More rapid reversal learning following overtraining in the rat is evidence that behavioural and cognitive flexibility are dissociable

The research was undertaken by SSD, in partial fulfillment of the requirements of a PhD degree under the joint supervision of VJB and DST and supported by The University of St Andrews (QR Block Grant).Cognitive flexibility is a term used to describe the brain processes underlying the phenomenon of adaptive change in behaviour in response to changed contingencies in the internal or external environment. Cognitive flexibility is often assessed in complex tasks measuring perceptual attentional shifting or response or task switching, but, arguably, reversal learning is a simple assay of cognitive flexibility. Reversal learning requires the detection of a changed outcome, the cessation of a previously-rewarded response and the selection of an alternative, previously-unrewarded, response. This study addressed the issue of the relationship between reversal learning and cognitive flexibility. In a single testing session, rats completed a series of 2-alternative forced-choice discriminations between digging bowls. The bowls differed according to both the medium within the bowl and the odor of the bowl. Having learned which cue (one of the odors or one of the digging media) indicated the food-baited bowl, half the rats were given additional trials of “over-training”. To test reversal learning, the meaning of the cues predictive of reward/non-reward was then switched. There was a robust effect of over-training, with over-trained rats performing reversal learning in fewer trials than rats trained to criterion only. The pattern of errors supported the hypothesis that more rapid reversing results from the formation of an attentional set. This is the same attentional mechanism that results in less rapid shifting or switching. We conclude that the behavioural flexibility demonstrated in reversal learning does not provide a scale on which cognitive flexibility can be measured.PostprintPeer reviewe

Effects of lesions of the subthalamic nucleus/zona incerta area and dorsomedial striatum on attentional set-shifting in the rat

This work was supported by The Wellcome Trust (project Grant 051945/z/97). Andrew Blackwell was in receipt of a BBSRC Studentship.Patients with Parkinson’s disease show cognitive impairments, including difficulty in shifting attention between perceptual dimensions of complex stimuli. Inactivation of the subthalamic nucleus (STN) has been shown to be effective in ameliorating the motor abnormalities associated with striatal dopamine depletion, but it is possible that STN inactivation might result in additional, perhaps attentional, deficits. This study examined the effects of: dopamine depletion from the dorsomedial striatum (DMS); lesions of the STN area; and the effects of the two lesions together, on the ability to shift attentional set in the rat. In a single session, rats performed the intradimensional/extradimensional (ID/ED) test of attentional set-shifting. This comprises a series of seven, two-choice discriminations, including acquisitions of novel discriminations in which the relevant stimulus is either in the currently-attended dimension (ID) or the currently-unattended dimension (ED shift) and reversals following each acquisition stage. Bilateral lesions were made by injection of 6-hydroxydopamine into the DMS, resulting in a selective impairment in reversal learning. Large bilateral ibotenic acid lesions centred on the STN resulted in an increase in trials to criterion in the initial stages, but learning rate improved within the session. There was no evidence of a ‘cost’ of set-shifting – the ED stage was completed in fewer trials than the ID stage – and neither was there a cost of reversal learning. Strikingly, combined lesions of both regions did not resemble the effects of either lesion alone and resulted in no apparent deficits.Publisher PDFPeer reviewe

Precocial juvenile lizards show adult level learning and behavioural flexibility

This project was funded by an ARC Discovery grant (DP130102998) to M.J.W. and R.W.B. and by Macquarie University.In altricial species, young rely on parental care and brain maturation mainly occurs after birth. In precocial species, young are born at a more advanced developmental stage in need of less or no parental care and brain development is mostly completed at the time of birth. We therefore predicted early maturation of learning ability in precocial species. We used a series of visual discrimination and reversal stages to investigate the ability of the precocial eastern blue-tongue lizard, Tiliqua scincoides scincoides, a long-lived Australian lizard species with slow-developing young, to respond to changes in stimulus relevance and test for behavioural flexibility. To test whether age affects learning in this species, we compared juveniles (23–56 days) with adults (sexually mature, at least 2 years). In accordance with our expectations, adults and juveniles performed similarly well in all stages, suggesting that juveniles of this precocial species learn at adult levels from an early age. Both age classes performed well during reversals showing good behavioural flexibility. This is the first study in lizards to directly compare juvenile and adult behavioural flexibility. Importantly, we demonstrate that precocial lizards can begin life with an advanced cognitive ability already in place.In altricial species, young rely on parental care and brain maturation mainly occurs after birth. In precocial species, young are born at a more advanced developmental stage in need of less or no parental care and brain development is mostly completed at the time of birth. We therefore predicted early maturation of learning ability in precocial species. We used a series of visual discrimination and reversal stages to investigate the ability of the precocial eastern blue-tongue lizard, Tiliqua scincoides scincoides, a long-lived Australian lizard species with slow-developing young, to respond to changes in stimulus relevance and test for behavioural flexibility. To test whether age affects learning in this species, we compared juveniles (23–56 days) with adults (sexually mature, at least 2 years). In accordance with our expectations, adults and juveniles performed similarly well in all stages, suggesting that juveniles of this precocial species learn at adult levels from an early age. Both age classes performed well during reversals showing good behavioural flexibility. This is the first study in lizards to directly compare juvenile and adult behavioural flexibility. Importantly, we demonstrate that precocial lizards can begin life with an advanced cognitive ability already in place.In altricial species, young rely on parental care and brain maturation mainly occurs after birth. In precocial species, young are born at a more advanced developmental stage in need of less or no parental care and brain development is mostly completed at the time of birth. We therefore predicted early maturation of learning ability in precocial species. We used a series of visual discrimination and reversal stages to investigate the ability of the precocial eastern blue-tongue lizard, Tiliqua scincoides scincoides, a long-lived Australian lizard species with slow-developing young, to respond to changes in stimulus relevance and test for behavioural flexibility. To test whether age affects learning in this species, we compared juveniles (23–56 days) with adults (sexually mature, at least 2 years). In accordance with our expectations, adults and juveniles performed similarly well in all stages, suggesting that juveniles of this precocial species learn at adult levels from an early age. Both age classes performed well during reversals showing good behavioural flexibility. This is the first study in lizards to directly compare juvenile and adult behavioural flexibility. Importantly, we demonstrate that precocial lizards can begin life with an advanced cognitive ability already in place.In altricial species, young rely on parental care and brain maturation mainly occurs after birth. In precocial species, young are born at a more advanced developmental stage in need of less or no parental care and brain development is mostly completed at the time of birth. We therefore predicted early maturation of learning ability in precocial species. We used a series of visual discrimination and reversal stages to investigate the ability of the precocial eastern blue-tongue lizard, Tiliqua scincoides scincoides, a long-lived Australian lizard species with slow-developing young, to respond to changes in stimulus relevance and test for behavioural flexibility. To test whether age affects learning in this species, we compared juveniles (23–56 days) with adults (sexually mature, at least 2 years). In accordance with our expectations, adults and juveniles performed similarly well in all stages, suggesting that juveniles of this precocial species learn at adult levels from an early age. Both age classes performed well during reversals showing good behavioural flexibility. This is the first study in lizards to directly compare juvenile and adult behavioural flexibility. Importantly, we demonstrate that precocial lizards can begin life with an advanced cognitive ability already in place.PostprintPeer reviewe

Kinetics and Phenotype of Vaccine-Induced CD8+ T-Cell Responses to Toxoplasma gondii

Multiple studies have established that the ability of CD8+ T cells to act as cytolytic effectors and produce gamma interferon is important in mediating resistance to the intracellular parasite Toxoplasma gondii. To better understand the generation of the antigen-specific CD8+ T-cell responses induced by T. gondii, mice were immunized with replication-deficient parasites that express the model antigen ovalbumin (OVA). Class I tetramers specific for SIINFEKL were used to track the OVA-specific endogenous CD8+ T cells. The peak CD8+ T-cell response was found at day 10 postimmunization, after which the frequency and numbers of antigen-specific cells declined. Unexpectedly, replication-deficient parasites were found to induce antigen-specific cells with faster kinetics than replicating parasites. The generation of optimal numbers of antigen-specific CD8+ effector T cells was found to require CD4+ T-cell help. At 7 days following immunization, antigen-specific cells were found to be CD62Llow, KLRG1+, and CD127low, and they maintained this phenotype for more than 70 days. Antigen-specific CD8+ effector T cells in immunized mice exhibited potent perforin-dependent OVA-specific cytolytic activity in vivo. Perforin-dependent cytolysis appeared to be the major cytolytic mechanism; however, a perforin-independent pathway that was not mediated via Fas-FasL was also detected. This study provides further insight into vaccine-induced cytotoxic T-lymphocyte responses that correlate with protective immunity to T. gondii and identifies a critical role for CD4+ T cells in the generation of protective CD8+ T-cell responses

Escitalopram restores reversal learning impairments in rats with lesions of orbital frontal cortex

This study was funded by H. Lundbeck A/S.The term ‘cognitive structures’ is used to describe the fact that mental models underlie thinking, reasoning and representing. Cognitive structures generally improve the efficiency of information processing by providing a situational framework within which there are parameters governing the nature and timing of information and appropriate responses can be anticipated. Unanticipated events that violate the parameters of the cognitive structure require the cognitive model to be updated, but this comes at an efficiency cost. In reversal learning a response that had been reinforced is no longer reinforced, while an alternative is now reinforced, having previously not been (A+/B− becomes A−/B+). Unanticipated changes of contingencies require that cognitive structures are updated. In this study, we examined the effect of lesions of the orbital frontal cortex (OFC) and the effects of the selective serotonin reuptake inhibitor (SSRI), escitalopram, on discrimination and reversal learning. Escitalopram was without effect in intact rats. Rats with OFC lesions had selective impairment of reversal learning, which was ameliorated by escitalopram. We conclude that reversal learning in OFC-lesioned rats is an easily administered and sensitive test that can detect effects of serotonergic modulation on cognitive structures that are involved in behavioural flexibility.Publisher PD

Assessment of intradimensional/extradimensional attentional set-shifting in rats

The initial development of the attentional set-shifting task was supported by The Wellcome Trust (Project Grant 051945/Z/97/Z) and a Biotechnology and Biological Sciences Research Council (UK) Studentship to Jennifer M. Birrell.The rat intradimensional/extradimensional (ID/ED) task, first described by Birrell and Brown 18 years ago, has become the predominant means by which attentional set-shifting is investigated in rodents: the use of rats in the task has been described in over 135 publications by researchers from nearly 90 universities and pharmaceutical companies. There is variation in the protocols used by different groups, including differences in apparatus, stimuli (both stimulus dimensions and exemplars within), and also the methodology. Nevertheless, most of these variations seem to be of little consequence: there is remarkable similarity in the profile of published data, with consistency of learning rates and in the size and reliability of the set-shifting and reversal ‘costs’. However, we suspect that there may be inconsistent data that is unpublished or perhaps ‘failed experiments’ that may have been caused by unintended deviations from effective protocols. The purpose of this review is to describe our approach and the rationale behind certain aspects of the protocol, including common pitfalls that are encountered when establishing an effective local protocol.PostprintPeer reviewe

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies

Gene therapy is a therapeutic approach that is designed to correct specific molecular defects that contribute to the cause or progression of cancer. Genes that are mutated or deleted in cancers include the cancer susceptibility genes p53 and BRCA1. Because mutational inactivation of gene function is specific to tumor cells in these settings, cancer gene correction strategies may provide an opportunity for selective targeting without significant toxicity for normal nontumor cells. Both p53 and BRCA1 appear to inhibit cancer cells that lack mutations in these genes, suggesting that the so-called gene correction strategies may have broader potential than initially believed. Increasing knowledge of cancer genetics has identified these and other genes as potential targets for gene replacement therapy. Initial patient trials of p53 and BRCA1 gene therapy have provided some indications of potential efficacy, but have also identified areas of basic and clinical research that are needed before these approaches may be widely used in patient care

Subproblem learning and reversal of a multidimensional visual cue in a lizard : evidence for behavioural flexibility?

This project was funded by an ARC Discovery grant (DP130102998) to Martin Whiting and Richard Byrne and by Macquarie University.Behavioural flexibility, the ability to adjust behaviour to environmental change by adapting existing skills to novel situations, is key to coping with, for example, complex social interactions, seasonal changes in food availability or detecting predators. We tested the tree skink, Egernia striolata, a family-living skink from eastern Australia, in a set-shifting paradigm of eight colour/shape discriminations including reversals, an intradimensional acquisition of a new colour/shape and extradimensional shift from colour to shape (and vice versa). Skinks could learn to discriminate between colour/shape pairs and reverse this initial stimulus–reward association; however, they showed no significant decrease in the probability of making a correct choice in the extradimensional shift suggesting that they did not form an attentional set. Subjects appear to have learnt each stage as a new problem instead of generalizing stimuli into specific dimensions (set formation). In conclusion, tree skinks solved a discrimination reversal by focusing their attention towards visual stimuli and flexibly adjusting their choice behaviour accordingly. These lizards learned to use multidimensional visual stimuli to find a food reward but did not generalize stimuli into dimensions. Furthermore, this study is the first to test for set shifting in a lizard species and thereby allows us to extend set-shifting theory to a new taxon for comparison with primates, rodents, a bird and a turtle.PostprintPeer reviewe

Measurement of Adenovirus-Based Vector Heterogeneity

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities