Phenotyping Young GluA1 Deficient Mice – A Behavioral Characterization in a Genetic Loss-of-Function Model

Alterations of glutamatergic neurotransmission have been implicated in neurodevelopmental and neuropsychiatric disorders. Mice lacking the GluA1 AMPA receptor subunit, encoded by the Gria1 gene, display multiple phenotypical features associated with glutamatergic dysfunction. While the phenotype of adult GluA1 deficient (Gria1-/- ) mice has been studied comprehensively, there are relevant gaps in knowledge about the course and the onset of behavioral alterations in the Gria1 knockout mouse model during post-weaning development. Based on former investigations in young wild-type mice, we exposed female and male adolescent Gria1-/- mice to a behavioral home-cage based testing battery designed for the purpose of severity assessment. Data obtained from mice with a constitutive loss of GluA1 were compared with those from wild-type littermates. We identified several genotype-dependent behavioral alterations in young Gria1-/- mice. While the preference for sweetness was not affected by genotype during adolescence, Gria1-/- mice displayed limited burrowing performance, and reached lower nest complexity scores. Analysis of home-cage based voluntary wheel running performance failed to confirm genotype-dependent differences. In contrast, when exposed to the open field test, Gria1-/- mice showed pronounced hyperlocomotion in early and late adolescence, and female Gria1 -/- mice exhibited thigmotaxis when prepubescent. We found increased corticosterone metabolite levels in fecal samples of adolescent Gria1-/- mice with females exhibiting increased adrenocortical activity already in prepubescence. Considering the course of behavioral modifications in early and late adolescence, the results do not support a persistent level of distress associated with GluA1 deficiency in the line. In contrast, the laboratory-specific readouts indicate transient, mild impairments of behavioral patterns relevant to animal welfare, and suggest a mild overall burden of the line

Development of behavioral patterns in young C57BL/6J mice: a home cage-based study

Evidence exists that behavioral patterns only stabilize once mice reach adulthood. Detailed information about the course of behavioral patterns is of particular relevance for neuroscientific research and for the assessment of cumulative severity in genetically modified mice. The analysis considered five age groups focusing on behavioral assessments in the animals’ familiar home cage environment during the adolescence phase. We confirmed age- and sex-specific differences for several of the behavioral parameters and fecal corticosterone metabolites. Interestingly, an age-dependent decline in saccharin preference was detected in female mice. Regardless of sex, relevant levels of burrowing activity were only observed during later developmental phases. The development of nest complexity following the offer of new material was affected by age in female mice. In female and male mice, an age-dependency was evident for wheel running reaching a peak at P 50. A progressive increase with age was also observed for Open field activity. The data sets provide guidance for behavioral studies and for development of composite measure schemes for evidence-based severity assessment in young mice. Except for the burrowing test, the different behavioral tests can be applied in different age groups during post-weaning development. However, age- and sex-specific characteristics need to be considered

Cellular localization of Y-box binding protein 1 in brain tissue of rats, macaques, and humans

Background: The Y-box binding protein 1 (YB-1) is considered to be one of the key regulators of transcription and translation. However, so far only limited knowledge exists regarding its cellular distribution in the adult brain. Results: Analysis of YB-1 immunolabelling as well as double-labelling with the neuronal marker NeuN in rat brain tissue revealed a predominant neuronal expression in the dentate gyrus, the cornu ammonis pyramidal cell layer, layer III of the piriform cortex as well as throughout all layers of the parahippocampal cortex. In the hilus of the hippocampus single neurons expressed YB-1. The neuronal expression pattern was comparable in the hippocampus and parahippocampal cortex of adult macaques and humans. Double-labelling of YB-1 with the endothelial cell marker Glut-1, the multidrug transporter P-glycoprotein, and the astrocytic marker GFAP did not indicate a co-localization. Following status epilepticus in rats, no induction of YB-1 occurred in brain capillary endothelial cells and neurons. Conclusion: In conclusion, our study demonstrates that YB-1 is predominantly expressed in neurons in the adult brain of rats, macaques and humans. Lack of a co-localization with Glut-1 and P-glycoprotein argues against a direct role of YB-1 in the regulation of blood-brain barrier P-glycoprotein

The impact of tethered recording techniques on activity and sleep patterns in rats

Funding Information: The project was supported by grants of Deutsche Forschungsgemeinschaft (FOR 2591, GZ: PO681/9-1 and 9-2). The authors thank Sarah Glisic, Helen Stirling, Claudia Siegl, Sieglinde Fischlein, Andreas Kutschka and Isabella Waclawczyk for their excellent technical assistance. The authors thank Helen Stirling for language revision. Open Access funding enabled and organized by Projekt DEALPeer reviewedPublisher PD

Molecular alterations of the TLR4-signaling cascade in canine epilepsy

Background: Cumulating evidence from rodent models points to a pathophysiological role of inflammatory signaling in the epileptic brain with Toll-like receptor-4 signaling acting as one key factor. However, there is an apparent lack of information about expression alterations affecting this pathway in canine patients with epilepsy. Therefore, we have analyzed the expression pattern of Toll-like receptor 4 and its ligands in brain tissue of canine patients with structural or idiopathic epilepsy in comparison with tissue from laboratory dogs or from owner-kept dogs without neurological diseases. Results: The analysis revealed an overexpression of Toll-like receptor-4 in the CA3 region of dogs with structural epilepsy. Further analysis provided evidence for an upregulation of Toll-like receptor-4 ligands with high mobility group box-1 exhibiting increased expression levels in the CA1 region of dogs with idiopathic and structural epilepsy, and heat shock protein 70 exhibiting increased expression levels in the piriform lobe of dogs with idiopathic epilepsy. In further brain regions, receptor and ligand expression rates proved to be either in the control range or reduced below control levels. Conclusions: Our study reveals complex molecular alterations affecting the Toll-like receptor signaling cascade, which differ between epilepsy types and between brain regions. Taken together, the data indicate that multi-targeting approaches modulating Toll-like receptor-4 signaling might be of interest for management of canine epilepsy. Further studies are recommended to explore respective molecular alterations in more detail in dogs with different etiologies and to confirm the role of the pro-inflammatory signaling cascade as a putative target

Pregabalin Add-On vs. Dose Increase in Levetiracetam Add-On Treatment: A Real-Life Trial in Dogs With Drug-Resistant Epilepsy

Epilepsy is a common neurological disorder affecting 0.6–0.75% of dogs in veterinary practice. Treatment is frequently complicated by the occurrence of drug-resistant epilepsy and cluster seizures in dogs with idiopathic epilepsy. Only few studies are available to guide treatment choices beyond licensed veterinary drugs. The aim of the study was to compare antiseizure efficacy and tolerability of two add-on treatment strategies in dogs with drug-resistant idiopathic epilepsy. The study design was a prospective, open-label, non-blinded, comparative treatment trial. Treatment success was defined as a 3-fold extension of the longest baseline interseizure interval and to a minimum of 3 months. To avoid prolonged adherence to a presumably ineffective treatment strategy, dog owners could leave the study after the third day with generalized seizures if the interseizure interval failed to show a relevant increase. Twenty-six dogs (mean age 5.5 years, mean seizure frequency 4/month) with drug-resistant idiopathic epilepsy and a history of cluster seizures were included. Dogs received either add-on treatment with pregabalin (PGB) 4 mg/kg twice daily (14 dogs) or a dose increase in levetiracetam (LEV) add-on treatment (12 dogs). Thirteen dogs in the PGB group had drug levels within the therapeutic range for humans. Two dogs in the PGB group (14.3%; 2/14) and one dog in the LEV group (8.3%; 1/12) achieved treatment success with long seizure-free intervals from 122 to 219 days but then relapsed to their early seizure frequency 10 months after the study inclusion. The overall low success rates with both treatment strategies likely reflect a real-life situation in canine drug-resistant idiopathic epilepsy in everyday veterinary practice. These results delineate the need for research on better pharmacologic and non-pharmacologic treatment strategies in dogs with drug-resistant epilepsy

Grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats-A systematic review

Several studies suggested an informative value of behavioral and grimace scale parameters for the detection of pain. However, the robustness and reliability of the parameters as well as the current extent of implementation are still largely unknown. In this study, we aimed to systematically analyze the current evidence-base of grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats. The following platforms were searched for relevant articles: PubMed, Embase via Ovid, and Web of Science. Only full peer-reviewed studies that describe the grimace scale, burrowing, and/or nest building as pain parameters in the post-surgical phase in mice and/or rats were included. Information about the study design, animal characteristics, intervention characteristics, and outcome measures was extracted from identified publications. In total, 74 papers were included in this review. The majority of studies have been conducted in young adult C57BL/6J mice and Sprague Dawley and Wistar rats. While there is an apparent lack of information about young animals, some studies that analyzed the grimace scale in aged rats were identified. The majority of studies focused on laparotomy-associated pain. Only limited information is available about other types of surgical interventions. While an impact of surgery and an influence of analgesia were rather consistently reported in studies focusing on grimace scales, the number of studies that assessed respective effects was rather low for nest building and burrowing. Moreover, controversial findings were evident for the impact of analgesics on post-surgical nest building activity. Regarding analgesia, a monotherapeutic approach was identified in the vast majority of studies with non-steroidal anti-inflammatory (NSAID) drugs and opioids being most commonly used. In conclusion, most evidence exists for grimace scales, which were more frequently used to assess post-surgical pain in rodents than the other behavioral parameters. However, our findings also point to relevant knowledge gaps concerning the post-surgical application in different strains, age levels, and following different surgical procedures. Future efforts are also necessary to directly compare the sensitivity and robustness of different readout parameters applied for the assessment of nest building and burrowing activities

Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling.

ABSTRACT Increased expression of drug efflux transporters at the bloodbrain barrier accompanies epileptic seizures and complicates therapy with antiepileptic drugs. This study is concerned with identifying mechanistic links that connect seizure activity to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures increase brain extracellular glutamate, which signals through an N-methyl-D-aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor; no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy and possibly other central nervous system disorders. Up to 40% of epileptic patients respond poorly if at all to conventional pharmacotherapy, and impaired drug uptake into the brain is considered to be one important contributor to therapeutic failure The present study is concerned with mechanistic links that connect seizure activity to increased P-glycoprotein expression. Our goals are to identify therapeutic targets that can be manipulated to prevent seizure-induced transporter overexpression and to improve pharmacotherapy with antiepileptic drugs. The combined in vitro/in vivo experiments are focuse

Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling.

ABSTRACT Increased expression of drug efflux transporters at the bloodbrain barrier accompanies epileptic seizures and complicates therapy with antiepileptic drugs. This study is concerned with identifying mechanistic links that connect seizure activity to increased P-glycoprotein expression at the blood-brain barrier. In this regard, we tested the hypothesis that seizures increase brain extracellular glutamate, which signals through an N-methyl-D-aspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) in brain capillaries to increase blood-brain barrier P-glycoprotein expression. Consistent with this hypothesis, exposing isolated rat or mouse brain capillaries to glutamate for 15 to 30 min increased P-glycoprotein expression and transport activity hours later. These increases were blocked by 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801), an NMDA receptor antagonist, and by celecoxib, a selective COX-2 inhibitor; no such glutamate-induced increases were seen in brain capillaries from COX-2-null mice. In rats, intracerebral microinjection of glutamate caused locally increased P-glycoprotein expression in brain capillaries. Moreover, using a pilocarpine status epilepticus rat model, we observed seizure-induced increases in capillary P-glycoprotein expression that were attenuated by administration of indomethacin, a COX inhibitor. Our findings suggest that brain uptake of some antiepileptic drugs can be enhanced through COX-2 inhibition. Moreover, they provide insight into one mechanism that underlies drug resistance in epilepsy and possibly other central nervous system disorders. Up to 40% of epileptic patients respond poorly if at all to conventional pharmacotherapy, and impaired drug uptake into the brain is considered to be one important contributor to therapeutic failure The present study is concerned with mechanistic links that connect seizure activity to increased P-glycoprotein expression. Our goals are to identify therapeutic targets that can be manipulated to prevent seizure-induced transporter overexpression and to improve pharmacotherapy with antiepileptic drugs. The combined in vitro/in vivo experiments are focuse