Short-term stimulations of the entopeduncular nucleus induce cerebellar changes of c-Fos expression in an animal model of paroxysmal dystonia.

Deep brain stimulation (DBS) of the globus pallidus internus (entopeduncular nucleus, EPN, in rodents) is important for the treatment of drug-refractory dystonia. The pathophysiology of this movement disorder and the mechanisms of DBS are largely unknown. Insights into the mechanisms of DBS in animal models of dystonia can be helpful for optimization of DBS and add-on therapeutics. We recently found that short-term EPN-DBS with 130 Hz (50 µA, 60 µs) for 3 h improved dystonia in dtsz hamsters and reduced spontaneous excitatory cortico-striatal activity in brain slices of this model, indicating fast effects on synaptic plasticity. Therefore, in the present study, we examined if these effects are related to changes of c-Fos, a marker of neuronal activity, in brains derived from dtsz hamsters after these short-term DBS or sham stimulations. After DBS vs. sham, c-Fos intensity was increased around the electrode, but the number of c-Fos+ cells was not altered within the whole EPN and projection areas (habenula, thalamus). DBS did not induce changes in striatal and cortical c-Fos+ cells as GABAergic (GAD67+ and parvalbumin-reactive) neurons in motor cortex and striatum. Unexpectedly, c-Fos+ cells were decreased in deep cerebellar nuclei (DCN) after DBS, suggesting that cerebellar changes may be involved in antidystonic effects already during short-term DBS. However, the present results do not exclude functional changes within the basal ganglia-thalamo-cortical network, which will be further investigated by long-term EPN stimulations. The present study indicates that the cerebellum deserves attention in ongoing examinations on the mechanisms of DBS in dystonia.

Electrophysiological insights into deep brain stimulation of the network disorder dystonia.

Deep brain stimulation (DBS), a treatment for modulating the abnormal central neuronal circuitry, has become the standard of care nowadays and is sometimes the only option to reduce symptoms of movement disorders such as dystonia. However, on the one hand, there are still open questions regarding the pathomechanisms of dystonia and, on the other hand, the mechanisms of DBS on neuronal circuitry. That lack of knowledge limits the therapeutic effect and makes it hard to predict the outcome of DBS for individual dystonia patients. Finding electrophysiological biomarkers seems to be a promising option to enable adapted individualised DBS treatment. However, biomarker search studies cannot be conducted on patients on a large scale and experimental approaches with animal models of dystonia are needed. In this review, physiological findings of deep brain stimulation studies in humans and animal models of dystonia are summarised and the current pathophysiological concepts of dystonia are discussed.

Fenbendazole treatment against Dentostomella translucida in Syrian golden hamsters.

Dentostomella translucida is an oxyurid nematode that was first discovered in the Mongolian gerbil but has also been detected in other wild and housed rodents. In conventional laboratory animals, oxyurid nematode parasites are widespread infections. A proven treatment strategy for pinworm eradication is the oral application of benzimidazoles, such as fenbendazole. In general, this drug is regarded as safe with minimal side effects. Nevertheless, in Sprague Dawley rats, a significantly reduced litter size could be seen after longer treatment with fenbendazole. Even though Dentostomella translucida was already described in Syrian golden hamsters (Mesocricetus auratus), data on treatment with fenbendazole and its effects on reproduction is lacking. Therefore, the main purposes of the study were (1) the verification of the effectiveness of fenbendazole as medicated feed (150 ppm) against this parasite in naturally infected Syrian golden hamsters in conventional husbandry and (2) monitoring of possible effects on reproduction during the treatment. Results show that fenbendazole treatment was highly effective against Dentostomella translucida, as numbers of pinworm eggs in the faeces were significantly reduced already after the first week of treatment in all animals. After four weeks of treatment, eggs were eradicated entirely. Interestingly, the average weaning weight was significantly reduced during treatment, but the litters were in good health.

In vivo optogenetic inhibition of striatal parvalbumin-reactive interneurons induced genotype-specific changes in neuronal activity without dystonic signs in male DYT1 knock-in mice.

The pathophysiology of early-onset torsion dystonia (TOR1A/DYT1) remains unclear. Like 70% of human mutation carriers, rodent models with ΔGAG mutation such as DYT1 knock-in (KI) mice do not show overt dystonia but have subtle sensorimotor deficits and pattern of abnormal synaptic plasticity within the striatal microcircuits. There is evidence that dysfunction of striatal parvalbumin-reactive (Parv+) fast-spiking interneurons (FSIs) can be involved in dystonic signs. To elucidate the relevance of these GABAergic interneurons in the pathophysiology of DYT1 dystonia, we used in vivo optogenetics to specifically inhibit Parv+ and to detect changes in motor behavior and neuronal activity. Optogenetic fibers were bilaterally implanted into the dorsal striatum of male DYT1 KI mice and wild-type (WT) littermates expressing halorhodopsin (eNpHR3.0) in Parv+ interneurons. While stimulations with yellow light pulses for up to 60 min at different pulse durations and interval lengths did not induce abnormal movements, such as dystonic signs, immunohistochemical examinations revealed genotype-dependent differences. In contrast to WT mice, stimulated DYT1 KI showed decreased striatal neuronal activity, that is, less c-Fos reactive neurons, and increased activation of cholinergic interneurons after optogenetic inhibition of Parv+ interneurons. These findings suggest an involvement of Parv+ interneurons in an impaired striatal network in DYT1 KI mice, but at least short-term inhibition of these GABAergic interneurons is not sufficient to trigger a dystonic phenotype, similar to previously shown optogenetic activation of cholinergic interneurons.

Sensitive and simultaneous quantification of 16 neurotransmitters and metabolites in murine microdialysate by fast liquid chromatography-tandem mass spectrometry.

The sensitive and simultaneous measurement of multiple neurotransmitters in microdialysate (MD) of freely moving mice is a prerequisite to study neurochemical imbalances in specific brain regions. The quantitative analysis of 16 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (l-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), homovanillinic acid (HVA), acetylcholine (ACh), deoxy carnitine (iso-ACh), choline (Ch), and É£-aminobutyric acid (GABA), adenosine (ADE), glutamine (Gln), and glutamic acid (Glu) was achieved within a chromatographic separation time of 6.5 min by the application of a biphenyl column coupled to an API-QTrap 5500 (AB SCIEX) mass spectrometer. Optimized chromatographic separation as well as high sensitivity allow the simultaneous analysis and precise quantification of 16 neurotransmitters and metabolites in artificial cerebrospinal fluid (CSF). Sample preparation procedure consisted of simply adding isotopically labeled internal standard solution to the microdialysis sample. The limits of detection in aCSF ranged from 0.025 pg (Ch) to 9.75 pg (Gln) and 85.5 pg (HVA) on column. Recoveries were between 83 and 111% for neurotransmitter concentrations from 0.6 to 45 ng/ml or 200 ng/ml with a mean intra-day and inter-day coefficient of variation of 7.6% and 11.2%, respectively. Basal extracellular concentrations of the following analytes: 5-HT, 5-HIAA, ME, DA, 3-MT, HVA, ACh, iso-ACh, Ch, GABA, ADE, Gln, and Glu were determined in the striatum of mice with a MD flow rate of 0.5 µl/min. This LC-MS/MS method leads to an accurate quantification of ACh and its isobaric structure iso-ACh, which were detected in the MD samples at ratios of 1:8.6. The main advantage of the high sensitivity is the miniaturization of the MD protocol with short sample collection times and volumes down to 5 µl, which makes this method suitable for pharmacological intervention and optogenetic studies to detect neurochemical changes in vivo.

Deep brain stimulation in animal models of dystonia.

During the last decades deep brain stimulation (DBS) has become an important treatment option for a variety of neurological disorders such as drug-intractable dystonia. Yet, the mechanisms of action of DBS are still largely unknown. Dystonia is a heterogenous movement disorder characterized by involuntary muscle contractions causing abnormal movements, postures, or both. The underlying pathophysiological processes remain unclear, but a dysfunction of the basal ganglia circuit is critically involved as supported by the effectiveness of DBS of the globus pallidus internus (GPi) in various types of dystonia. However, the degree of clinical improvement differs among the types of dystonia, as well as from patient to patient, and the delayed response to GPi-DBS in dystonia patients hampers the adjustment and optimization of stimulation parameters. Preclinical studies in suitable animal models can contribute decisively to detect the underlying mechanisms of DBS and biomarkers, to identify new possible stimulation targets and to optimize stimulation patterns. In this review, we give an overview of previous research on DBS in animal models of dystonia. With regard to the aims of research we discuss the opportunities and limitations concerning different available animal models of dystonia and technical challenges.

Deep brain stimulation for movement disorder treatment: exploring frequency-dependent efficacy in a computational network model.

A large-scale computational model of the basal ganglia network and thalamus is proposed to describe movement disorders and treatment effects of deep brain stimulation (DBS). The model of this complex network considers three areas of the basal ganglia region: the subthalamic nucleus (STN) as target area of DBS, the globus pallidus, both pars externa and pars interna (GPe-GPi), and the thalamus. Parkinsonian conditions are simulated by assuming reduced dopaminergic input and corresponding pronounced inhibitory or disinhibited projections to GPe and GPi. Macroscopic quantities are derived which correlate closely to thalamic responses and hence motor programme fidelity. It can be demonstrated that depending on different levels of striatal projections to the GPe and GPi, the dynamics of these macroscopic quantities (synchronisation index, mean synaptic activity and response efficacy) switch from normal to Parkinsonian conditions. Simulating DBS of the STN affects the dynamics of the entire network, increasing the thalamic activity to levels close to normal, while differing from both normal and Parkinsonian dynamics. Using the mentioned macroscopic quantities, the model proposes optimal DBS frequency ranges above 130 Hz.

The software defined implantable modular platform (STELLA) for preclinical deep brain stimulation research in rodents.

Context.Long-term deep brain stimulation (DBS) studies in rodents are of crucial importance for research progress in this field. However, most stimulation devices require jackets or large head-mounted systems which severely affect mobility and general welfare influencing animals' behavior.Objective.To develop a preclinical neurostimulation implant system for long-term DBS research in small animal models.Approach.We propose a low-cost dual-channel DBS implant called software defined implantable platform (STELLA) with a printed circuit board size of Ø13 × 3.3 mm, weight of 0.6 g and current consumption of 7.6µA/3.1 V combined with an epoxy resin-based encapsulation method.Main results.STELLA delivers charge-balanced and configurable current pulses with widely used commercial electrodes. Whilein vitrostudies demonstrate at least 12 weeks of error-free stimulation using a CR1225 battery, our calculations predict a battery lifetime of up to 3 years using a CR2032. Exemplary application for DBS of the subthalamic nucleus in adult rats demonstrates that fully-implanted STELLA neurostimulators are very well-tolerated over 42 days without relevant stress after the early postoperative phase resulting in normal animal behavior. Encapsulation, external control and monitoring of function proved to be feasible. Stimulation with standard parameters elicited c-Fos expression by subthalamic neurons demonstrating biologically active function of STELLA.Significance.We developed a fully implantable, scalable and reliable DBS device that meets the urgent need for reverse translational research on DBS in freely moving rodent disease models including sensitive behavioral experiments. We thus add an important technology for animal research according to 'The Principle of Humane Experimental Technique'-replacement, reduction and refinement (3R). All hardware, software and additional materials are available under an open source license.

Mechanisms of pallidal deep brain stimulation: Alteration of cortico-striatal synaptic communication in a dystonia animal model.

Pallidal deep brain stimulation (DBS) is an important option for patients with severe dystonias, which are thought to arise from a disturbance in striatal control of the globus pallidus internus (GPi). The mechanisms of GPi-DBS are far from understood. Although a disturbance of striatal function is thought to play a key role in dystonia, the effects of DBS on cortico-striatal function are unknown. We hypothesised that DBS, via axonal backfiring, or indirectly via thalamic and cortical coupling, alters striatal function. We tested this hypothesis in the dtsz hamster, an animal model of inherited generalised, paroxysmal dystonia. Hamsters (dystonic and non-dystonic controls) were bilaterally implanted with stimulation electrodes in the GPi. DBS (130 Hz), and sham DBS, were performed in unanaesthetised animals for 3 h. Synaptic cortico-striatal field potentials, as well as miniature excitatory postsynaptic currents (mEPSC) and firing properties of medium spiny striatal neurones were recorded in brain slice preparations obtained immediately after EPN-DBS. The main findings were as follows: a. DBS increased cortico-striatal evoked responses in healthy, but not in dystonic tissue. b. Commensurate with this, DBS increased inhibitory control of these evoked responses in dystonic, and decreased inhibitory control in healthy tissue. c. Further, DBS reduced mEPSC frequency strongly in dystonic, and less prominently in healthy tissue, showing that also a modulation of presynaptic mechanisms is likely involved. d. Cellular properties of medium-spiny neurones remained unchanged. We conclude that DBS leads to dampening of cortico-striatal communication, and restores intrastriatal inhibitory tone.

Systematic Review of the Pharmacological Evidence for the Selection of Antimicrobials in Bacterial Infections of the Central Nervous System in Dogs and Cats.

Bacterial meningitis in dogs and cats is a rare disease associated with a high lethality rate. The spectrum of causative bacteria includes a diverse set of gram positive, gram negative and anaerobic species. Currently, no veterinary medicinal product is approved for this indication in these species in Europe. The objective of this review was to collect the available pharmacokinetic data for antibiotics approved in dogs and cats to enable a preliminary analysis of their potential effectiveness for the treatment of bacterial meningitis. This analysis yielded data for 13 different antibiotics in dogs and two in cats. Additionally, data about frequently recommended cephalosporines not approved in dogs and cats were included. The collected data was used to assess the potential of the respective antibiotics to attain certain simple pharmacokinetic-pharmacodynamic (PK-PD) indexes in the cerebrospinal fluid (CSF). A more sophisticated investigation using modern methods was not possible due to the limited data available. For this purpose, data about the sensitivity of four bacterial species commonly associated with meningitis in dogs and cats to these antibiotics were included. The analysis provided evidence for the potential effectiveness of ampicillin, doxycycline, enrofloxacin, ceftriaxone and cefoxitin against bacteria frequently detected in bacterial meningitis in dogs. Data were not available or insufficient for the assessment of several antibiotics, including frequently recommended substances like metronidazole and trimethoprim-sulphonamide. Little evidence is available for the use of antibiotics in cats afflicted with this disease, highlighting the need for further research to obtain data for evidence based therapeutic recommendations.

Deep brain stimulation by optimized stimulators in a phenotypic model of dystonia: Effects of different frequencies.

Deep brain stimulation (DBS) of the globus pallidus internus (GPi, entopeduncular nucleus, EPN, in rodents) has become important for the treatment of generalized dystonia, a severe and often intractable movement disorder. It is unclear if lower frequencies of GPi-DBS or stimulations of the subthalamic nucleus (STN) are of advantage. In the present study, the main objective was to examined the effects of bilateral EPN-DBS at different frequencies (130 Hz, 40 Hz, 15 Hz) on the severity of dystonia in the dtsz mutant hamster. In addition, STN stimulations were done at a frequency, proven to be effective by the present EPN-DBS in dystonic hamsters. In order to obtain precise bilateral electrical stimuli with magnitude of 50 µA, a pulse width of 60 µs and defined frequencies, it was necessary to develop a new optimized stimulator prior to the experiments. Since the individual highest severity of dystonic episodes is known to be reached within three hours after induction in dtsz hamsters, the duration of DBS was 180 min. During DBS with 130 Hz the severity of dystonia was significantly lower within the third hour than without DBS in the same animals (p < 0.05). DBS with 40 Hz tended to exert antidystonic effects after three hours, while 15 Hz stimulations of the EPN and 130 Hz stimulations of the STN failed to show any effects on the severity. DBS of the EPN at 130 Hz was most effective against generalized dystonia in the dtsz mutant. The response to EPN-DBS confirms that the dtsz mutant is suitable to further investigate the effects of long-term DBS on severity of dystonia and neuronal network activities, important to give insights into the mechanisms of DBS.

Development of a fast liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in murine microdialysate.

The continuous measurement of multiple neurotransmitters in microdialysate of freely moving mice to study neurochemical changes in specific brain regions requires a rapid and very sensitive quantitative analytical method. The quantitative analysis of 11 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (L-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), acetylcholine (ACh), choline (Ch), and γ-aminobutyric acid (GABA), was performed using a biphenyl column coupled to an API-QTrap 3200 (AB SCIEX) mass spectrometer in positive electrospray ionization mode. To the microdialysate samples, 0.5 ng of isotopically labeled standard was added for analyte quantification. A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous analysis of monoamines, their precursor, and metabolites, as well as ACh, Ch, and GABA in murine microdialysate within 7.0 min. The limit of detection in artificial CSF ranged from 0.005 ng/mL (ME) to 0.75 ng/mL (NE and GABA). A comprehensive pre-analytical protocol was validated. Recovery was between 87 and 117% for neurotransmitter concentrations from 0.6 to 45 ng/mL with an inter-day accuracy of below 20%. Basal neurotransmitter values were determined in the striatum of mice over a time period of 3 h. This LC-MS/MS method, including a short and gentle sample preparation, is suitable for simultaneous measurements of neurotransmitters in murine cerebral microdialysate and enables the determination of basal neurotransmitter levels in specific brain regions to detect disease-related and drug-induced neurochemical changes.Graphical abstract.

Striatal and cortical metabotropic glutamate 5 receptor expression and behavioral effects of the positive allosteric modulator CDPPB in a model of DYT1 dystonia.

The metabotropic glutamate 5 (mGlu5) receptor is critically involved in corticostriatal plasticity which is disturbed in various animal models of dystonia. Recently, the positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) exerted prodyskinetic effects in a phenotypic model of episodic dystonia. In the DYT1 knock-in (KI) mouse, a model for a persistent type of dystonia, previous ex vivo electrophysiological experiments indicated that mGlu5 receptors are involved in abnormal striatal plasticity. Therefore, in the present study we examined the mGlu5 receptor expression in the striatum and cortex of DYT1 KI mice in comparison with wildtype littermates. By immunohistochemistry (IHC) we found a lower expression of mGlu5 receptors in the cortex (16%) and ventral striatum (10%) but not in the whole striatum of DYT1 KI mice, while mRNA levels were merely lower in the striatum of DYT1 KI mice (43%). However, mGlu5 receptor protein levels measured by western blotting showed no significant differences in tissue of the whole striatum and in the cortex between both genotypes. Since DYT1 KI mice do not exhibit dystonic symptoms, we investigated if CDPPB provokes dystonia or dyskinesia. CDPPB (10, 20 and 30 mg/kg intraperitoneal, i.p.) did not induce abnormal movements and the locomotor activity did not differ between DYT1 KI and wildtype mice. The present data do not provide evidence for a crucial role of the mGlu5 receptor in the pathophysiology of DYT1 dystonia, but corticostriatal changes are in line with the hypothesis of maladaptive plasticity in dystonia.

Reasons for antimicrobial treatment failures and predictive value of in-vitro susceptibility testing in veterinary practice: An overview.

The choice of the most suitable antimicrobial agent for the treatment of an animal suffering from a bacterial infection is a complex issue. The results of bacteriological diagnostics and the in-vitro antimicrobial susceptibility testing (AST) provide guidance of potentially suitable antimicrobials. However, harmonized AST methods, veterinary-specific interpretive criteria and quality control ranges, which are essential to conduct AST in-vitro and to evaluate the corresponding results lege artis, are not available for all antimicrobial compounds, bacterial pathogens, animal species and sites of infection of veterinary relevance. Moreover, the clinical benefit of an antimicrobial agent (defined as its in vivo efficacy) is not exclusively dependent on the in-vitro susceptibility of the target pathogen. Apart from the right choice of an antibacterial drug with suitable pharmacokinetic properties and an appropriate pharmaceutical formulation, the success of treatment depends substantially on its adequate use. Even if this is ensured and in-vitro susceptibility confirmed, an insufficient improvement of clinical signs might be caused by biofilm-forming bacteria, persisters, or specific physicochemical conditions at the site of infection, such as pH value, oxygen partial pressure and perfusion rate. This review summarizes relevant aspects that have an impact on the predictive value of in-vitro AST and points out factors, potentially leading to an ineffective outcome of antibacterial treatment in veterinary practice. Knowing the reasons of inadequate beneficial effects can help to understand possible discrepancies between in-vitro susceptibility and in vivo efficacy and aid in undertaking strategies for an avoidance of treatment failures.

Subtle changes in striatal muscarinic M1 and M4 receptor expression in the DYT1 knock-in mouse model of dystonia.

In early-onset generalized torsion dystonia, caused by a GAG deletion in TOR1A (DYT1), enhanced striatal cholinergic activity has been suggested to be critically involved. Previous studies have shown increased acetylcholine levels in the striatum of DYT1 knock-in (KI) mice. Ex vivo data indicated that muscarinic receptor antagonists normalize the activity of striatal cholinergic interneurons. Currently receptor subtype specific antagonists are developed for therapy, however, it is yet unknown whether the levels of targeted receptors are unaltered. In the present study, we firstly examined the expression of M1 and M4 receptors in DYT1 KI mice in comparison to wildtype mice. While no changes in mRNA were found in the motor cortex, the expression of M1 was higher in the striatum of DYT1 KI. However, M1 protein did not differ in striatum and cortex between the animal groups as shown by immunohistochemistry and western blot. M4 receptor protein, unaltered in the cortex, was slightly lower in lateral subparts of the striatum, but unchanged in somata of cholinergic interneurons and substance P immunoreactive projection neurons. Functional alterations of the cholinergic system and of aberrant striatal plasticity, demonstrated by previous studies, seem not to be related to overt changes in M1 and M4 expression. This critically informs the ongoing development of respective antagonists for therapy of dystonia.

Systematic analysis to assess the scientific validity of the international residue limits for caffeine and theophylline in horse-racing.

Based on their performance-enhancing potential, caffeine and theophylline are prohibited substances in equine sports. Residues in horses can be caused by wilful application or by unintended uptake of contaminated feed. The International Federation of Horseracing Authorities recently introduced international residue limits (IRLs) to facilitate the discrimination between pharmacological relevant and irrelevant concentrations in doping samples. The objective of this study was to investigate the scientific validity of these IRLs. A systematic analysis was performed to assess the IRLs by different statistical approaches using published pharmacokinetic data. 31 out of 218 potentially relevant publications met the inclusion criteria. Thereby, both IRLs were found to be appropriate for the exclusion of the presence of a relevant pharmacological effect after a wilful application. The IRL of theophylline was also determined to be suitable for the prevention of positive doping tests caused by the ingestion of contaminated feed. In contrast, the IRL of caffeine is not suitable to prevent positive doping test caused by the ingestion of more than 10 mg caffeine per day per horse with contaminated feed. The lack of corresponding regulation for paraxanthine, a major active metabolite of caffeine and theophylline, was recognised as a substantial shortcoming of the current system, rendering both IRLs incomplete.

Residue concentration of cefquinome taking into account different milk fractions and comparing the performance of two screening tests.

This Research Communication describes the residue concentration of a dry cow antibiotic in two different milk fractions and describes effects of milk fraction and milk composition on the test performance of a rapid screening and a microbial inhibitor test. Thirteen dry cows were treated with an intramammary dry cow antibiotic containing 150 mg cefquinome. Quarter foremilk and stripping samples were collected on the first 10 d postpartum. All milk samples were analyzed for milk composition by the local Dairy Herd Improvement Association and were tested for antibiotic residues using the rapid screening test Milchtest BL and the microbial inhibitor test Delvotest BR Brilliant Plates. The residue concentration of cefquinome was determined in foremilk and stripping samples from milkings 1, 2, 3, 5, and 7 after calving using high performance liquid chromatography - tandem mass spectrometry. The logarithm of cefquinome concentration (logCef) was higher in foremilk than in stripping samples and higher in milk samples with lower lactose content. Furthermore, logCef decreased with the number of milkings (P < 0.001). The Milchtest BL was more likely to be not evaluated (i.e. no test and control line or no control line appeared) in stripping samples and milk samples with higher protein content. In the Delvotest BR Brilliant Plates milk samples with higher protein content were more likely to have a false positive result (i.e. the screening test result was positive, but the HPLC-MS/MS result was below the detection limit of the screening test). These results indicate that foremilk is the recommended milk fraction to be tested for residues of cefquinome and that a high protein content can be a cause of test failure and false positive results when milk during the first 10 d postpartum is tested for antibiotic residues using screening tests.

Expression of metabotropic glutamate 5 receptors in the striatum and cortex and effects of modulators on the severity of dystonia in the phenotypic dtsz model.

The metabotropic glutamate 5 (mGlu5) receptor has been suggested as therapeutic target for L-Dopa-induced dyskinesia which is often associated with dystonic symptoms. Therefore, we investigated the acute effects of the non-competitive mGlu5 receptor antagonist fenobam as well as the positive modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the severity of inherited dystonia in the mutant dtsz hamster, a phenotypic model with age-dependent episodes of dystonia. Fenobam did not exert significant antidystonic effects (20-50 mg/kg intraperinoneal, i.p.). CDPPB (10, 20 mg/kg i.p.) which was expected to worsen dystonia also failed to show any effects on the severity of dystonia. Interestingly, CDPPB caused axial dyskinesia in addition to the dystonic symptoms in mutant hamsters. This adverse effect could not be observed in non-dystonic control hamsters, indicating possible changes in the expression of mGlu5 receptors in dystonic hamsters. The mGlu5 receptor mRNA did not differ between the dtsz mutant and control hamsters, while immunohistochemical studies indicated that the mGlu5 receptor expression was about 35% higher in striatum and cortex of mutant hamsters at the age of high dystonia severity scores, notably not after spontaneous remission of dystonia, compared to age-matched controls. This difference in mGlu5 receptor protein may be due to altered protein conformation instead of protein level, as western blots revealed similar amounts of monomeric and dimeric protein in mutant hamsters versus control. Thus, the present data do not provide clear evidence for an important role of the mGlu5 receptor in the pathophysiology and as a therapeutic target for types of inherited dystonia.

Optogenetic augmentation of the hypercholinergic endophenotype in DYT1 knock-in mice induced erratic hyperactive movements but not dystonia.

BACKGROUND: The most prevalent inherited form of generalized dystonia is caused by a mutation in torsinA (DYT1, ∆GAG) with incomplete penetrance. Rodent models with mutated torsinA do not develop dystonic symptoms, but previous ex vivo studies indicated abnormal excitation of cholinergic interneurons (ChI) and increased striatal acetylcholine. METHODS: We used in vivo optogenetics to exacerbate this endophenotype in order to determine its capacity to trigger dystonic symptoms in freely behaving mice. Tor1a+/Δgag DYT1 mice and wildtype littermates expressing channelrhodopsin2 under the Chat promotor were implanted bilaterally with optical LED cannulae and stimulated with blue light pulses of varied durations. FINDINGS: Six months old DYT1 KI mice but not wildtype controls responded with hyperactivity to blue light specifically at 25 ms pulse duration, 10 Hz frequency. Neuronal activity (c-Fos) in cholinergic interneurons was increased immediately after light stimulation and persisted only in DYT1 KI over 15 min. Substance P was increased specifically in striosome compartments in naïve DYT1 KI mice compared to wildtype. Under optogenetic stimulation substance P increased in wildtype to match levels in Dyt1 KI, and acetylcholinesterase was elevated in the striatum of stimulated DYT1 KI. No signs of dystonic movements were observed under stimulation of up to one hour in both genotypes and age groups, and the sensorimotor deficit previously observed in 6 months old DYT1 KI mice persisted under stimulation. INTERPRETATION: Overall this supports an endophenotype of dysregulated cholinergic activity in DYT1 dystonia, but depolarizing cholinergic interneurons was not sufficient to induce overt dystonia in DYT1 KI mice.

Behavioral phenotyping of calcium channel (CACN) subunit α2δ3 knockout mice: Consequences of sensory cross-modal activation.

Sensory cross-activation is still ill-defined and research concerning the consequences of sensory mergence on normal brain function is very limited. Human studies describe behavioral benefits of people with synesthesia- a peculiar form of perception possibly due to cross-modal activation- regarding sensory and memory abilities. Here, we studied behavioral alterations in calcium channel (CACN) subunit α2δ3 knockout (KO) mice exhibiting pain-induced cortical cross-modal activation. Knockout mice exhibited an increased response upon touch of a pinna and impaired audition, while elementary olfaction, vision, somatosensation and motor function were not altered. In contrast to synesthetic humans for whom enhanced memory function had been described, α2δ3 KO mice might have developed defects for object-based memory. However, in a task requiring use of multiple modalities, mutant mice revealed an enhanced performance compared to wild-type controls. Furthermore, several tests revealed evidence for increased anxiety-like behavior of α2δ3 KO animals. In summary, deficits in single sensory abilities and a potential gain in processing simultaneous sensory information in α2δ3 KO mice might represent behavioral correlates of sensory cross-activation. Further, our data suggest a role of CACNα2δ3 within the functionality of the sensory system, but not the motor system and general health.