S. Clare Stanford

The Open Field Test: reinventing the wheel:

A molecular geneticist advised me recently that no study of a new strain of mutant mice would survive peer review unless the Open Field Test has been used to profile their locomotor (ambulatory) activity. This would certainly help to explain why the number of publications extracted by keywords ‘open field’ with ‘locomotor activity’ has increased fourfold in the last 10 years, but it’s worrying. Although it is not at all certain what the Open Field Test actually measures, it is absolutely clear that it does not give a simple index of the status of motor output. It is easy to see why the Open Field Test is so alluring. The modest approach is to score the number of times the rodent crosses a matrix of lines that has been painted on the floor of the arena. Higher-tech labs have the option of using activity meters, which record the number of times the animal intercepts parallel beams of light that span the arena. When two adjacent infrared beams are intercepted, the animal is deemed to have engaged in locomotor activity rather than vegetative motor activities, which are scored as single beam breaks. The procedure is apparently so straightforward that neither the rodents nor the humans require specialist training. However, a review, written 30 years ago by Walsh and Cummins (1976) should be compulsory reading for anyone contemplating using the Open Field Test. They discuss its shortcomings in detail. High on their list was the variability in the protocols, leading them to question: ‘When is an open field not an open field?’ Judging from a random sample of recent studies that have used this test, nothing has changed. As in those days, the enclosure is still made of just about anything: Plexiglas, anodized aluminium, polypropylene or even painted (black or white) plywood. It can be any size or shape: round, square, rectangular or a circular corridor. Scoring can start either immediately, several minutes or several hours after the animals have been placed in the apparatus. The behaviour can be scored for as long as you choose, e.g. 2min or 4h. Husbandry, litter size, animals’ age, prior handling, ambient odours, whereabouts the animal is placed in the field and light intensity will all differ from lab to lab and influence experimental outcome, but details are rarely specified. Of particular relevance is the problem that differences in ambulation across different genotypes can change (even reverse) with repeated testing. As a consequence, a single snapshot could be misleading. The possibility of secondorder changes in behaviour, resulting fro genotype and environment, adds to the s 2005). At best, this wide range of experimenta ficult to compare one study with another. uninterpretable. ‘The dangers of unqualifi genetic, experimental and testing backgro ated’ (Walsh and Cummins, 1976). It is worth reflecting that the Open Fi designed to score defaecation as a measur ality’ (Hall, 1934). Later, it was used to sc induced by interventions such as elect administration of psychotropic drugs. Bro the Maudsley inbred (reactive and non-r the effects of some environmental varia cluded that movement in the open field wa exploratory drive (Broadhurst, 1957, 1958) Subsequent refinements of the test incl versus oblique movements around the fi Animals that spent the greatest time in th field are regarded as less fearful (or ‘an prefer the perimeter. It was generally agre and penetration of the central zone of the most informative measures from this tes was acknowledged that differences in anim could distort other behavioural measures and Stanford, 1989). Given that individual differences in l found measures of emotionality, it follows tionality will confound measures of locom 1997). Yet, these days, this complicatio ignored. There are numerous reports of di of line crosses or beam breaks in genetic types but, in the absence of other behavi no way of knowing whether these are e motor output, exploratory drive or the free The only reliable way to measure anim motor activity is to record their movement home cage. (Broadhurst (1957) and other 2000) have noted the lack of correlation the open field and cage activity, confirm

Prozac: panacea or puzzle?

Selective 5-HT (serotonin) reuptake inhibitors (SSRIs) lack many of the adverse side-effects of older antidepressants. One of these compounds, fluoxetine (Prozac), has acquired manifest public awareness that is not evident with other SSRIs. Here, Clare Stanford compares the basic pharmacology of SSRIs to determine whether fluoxetine has any atypical features, and thus provide scientific justification for the attention focused on this compound. Ironically, fluoxetine might be distinctive in that it is the least selective SSRI and has marked effects on catecholamine function in the brain.

Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers

This article updates the guidance published in 2015 for authors submitting papers to British Journal of Pharmacology (Curtis et al., 2015) and is intended to provide the rubric for peer review. Thus, it is directed towards authors, reviewers and editors. Explanations for many of the requirements were outlined previously and are not restated here. The new guidelines are intended to replace those published previously. The guidelines have been simplified for ease of understanding by authors, to make it more straightforward for peer reviewers to check compliance and to facilitate the curation of the journals efforts to improve standards.

A comparison of InVivoStat with other statistical software packages for analysis of data generated from animal experiments

InVivoStat is a free-to-use statistical software package for analysis of data generated from animal experiments. The package is designed specifically for researchers in the behavioural sciences, where exploiting the experimental design is crucial for reliable statistical analyses. This paper compares the analysis of three experiments conducted using InVivoStat with other widely used statistical packages: SPSS (V19), PRISM (V5), UniStat (V5.6) and Statistica (V9). We show that InVivoStat provides results that are similar to those from the other packages and, in some cases, are more advanced. This investigation provides evidence of further validation of InVivoStat and should strengthen users’ confidence in this new software package.

Performance Deficits of NK1 Receptor Knockout Mice in the 5-Choice Serial Reaction-Time Task: Effects of d-Amphetamine, Stress and Time of Day

Background The neurochemical status and hyperactivity of mice lacking functional substance P-preferring NK1 receptors (NK1R-/-) resemble abnormalities in Attention Deficit Hyperactivity Disorder (ADHD). Here we tested whether NK1R-/- mice express other core features of ADHD (impulsivity and inattentiveness) and, if so, whether they are diminished by d-amphetamine, as in ADHD. Prompted by evidence that circadian rhythms are disrupted in ADHD, we also compared the performance of mice that were trained and tested in the morning or afternoon. Methods and Results The 5-Choice Serial Reaction-Time Task (5-CSRTT) was used to evaluate the cognitive performance of NK1R-/- mice and their wildtypes. After training, animals were tested using a long (LITI) and a variable (VITI) inter-trial interval: these tests were carried out with, and without, d-amphetamine pretreatment (0.3 or 1 mg/kg i.p.). NK1R-/- mice expressed greater omissions (inattentiveness), perseveration and premature responses (impulsivity) in the 5-CSRTT. In NK1R-/- mice, perseveration in the LITI was increased by injection-stress but reduced by d-amphetamine. Omissions by NK1R-/- mice in the VITI were unaffected by d-amphetamine, but premature responses were exacerbated by this psychostimulant. Omissions in the VITI were higher, overall, in the morning than the afternoon but, in the LITI, premature responses of NK1R-/- mice were higher in the afternoon than the morning. Conclusion In addition to locomotor hyperactivity, NK1R-/- mice express inattentiveness, perseveration and impulsivity in the 5-CSRTT, thereby matching core criteria for a model of ADHD. Because d-amphetamine reduced perseveration in NK1R-/- mice, this action does not require functional NK1R. However, the lack of any improvement of omissions and premature responses in NK1R-/- mice given d-amphetamine suggests that beneficial effects of this psychostimulant in other rodent models, and ADHD patients, need functional NK1R. Finally, our results reveal experimental variables (stimulus parameters, stress and time of day) that could influence translational studies.

Increased noradrenaline efflux induced by local infusion of fluoxetine in the rat frontal cortex

In microdialysis experiments in vivo, local infusion of either the selective serotonin reuptake inhibitor, fluoxetine, or the selective noradrenaline uptake inhibitor, desipramine, increased noradrenaline efflux in rat frontal cortex. Synaptosomal uptake of [3H]noradrenaline was used to test whether inhibition of uptake could contribute to this effect of fluoxetine. Low concentrations of fluoxetine were less effective than desipramine at inhibiting [3H]noradrenaline uptake; both compounds were more potent than the selective serotonin reuptake inhibitor, citalopram. To investigate whether this inhibition of uptake involved an action on noradrenergic neurones, experiments compared the effects of a noradrenergic lesion, induced by the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), on the inhibition of uptake by fluoxetine, desipramine and citalopram. The lesion reduced [3H]noradrenaline uptake in the presence of fluoxetine and citalopram but increased it in the presence of desipramine. The results suggest both that inhibition of noradrenaline uptake could contribute to the actions of fluoxetine and that a non-noradrenergic mechanisms is a target for this action.

Evidence from microdialysis and synaptosomal studies of rat cortex for noradrenaline uptake sites with different sensitivities to SSRIs

1 Microdialysis of the frontal cortex of freely‐moving rats and uptake of [3H]noradrenaline into cortical synaptosomes were used to evaluate changes in efflux of noradrenaline in vivo and uptake of [3H]noradrenaline in vitro, respectively, induced by the selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram, and the tricyclic antidepressant, desipramine. 2 Noradrenaline efflux was increased during local infusion into the cortex of each of these drugs. All three agents also inhibited synaptosomal uptake of [3H]noradrenaline; this inhibition was unaffected by a substantial (50%) lesion of central 5‐hydroxytrytaminergic neurones induced by intracerebroventricular infusion of 5,7‐DHT (150 μg). 3 A noradrenergic lesion (70%), induced by pretreatment with the selective neurotoxin, N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4, 40 mg kg−1 i.p.), 5 days earlier, abolished the increase in noradrenaline efflux caused by local infusion of fluoxetine. In contrast, the desipramine‐induced increase in efflux was greater than in non‐lesioned rats whereas the effect of citalopram on noradrenaline efflux was unaffected by DSP‐4 pretreatment. 4 The combined results of all these experiments suggest that there could be more than one, functionally distinct, noradrenaline uptake site in rat frontal cortex which can be distinguished by their different sensitivities to desipramine and the SSRIs, fluoxetine and citalopram.

Disruption of noradrenergic transmission and the behavioural response to a novel environment in NK1R-/- mice.

The behaviour of neurokinin‐1‐receptor gene knockout (NK1R–/–) mice, which lack functional, substance P‐preferring receptors, resembles that of NK1R+/+ mice treated with an antidepressant. Because all antidepressants increase central monoamine transmission, we have investigated whether noradrenergic transmission is increased in NK1R–/– mice and, if so, whether this could influence their behaviour. In anaesthetized subjects, the concentration of extracellular noradrenaline in NK1R–/– mice was two–fourfold greater than in NK1R+/+ mice. Systemic administration of the α2‐adrenoceptor antagonist, 2‐(2,3‐dihydro‐2‐methoxy‐1,4‐benzodioxan‐2‐yl)‐4,5‐dihydro‐1H‐imidazoline (RX 821002), in anaesthetized or freely moving animals increased extracellular noradrenaline in NK1R+/+ mice only. This suggests that the function of α2a‐autoreceptors, which modulate noradrenergic transmission, is impaired in NK1R–/– mice. Consistent with this, [35S]GTPγS binding to activated α2a‐adrenoceptors was lower (−70%) in the locus coeruleus, but not the frontal cortex, of NK1R–/– mice compared with their NK1R+/+ counterparts. RX 821002‐pretreatment, followed by retrodialysis of the noradrenaline reuptake inhibitor, desipramine, into the frontal cortex of anaesthetized mice increased extracellular noradrenaline to the same extent in the two genotypes. Western blots confirmed that there was no difference in the amount of noradrenaline transporter protein in NK1R–/– and NK1R+/+ mice. Finally, the effects of RX 821002 on certain behaviours in a light/dark exploration box were blunted in NK1R–/– mice, but there was no consistent effect on anxiety‐like behaviour in the two genotypes. It is concluded that the greater basal efflux of noradrenaline in NK1R–/– mice is explained by increased transmitter release, coupled with desensitization of somatodendritic α2a‐adrenoceptors. These changes could contribute to the difference in the behavioural phenotypes.

Deficits in exploratory behaviour in socially isolated rats are not accompanied by changes in cerebral cortical adrenoceptor binding

Social isolation of rats produces marked deficits in exploratory behaviour in a novel environment. To test our hypothesis that adrenoceptors are modified by social isolation we have measured alpha 2- and beta-adrenoceptor binding in the cerebral cortex of rats isolated for 21 days starting immediately after weaning. After this period the animals showed a significant reduction in exploration in the elevated X-maze and increased avoidance of bright light in a two-compartment shuttlebox. There were no changes in adrenoceptor binding at this time, however. The implications of these findings are discussed.

Genetic association of the tachykinin receptor 1 TACR1 gene in bipolar disorder, attention deficit hyperactivity disorder, and the alcohol dependence syndrome.

Single nucleotide polymorphisms (SNPs) in the tachykinin receptor 1 gene (TACR1) are nominally associated with bipolar affective disorder (BPAD) in a genome‐wide association study and in several case‐control samples of BPAD, alcohol dependence syndrome (ADS) and attention‐deficit hyperactivity disorder (ADHD). Eighteen TACR1 SNPs were associated with BPAD in a sample (506 subjects) from University College London (UCL1), the most significant being rs3771829, previously associated with ADHD. To further elucidate the role of TACR1 in affective disorders, rs3771829 was genotyped in a second BPAD sample of 593 subjects (UCL2), in 997 subjects with ADS, and a subsample of 143 individuals diagnosed with BPAD and comorbid alcohol dependence (BPALC). rs3771829 was associated with BPAD (UCL1 and UCL2 combined: P = 2.0 × 10−3), ADS (P = 2.0 × 10−3) and BPALC (P = 6.0 × 10−4) compared with controls screened for the absence of mental illness and alcohol dependence. DNA sequencing in selected cases of BPAD and ADHD who had inherited TACR1‐susceptibility haplotypes identified 19 SNPs in the promoter region, 5′ UTR, exons, intron/exon junctions and 3′ UTR of TACR1 that could increase vulnerability to BPAD, ADS, ADHD, and BPALC. Alternative splicing of TACR1 excludes intron 4 and exon 5, giving rise to two variants of the neurokinin 1 receptor (NK1R) that differ in binding affinity of substance P by 10‐fold. A mutation in intron four, rs1106854, was associated with BPAD, although a regulatory role for rs1106854 is unclear. The association with TACR1 and BPAD, ADS, and ADHD suggests a shared molecular pathophysiology between these affective disorders.