with this mutation have higher body mass index and prevalence of type 2 diabetes. We further show that the effects of LPA following fasting are under the control of hypothalamic agouti-related peptide (AgRP) neurons. Depletion of AgRP-expressing cells in adult mice decreases fasting-induced elevation of circulating LPAs, as well as cortical excitability, while blunting hyperphagia. These findings reveal a direct influence of circulating LPAs under the control of hypothalamic AgRP neurons on cortical excitability, unmasking an alternative non-neuronal route by which the hypothalamus can exert a robust impact on the cortex and thereby affect food intake. Hypothalamic AgRP neurons are shown to control peripheral and central levels of lysophospholipids in association with food deprivation, which leads to cortical excitability, hyperphagia and body weight gain.