Refinements to rodent head fixation and fluid/food control for neuroscience.

The use of head fixation in mice is increasingly common in research, its use having initially been restricted to the field of sensory neuroscience. Head restraint has often been combined with fluid control, rather than food restriction, to motivate behaviour, but this too is now in use for both restrained and non-restrained animals. Despite this, there is little guidance on how best to employ these techniques to optimise both scientific outcomes and animal welfare. This article summarises current practices and provides recommendations to improve animal wellbeing and data quality, based on a survey of the community, literature reviews, and the expert opinion and practical experience of an international working group convened by the UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Topics covered include head fixation surgery and post-operative care, habituation to restraint, and the use of fluid/food control to motivate performance. We also discuss some recent developments that may offer alternative ways to collect data from large numbers of behavioural trials without the need for restraint. The aim is to provide support for researchers at all levels, animal care staff, and ethics committees to refine procedures and practices in line with the refinement principle of the 3Rs.

Reprint of: Production of Superoxide Radicals and Hydrogen Peroxide by NADH- Ubiquinone Reductase and Ubiquinol-Cytochrome c Reductase from Beef-Heart Mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2-at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2-, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2- and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2-, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2- and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2-with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1.

The effects of perinatal SSRI exposure on anxious behavior and neurobiology in rodent and human offspring.

While the postpartum period is typically associated with increased positive affect, many women will develop a depressive- or anxiety-related disorder during this time, which can degrade the mother-infant bond and lead to detrimental consequences for the infant. Given the potential for negative consequences, effective treatments have been critical, with selective serotonin reuptake inhibitors (SSRIs) being the most commonly-prescribed pharmaceutical agents to treat postpartum depression and anxiety. However, SSRIs can readily cross the placenta and are present in breast milk, so they might, therefore, unintentionally interact with the developing fetus/infant. There is already experimental evidence that perinatal SSRI exposure has a number of long-term effects on offspring, but this review focuses on the current literature examining the timing and consequences of perinatal SSRI exposure specifically on anxiety-like behaviors in rodents and humans, with an emphasis on the anxiety-related brain regions of the amygdala and hippocampus. This review also discusses discrepancies between the rodent and human literatures and how they might inform future studies. Finally, some key factors to consider when examining the role of perinatal SSRIs on offspring anxiety will be discussed, such as the duration of SSRI exposure and the potential neuroprotective effects of SSRIs. Given the extensive prescribing of SSRIs, the potential health consequences of perinatal SSRI exposure, and the discrepancies in the literature, it will be necessary to critically examine the factors underlying offspring anxiety outcomes.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Differential postpartum sensitivity to the anxiety-modulating effects of offspring contact is associated with innate anxiety and brainstem levels of dopamine beta-hydroxylase in female laboratory rats.

In female mammals, the postpartum period involves dramatic shifts in many socioemotional behaviors. This includes a suppression of anxiety-related behaviors that requires recent physical contact with offspring. Factors contributing to differences among females in their susceptibility to the anxiety-modulating effect of offspring contact are unknown, but could include their innate anxiety and brain monoaminergic activity. Anxiety behavior was assessed in a large group of nulliparous female rats and the least-anxious and most-anxious tertiles were mated. Anxiety was assessed again postpartum after females were permitted or prevented from contacting their offspring 4 h before testing. Levels of dopamine ß-hydroxylase (DBH, norepinephrine synthesizing enzyme) and tryptophan hydroxylase-2 (TPH2, serotonin synthesizing enzyme) were measured in the brainstem and dorsal raphe, respectively. It was found that anxiety-related behavior in the two groups did not differ when dams were permitted contact with offspring before testing. Removal of the offspring before testing, however, differentially affected anxiety based on dams' innate anxiety. Specifically, dams reverted back to their pre-mating levels of anxiety such that offspring removal slightly increased anxiety in the most-anxious females but greatly lowered anxiety in the least-anxious females. This reduction in anxiety in the least-anxious females after litter removal was associated with lower brainstem DBH. There was no relationship between females' anxiety and dorsal raphe TPH2. Thus, a primary effect of recent contact with offspring on anxiety-related behavior in postpartum rats is to shift females away from their innate anxiety to a more moderate level of responding. This effect is particularly true for females with the lowest anxiety, may be mediated by central noradrenergic systems, and has implications for their ability to attend to their offspring.

What should we do about student use of cognitive enhancers? An analysis of current evidence.

This article reviews current data on the use of cognition enhancers as study aids in the student population. It identifies gaps and uncertainties in the knowledge required to make a balanced assessment of the need for some form of regulation. The review highlights the weak evidence on the prevalence of use of such drugs, especially outside the US, and the ambiguous evidence for their efficacy in a healthy population. Risks are well documented for the commonly used drugs, but poorly appreciated by users. These include not only the side-effects of the drugs themselves, but risks associated with on-line purchase, which offers no guarantees of authenticity and which for some drugs is illegal. The case for urgent action to regulate use is often linked to the belief that new and more effective drugs are likely to appear in the near future. The evidence for this is weak. However, drugs are not the only possible route to neuroenhancement and action is needed to collect more data on the impact of existing drugs, as well as new technologies, in order to guide society in making a proportionate response to the issue. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates.

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.

Within-litter variance in early rat pup-mother interactions and adult offspring responses to novelty.

Siblings share similar genetics and environments, however, their behavior can be quite different. To determine if within-litter variance in neonatal-maternal interactions predict adult sibling behavioral variance, we observed mother-pup interactions during postnatal days 1-8 in four Sprague-Dawley rat litters and measured adult offspring behavioral responses to social and physical novelty. Our results indicate that pup and maternal behavior varied by at least twofold within each litter, and that specific pup behaviors within each litter (perioral contact) were associated with increased maternal licking. Furthermore, siblings that received more licks and made more perioral contact during postnatal days 1-8 had longer latencies to approach novel objects in adulthood than siblings that received less licking and made less perioral contact. This within-litter variance in postnatal mother and pup behavior and offspring adult behavior indicates that early social dynamics within families are an important area to examine to understand the development of sibling variance.

Effect of a cage divider permitting social stimuli on stress and food intake in rats.

The need to obtain data from individual laboratory animals has forced many researchers to singly-house small animals. This is costly to the researcher and isolation can adversely affect animal physiology and behavior which in turn may threaten the validity and generalization of experiment results to humans. We assessed the practical use of a housing device - dubbed "Buddy Barrier" (BB) - that allows social stimulation in a paired-housing situation while at the same time permitting the collection of individual measures that traditionally require individual-housing. To assess stress responses to the BB, adult male rats were single or pair-housed for several days with and without a BB in the cage. Fecal corticosterone metabolites (fCORT), food intake and body weight were monitored daily. Plasma CORT and adrenal catecholamine levels were assessed at the end of the housing manipulation. Stress hormone measures did not differ in paired vs. singly-housed rats and paired rats quickly habituated to introduction and removal of the BB. Barring a trend for paired rats to eat more in the first 4 h of the dark, there was no difference in 24 h intakes or body weight gain between singly and paired-housed rats. While the BB attenuated 24 h intakes in both groups, intakes normalized to non-BB conditions by the third BB reintroduction. A device such as the BB can enhance the welfare of animals by providing social enrichment without compromising the integrity of experimental protocols traditionally requiring single-housing. In times of lagging research funding it can also substantially reduce housing costs.

Shock temperature measurement using neutron resonance spectroscopy.

We report a direct measurement of temperature in a shocked metal using Doppler broadening of neutron resonances. The 21.1-eV resonance in 182W was used to measure the temperature in molybdenum shocked to approximately 63 GPa. An explosively launched aluminum flyer produced a planar shock in a molybdenum target that contained a 1-mm thick layer doped with 1.7 at. %(182)W. A single neutron pulse, containing resonant neutrons of less than 1 mus duration, probed the shocked material. Fits to the neutron time-of-flight data were used to determine the temperature of the shocked molybdenum.

Pharmacological and functional characterisation of dopamine D4 receptors in the rat retina.

In the retina, activation of dopamine receptors, particularly the D2-like family (D2, D3, D4 receptor subtypes), with quinpirole suppresses the light sensitive cAMP pool and inhibits melatonin synthesis in photoreceptor cells. We have characterised rat retinal D4 receptors using the D4 selective radioligand [(125)I] L-750667 which bound specifically and saturably to rat retinal membranes with high affinity (K(d) 0.06+/-0.02 nM) and exhibited a D4 receptor pharmacology. Comparison of the binding kinetics of [(125)I] L-750667 and [(3)H] spiperone revealed B(max) values of 134+/-27 fmol/mg and 219+/-47 fmol/mg respectively, indicating that the dopamine D4 receptor is a major component of D2-like dopamine receptors in the rat retina. Modulation of retinal cAMP levels by quinpirole was used to evaluate the functional relevance of rat retinal dopamine D4 receptors. Quinpirole (0.03-3 micro ) produced a dose-related decrease of the light sensitive cAMP pool which was reversed by haloperidol, clozapine and the D4 selective antagonist, L-745870 with a rank order of potency suggesting that the quinpirole effect is due to activation of the dopamine D4 receptors. The D2 selective ligand L-741626 had no effect on the quinpirole response confirming that the D4 receptor is the major receptor subtype mediating dopamine induced suppression of adenylate cyclase in the retina.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Biological profile of L-745,870, a selective antagonist with high affinity for the dopamine D4 receptor.

L-745,870,(3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H- pyrollo[2,3-b] pyridine, was identified as a selective dopamine D4 receptor antagonist with excellent oral bioavailability and brain penetration. L-745,870 displaced specific binding of 0.2 nM [3H] spiperone to cloned human dopamine D4 receptors with a binding affinity (Ki) of 0. 43 nM which was 5- and 20-fold higher than that of the standard antipsychotics haloperidol and clozapine, respectively. L-745,870 exhibited high selectivity for the dopamine D4 receptor (>2000 fold) compared to other dopamine receptor subtypes and had moderate affinity for 5HT2, sigma and alpha adrenergic receptors(IC50 < 300 nM). In vitro, L-745,870 (0.1-1 microM) exhibited D4 receptor antagonist activity, reversing dopamine (1 microM) mediated 1) inhibition of adenylate cyclase in hD4HEK and hD4CHO cells; 2) stimulation of [35S] GTPgammaS binding and 3) stimulation of extracellular acidification rate, but did not exhibit any significant intrinsic activity in these assays. Although standard antipsychotics increase dopamine metabolism or plasma prolactin levels in rodents, L-745,870 (

4-Heterocyclylpiperidines as selective high-affinity ligands at the human dopamine D4 receptor.

5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.

Schizophrenia and L-745,870, a novel dopamine D4 receptor antagonist.

The discovery of a novel high-affinity and selective dopamine D4 receptor antagonist, L-745,870, and the results of clinical trials with this compound are reviewed. Despite several lines of evidence which suggest that a selective D4 receptor antagonist may be an effective antipsychotic agent with a lower propensity to induce extrapyramidal side-effects, L-745,870 was ineffective as an antipsychotic in humans.

Metal ions in neuroscience.

Metal ions are believed to participate in many neurodegenerative conditions. In excitotoxic cell death there is convincing evidence for the participation of Ca(2+) and Zn(2+) ions although the exact molecular mechanisms by which these metals exert their effects are unclear. Only in one instance has the metal binding site of metalloenzymes been exploited for therapeutic purposes and this is the use of Li(+) in the treatment of bipolar affective disorder. Again the exact molecular target is not clear but is likely to involve a Mg(2+)-dependent enzyme of an intracellular signalling pathway. In Parkinson's disease, the selective loss of dopaminergic neurones in the substantia nigra may be caused by radical-mediated damage and there is good evidence to suggest that Fe(2+) or (3+) is important in promoting formation of radical species. The evidence that free radicals are important in mediating other neurodegenerative conditions is less strong but still substantial enough to suggest that removal of reactive oxygen species or preventing their formation may be a valid approach to therapy.

L-745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavioral tests.

This study examined the high-affinity, selective dopamine D4 receptor antagonist, L-745,870 (3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2, 3-b]pyridine) in rodent behavioral models used to predict antipsychotic potential and side-effect liabilities in humans. In contrast to the classical neuroleptic, haloperidol, and the atypical neuroleptic, clozapine, L-745,870 failed to antagonize amphetamine-induced hyperactivity in mice or impair conditioned avoidance responding in the rat at doses selectively blocking D4 receptors. Furthermore, L-745,870 failed to reverse the deficit in prepulse inhibition of acoustic startle responding induced by the nonselective dopamine D2/3/4 receptor agonist apomorphine, an effect which was abolished in rats pretreated with the D2/3 receptor antagonist, raclopride (0.2 mg/kg s.c.). L-745,870 had no effect on apomorphine-induced stereotypy in the rat but did induce catalepsy in the mouse, albeit at a high dose of 100 mg/kg, which is likely to occupy dopamine D2 receptors in vivo. High doses also impaired motor performance; in rats L-745,870 significantly reduced spontaneous locomotor activity (minimum effective dose = 30 mg/kg) and in mice, L-745,870 reduced the time spent on a rotarod revolving at 15 rpm (minimum effective dose = 100 mg/kg). Altogether these results suggest that dopamine D4 receptor antagonism is not responsible for the ability of clozapine to attenuate amphetamine-induced hyperactivity and conditioned avoidance responding in rodents. Furthermore, the lack of effect of L-745,870 in these behavioral tests is consistent with the inability of the compound to alleviate psychotic symptoms in humans.