Nanna Marie Lind

The use of pigs in neuroscience: Modeling brain disorders

The use of pigs in neuroscience research has increased in the past decade, which has seen broader recognition of the potential of pigs as an animal for experimental modeling of human brain disorders. The volume of available background data concerning pig brain anatomy and neurochemistry has increased considerably in recent years. The pig brain, which is gyrencephalic, resembles the human brain more in anatomy, growth and development than do the brains of commonly used small laboratory animals. The size of the pig brain permits the identification of cortical and subcortical structures by imaging techniques. Furthermore, the pig is an increasingly popular laboratory animal for transgenic manipulations of neural genes. The present paper focuses on evaluating the potential for modeling symptoms, phenomena or constructs of human brain diseases in pigs, the neuropsychiatric disorders in particular. Important practical and ethical aspects of the use of pigs as an experimental animal as pertaining to relevant in vivo experimental brain techniques are reviewed. Finally, current knowledge of aspects of behavioral processes including learning and memory are reviewed so as to complete the summary of the status of pigs as a species suitable for experimental models of diverse human brain disorders.

Validation of in vitro probabilistic tractography

Diffusion weighted imaging (DWI) and tractography allow the non-invasive study of anatomical brain connectivity. However, a gold standard for validating tractography of complex connections is lacking. Using the porcine brain as a highly gyrated brain model, we quantitatively and qualitatively assessed the anatomical validity and reproducibility of in vitro multi-fiber probabilistic tractography against two invasive tracers: the histochemically detectable biotinylated dextran amine and manganese enhanced magnetic resonance imaging. Post mortem DWI was used to ensure that most of the sources known to degrade the anatomical accuracy of in vivo DWI did not influence the tracking results. We demonstrate that probabilistic tractography reliably detected specific pathways. Moreover, the applied model allowed identification of the limitations that are likely to appear in many of the current tractography methods. Nevertheless, we conclude that DWI tractography can be a precise tool in studying anatomical brain connectivity.

Validation of a digital video tracking system for recording pig locomotor behaviour.

We are introducing a system for automatically tracking pig locomotor behaviour. Transposing methods for the video-based tracking of rodent behaviour engenders several problems. We have therefore improved existing methods, based on image-subtraction, to offer increased flexibility and accuracy in tracking large-sized animals in situations with a constantly changing background. The improved tracking algorithms introduce a reference frame, which does not include the animal and is automatically updated, and implementation of an automatic threshold detection algorithm. This makes the system more robust to the tracking environment, which could even be of the same colour as the animal, and allows the tracking environment to change during recording. We validated the system by estimating the repeatability, accuracy, and basic noise level, and tested the system in different levels of animal activity evoked by administration of apomorphine (APO) to minipigs in an open field test. Seven pigs each received the vehicle and three doses of APO (0.05, 0.1, and 0.3 mg/kg i.m.), and the locomotor behaviour of each session was recorded for 60-min. The calculated coefficient of repeatability was 0.6%, indicating high repeatability and the basic noise level of the tracking system was estimated to be 2%. Administration of the two lowest doses of APO was accompanied by increased locomotor activity of the pigs. Thus, this digital video-based tracking system for automatically tracking the spontaneous locomotor behaviour of pigs is highly reliable and accurate, and was able to detect well-known effects of APO in pig locomotor activity.

Evaluation of the Novel 5-HT4 Receptor PET Ligand [11C]SB207145 in the Göttingen Minipig

This study investigates 5-hydroxytryptamine 4 (5-HT4) receptor binding in the minipig brain with positron emission tomography (PET), tissue homogenate-binding assays, and autoradiography in vitro. The cerebral uptake and binding of the novel 5-HT4 receptor radioligand [11C]SB207145 in vivo was modelled and the outcome compared with postmortem receptor binding. Different models for quantification of [11C]SB207145 binding were evaluated: One-tissue and two-tissue compartment kinetic modelling, Logan arterial input, and three different reference tissue models. We report that the pig autoradiographic 5-HT4 receptor distribution resembles the human 5-HT4 receptor distribution with the highest binding in the striatum and no detectable binding in the cerebellum. We found that in the minipig brain [11C]SB207145 follows one-tissue compartment kinetics, and the simplified reference tissue model provides stable and precise estimates of the binding potential in all regions. The binding potentials calculated for striatum, midbrain, and cortex from the PET data were highly correlated with 5-HT4 receptor concentrations determined in brain homogenates from the same regions, except for hippocampus where PET-measurements significantly underestimate the 5-HT4 receptor binding, probably because of partial volume effects. This study validates the use of [11C]SB207145 as a promising PET radioligand for in vivo brain imaging of the 5-HT4 receptor in humans.

Behavioral response to novelty correlates with dopamine receptor availability in striatum of Göttingen minipigs

Behavioral response to novelty in rats has been linked both to dopamine transmission in the ventral striatum, and to propensity to self-administer psychostimulant drugs. In order to probe the relationship between behavioral response to novelty and dopamine systems we have developed a behavioral model for correlation with positron emission tomography (PET) of dopamine transmission in brain of Göttingen minipigs. In the present study, we measured exploration of a novel object by recording the number of contacts, and duration of contact with a novel object, in groups of six male and six female adult minipigs. We hypothesized that these novelty scores would correlate with the amphetamine-evoked dopamine release in ventral striatum, measured 2 weeks later in a PET study of the availability of binding sites for the dopamine D2/3 antagonist [11C]raclopride. There were significant correlations between duration of contact with a novel object and the amphetamine-evoked reductions in binding potential (DeltapB) in the left ventral striatum of the 12 animals; Comparison of results by gender revealed that the correlation was driven mainly by the male group, and was not present in the female group. We interpret these results to show that propensity to explore an unfamiliar object is relatively elevated in pigs with low basal occupancy of dopamine D2/3 receptors by endogenous dopamine, and with high amphetamine-induced occupancy of released dopamine in the male pigs.

Spontaneous Object Recognition in the Göttingen Minipig

Göttingen minipigs were tested in an object recognition procedure based on spontaneous exploration. Eight pigs were exposed to two similar objects in a sample trial and after a one-hour delay exposed to two objects, one familiar and one novel, in a test trial. The pigs explored the novel object significantly more than the familiar object in the test trial (p<0.05), thereby showing recognition of the familiar object. Furthermore, habituation of exploration of the familiar object between the sample trial and the test trial was found (p<0.05). The procedure can be useful for testing of spontaneous trial-unique memory in pigs.

The effect of the inter-phase delay interval in the spontaneous object recognition test for pigs

In the neuroscience community interest for using the pig is growing. Several disease models have been developed creating a need for validation of behavioural paradigms in these animals. Here, we report the effect of different inter-phase delay intervals on the performance of Göttingen minipigs in the spontaneous object recognition test. The test consisted of a sample and a test phase. First, the pigs explored two similar objects. After a 10-min, 1-h, or 24-h delay two different objects were presented; one familiar from the sample phase and one novel. An exploration-time difference between the novel and the familiar object was interpreted as recognition of the familiar object. We scored the exploration times both manually and automatically, and compared the methods. A strong discrimination between novel and familiar objects after a 10-min inter-phase delay interval and no discrimination after 24h were found in our set-up of the spontaneous object recognition test. After a 1-h delay, the pigs still showed a significant habituation to the familiar object, but no discrimination was observed. Discrimination between the two objects was mainly confined to the first half of the test phase, and we observed a high between-subject variation. Furthermore, automatic tracking was valid for determination of habituation and discrimination parameters but lead to an overestimation of individual measurements. We conclude that the spontaneous object recognition test for pigs is sensitive to increasing inter-phase delay intervals, and that automatic data acquisition can be applied.

Mapping the amphetamine-evoked dopamine release in the brain of the Göttingen minipig

The availability of dopamine D(2/3) binding sites in brain of six male and six female Göttingen minipigs was measured in a baseline condition and after challenge with amphetamine sulfate (1mg/kg, i.v.) in PET studies with [(11)C]raclopride. Maps of the binding potential (pB; B(max)/K(d)) of [(11)C]raclopride were spatially normalized and co-registered to a common stereotaxic coordinate system for pig brain. The pB maps were then analyzed by volume of interest and voxel-wise comparisons of gender and condition. The mean baseline pB tended to be 10-20% higher in striatum of the female group, but this gender difference was not significant. Variance of the mean baseline pB was higher in the males (44%) than in females (30%), but there was no correlation between pB and individual plasma cortisol or testosterone concentrations. Using statistical parametric mapping, we detected a focus in the right posterior putamen where the magnitude of the amphetamine-evoked decrease in pB was greater in the male than in the female group. Thus, the spatial pattern of reactivity of dopamine D(2/3) receptor availability to amphetamine challenge is not identical in male and female pigs. Within the entire population, the decline in pB evoked by amphetamine (Delta pB) was greater in the ventral striatum (-28%) than in the caudate nucleus (-17%), consistent with earlier reports in monkeys and humans. The magnitude of Delta pB correlated highly with the baseline pB values in all divisions of the striatum. Based upon the principles of competitive binding, the slope of this empirical relationship, f(i), is equal to the fraction of [(11)C]raclopride binding sites sensitive to endogenous dopamine; the magnitude of this fraction ranged from 0.29 in the caudate to 0.36 in the ventral striatum.

Discriminations, reversals, and extra-dimensional shifts in the Göttingen minipig

Göttingen minipigs were trained on a set-shifting procedure involving discriminations, reversals, and extra-dimensional shifts. The discriminations used were black-white discriminations and right-left discriminations. The initial visual and spatial discrimination seemed equally difficult, and only for the visual modality was reversal found to be more difficult than the initial discrimination. Visual reversal was more difficult than spatial reversal, and a larger number of perseverative sessions were found for visual reversal compared to spatial reversal. The acquisition of the extra-dimensional shift from the visual to the spatial dimension was not inferior to the learning of spatial reversal. Neither was the acquisition of the extra-dimensional shift from the spatial to the visual dimension inferior to the learning of visual reversal. Thus, no evidence was found for attention to stimulus dimensions in discrimination learning of the pigs.

Prepulse inhibition of the acoustic startle reflex in pigs and its disruption by d-amphetamine

Prepulse inhibition (PPI) of the startle reflex is an operational measure of sensorimotor gating. The dopamine receptor agonist-mediated disruption of PPI in rats is widely used as a model of the sensorimotor gating deficiencies demonstrated in schizophrenia patients. As a possible tool for validation of a pig model of psychosis, we wished to verify the existence of PPI in landrace pigs and investigate the potential disruption of PPI by d-amphetamine (AMPH) in these animals. PPI of the acoustic startle reflex and its potential disruption by AMPH were investigated using three doses 0.5-1.5mg/kg with a paradigm including two levels of prepulses (82 and 88dB) and a prepulse (PP) interval of 60 and 120ms. We found an average PPI of the startle reflex of 25.6% and both of the investigated PP intensities and PP intervals were equally effective in this PP-inhibitive paradigm. AMPH significantly disrupted PPI and, in spite of only the 0.5mg/kg dose proved statistically significant, the results indicate this to be dose-related. We have demonstrated the phenomenon of PPI of the startle reflex in landrace pigs and its disruption by d-amphetamine. Studies of sensorimotor gating defects could be a valuable additional tool in assessing pig models of neuropsychiatric disorders.