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In JoVE (1)
Other Publications (37)
- Behavioural Brain Research
- Behavioural Brain Research
- Behavioral Neuroscience
- Behavioral Neuroscience
- Journal of Neuroimmunology
- Behavioural Brain Research
- Annals of Neurology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Neuroscience
- Nature Genetics
- Behavioural Brain Research
- Behavioural Brain Research
- Mammalian Genome : Official Journal of the International Mammalian Genome Society
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Protocols
- Nature Protocols
- Nature Protocols
- Nature Protocols
- Nature Protocols
- Nature Protocols
- Nature Protocols
- Current Biology : CB
- The European Journal of Neuroscience
- Developmental Psychobiology
- Biological Psychiatry
- Behavioural Brain Research
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- PLoS Genetics
- Experimental Neurology
- European Neuropsychopharmacology : the Journal of the European College of Neuropsychopharmacology
- European Journal of Pain (London, England)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Neuroscience
- Hippocampus
- Neurobiology of Aging
Articles by Robert Deacon in JoVE
JoVE Neuroscience
Assessing Burrowing, Nest Construction, and Hoarding in Mice
Department of Experimental Psychology, University of Oxford
Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute “environmental enrichment” for the mice, and embody the “Refinement” aspect of the “3 Rs”18.
Other articles by Robert Deacon on PubMed
Hippocampal Cytotoxic Lesion Effects on Species-typical Behaviours in Mice
The behavioural effects of hippocampal lesions have been extensively documented in rats. However, paradigms developed for rats cannot be assumed to transfer straightforwardly to mice; the behaviour of the two species differs in many respects. Mice are currently the species of choice for targeted genetic manipulations. A number of these programs aim to modulate hippocampal function. The present studies were therefore designed to provide a behavioural profile of selective, cytotoxic hippocampal lesions in tasks appropriate for mice. The lesions abolished food hoarding from a source outside the home base, and reduced the tendency to displace food pellets from a tube inside the home cage (burrowing). Lesioned mice showed reductions of directed exploration (rearing and head dipping), but not locomotor activity, in a holeboard and open field, and explored the edges of their home cages less when the lids were removed. Nest construction was also impaired. These effects were not due to gross motor impairments, as formal tests revealed no deficiencies in co-ordination or strength. There were suggestions of changes in emotionality, although a more consistent finding was that lesioned mice were often slower to initiate behaviour in novel surroundings, which may be congruent with the other deficits we observed. These results may aid interpretation of the many genetic manipulations that target the hippocampus, and of neurodegenerative conditions that induce hippocampal pathology.
Effects of Cytotoxic Hippocampal Lesions in Mice on a Cognitive Test Battery
Mice received cytotoxic lesions which selectively removed all of the hippocampus and dentate gyrus except the most ventral portions. They were impaired on both spontaneous and rewarded discrete-trial alternation in T-mazes. Acquisition of reference memory for the location of a hidden platform in the Morris water maze was impaired in lesioned mice. On an elevated Y-maze reference memory task, in which only one arm was rewarded, lesioned mice showed no evidence of learning. In a Lashley III maze task, however, where maze rotation demonstrated that control performance was independent of distal spatial cues, acquisition in the lesioned mice was unimpaired. Control levels of continuous spontaneous alternation in a Y-maze were too low to reveal a hippocampal deficit. A small impairment in acquisition of a multiple-trial passive avoidance task was seen in lesioned mice, despite a small but significant increase in reactivity to the footshock. These results are largely consistent with findings in hippocampal lesioned rats on the same or similar tasks, and reflect a major impairment of spatial cognition, with relative sparing of non-spatial task performance.
Learning Impairments of Hippocampal-lesioned Mice in a Paddling Pool
Control mice rapidly learned to escape from shallow water in a paddling pool, which combined elements of the Morris water maze and the Barnes holeboard maze. The pool's transparent perimeter wall contained 12 exits, only 1 of which led to an escape tunnel. Learning was impaired in mice with cytotoxic lesions of the hippocampus. Probe trials suggested that the controls were using extramaze cues. When the exit was blocked, controls, but not hippocampals, spent more time searching in this previously correct sector. When the spatial location of the exit was changed, hippocampals escaped more quickly, as they showed no preference for the old location. These results may be useful in the assessment of hippocampal dysfunction, particularly in genetically manipulated mice.
Anxiolytic Effects of Cytotoxic Hippocampal Lesions in Rats
Rats with cytotoxic lesions of the hippocampus were given 3 anxiety tests: social interaction with a novel rat, the elevated zero-maze (a modification of the plus-maze), and hyponeophagia (eating familiar and novel foods in a novel place). Marked anxiolytic effects were seen in the social interaction and hyponeophagia tests, but not on the zero-maze. These results confirm and extend previous experiments that used traditional lesion techniques. The zero-maze result was consistent with other experiments using the plus-maze, in which intrahippocampal administrations of pharmacological agents were not anxiolytic, although variability in ethological tests may also be a factor. As the hyponeophagia test used an elevated apparatus, as in the zero- and plus-mazes, the lack of a lesion effect in the zero-maze was unlikely to have been due to an inability to relieve height-induced anxiety.
Maternal Antibody-mediated Dyslexia? Evidence for a Pathogenic Serum Factor in a Mother of Two Dyslexic Children Shown by Transfer to Mice Using Behavioural Studies and Magnetic Resonance Spectroscopy
The causes of dyslexia are unknown, but previous studies have suggested an immunological basis in some cases. We hypothesised that maternal antibodies, which cross the placenta and bind to fetal antigens, could be responsible, particularly when the dyslexia recurs in consecutive pregnancies. We injected serum samples from five mothers of two or more children with dyslexia into pregnant mice, and tested the offspring for behavioural abnormalities and cerebellar metabolites by magnetic resonance spectroscopy (MRS). Mice exposed in utero to serum factors from one woman with two dyslexic children, who had also had three spontaneous fetal losses, showed deficits in motor tests which correlated with cerebellar choline (Cho) and creatine (Cr) levels. These preliminary results are consistent with a role for maternal serum factors, probably antibodies, in causing some of the features of dyslexia, and possibly in other neurodevelopmental disorders.
Effects of Medial Prefrontal Cortex Cytotoxic Lesions in Mice
Mice (C57BL/6J strain, females) with cytotoxic lesions of the medial wall of the prefrontal cortex were given a battery of tests to assess emotional, species-typical, cognitive, motor and other behaviours. Lesioned mice showed a profile of reduced anxiety, both on a plus-maze, and a similar, novel test, the successive alleys. There was no evidence, however, for attenuation of anxiety in tests of hyponeophagia, and lesioned mice, like controls, preferred the black to the white area of an enclosed alley. Their locomotor activity tended to be higher than that of the controls, particularly when the test surroundings were novel or relatively so. Species-typical behaviours were similar to those of control mice, except lesioned mice displaced ('burrowed') less food pellets from a tube in their home cage. They were not impaired at learning a spatial Y-maze reference memory task, which is profoundly affected by cytotoxic hippocampal lesions in the same strain, or at learning a multi-trial passive avoidance test. Their strength and co-ordination in motor performance tests was also normal. The results show that cytotoxic medial prefrontal cortex lesions in mice produce a clear but restricted anxiolytic action. The marked reduction in burrowing, in the absence of any detectable impairment of motor ability, demonstrates the sensitivity of this behavioural index.
Maternal Neuronal Antibodies Associated with Autism and a Language Disorder
Neurodevelopmental disorders could be caused by maternal antibodies or other serum factors. We detected serum antibodies binding to rodent Purkinje cells and other neurons in a mother of three children: the first normal, the second with autism, and the third with a severe specific language disorder. We injected the serum (0.5-1.0 ml/day) into pregnant mice during gestation and found altered exploration and motor coordination and changes in cerebellar magnetic resonance spectroscopy in the mouse offspring, comparing with offspring of mice injected with sera from mothers of healthy children. This evidence supports a role for maternal antibodies in some forms of neurodevelopmental disorder.
A Within-subjects, Within-task Demonstration of Intact Spatial Reference Memory and Impaired Spatial Working Memory in Glutamate Receptor-A-deficient Mice
Gene-targeted mice lacking the AMPA receptor subunit glutamate receptor-A (GluRA) (GluR1) and wild-type controls were compared on a radial-maze task in which the same three of six arms were always baited, but in which the rewards of milk were not replaced within a trial. This procedure allowed not only a within-subjects but also a within-trials assessment of both spatial working memory (WM) and reference memory (RM) in GluRA-/- mice, using identical spatial cues. In experiment 1, the GluRA-/- mice made more WM and RM errors during task acquisition. However, separate groups of GluRA-/- and wild-type mice (experiment 2) acquired a purely RM version of the task at a similar rate, using a paradigm with which it was not possible to make WM errors (doors prevented mice from re-entering an arm that they had already visited on that trial). In contrast, mice with hippocampal lesions were dramatically impaired. These results are consistent with the possibility that the WM impairment in the GluRA-/- mice during experiment 1 produced interference that disrupted RM acquisition. A WM component was therefore introduced after RM acquisition in experiment 2 (i.e., the mice were no longer prevented from re-entering a previously visited arm). The GluRA-/- mice now made considerably more WM errors than did wild-type mice, but simultaneously, RM was only mildly and transiently impaired. These experiments provide additional evidence of a selective spatial WM deficit coexisting with intact spatial RM acquisition in GluRA-/- mice, suggesting that different neuronal mechanisms within the hippocampus may support these different kinds of information processing.
The Role of Hippocampal Glutamate Receptor-A-dependent Synaptic Plasticity in Conditional Learning: the Importance of Spatiotemporal Discontiguity
Gene-targeted mice lacking the AMPA receptor subunit glutamate receptor-A (GluR-A or GluR1) and mice with cytotoxic hippocampal lesions were compared with wild-type and sham-operated controls, respectively, on a conditional learning task using an elevated T-maze. Floor inserts (white perspex vs wire mesh) provided a conditional cue indicating in which goal arm a food reward was to be found. The relationship between the floor insert and the rewarded goal arm was constant throughout the experiment. Both lesioned and knock-out mice were able to acquire the task if the floor inserts extended throughout the entire maze, including the start arm and both goal arms. In contrast, both lesioned and knock-out mice were unable to acquire the task if the floor inserts were only present in the start arm of the maze. The absence of the conditional cue (the floor insert) at the time when the place-reward association was experienced thus critically determined whether or not the mice were impaired. We suggest that hippocampal GluR-A-dependent synaptic plasticity contributes to a memory system in rodents for encoding both the spatial and temporal contexts (the where and the when) associated with a particular event.
Human Endogenous Retrovirus Glycoprotein-mediated Induction of Redox Reactants Causes Oligodendrocyte Death and Demyelination
Human endogenous retroviruses (HERVs) constitute 8% of the human genome and have been implicated in both health and disease. Increased HERV gene activity occurs in immunologically activated glia, although the consequences of HERV expression in the nervous system remain uncertain. Here, we report that the HERV-W encoded glycoprotein syncytin is upregulated in glial cells within acute demyelinating lesions of multiple sclerosis patients. Syncytin expression in astrocytes induced the release of redox reactants, which were cytotoxic to oligodendrocytes. Syncytin-mediated neuroinflammation and death of oligodendrocytes, with the ensuing neurobehavioral deficits, were prevented by the antioxidant ferulic acid in a mouse model of multiple sclerosis. Thus, syncytin's proinflammatory properties in the nervous system demonstrate a novel role for an endogenous retrovirus protein, which may be a target for therapeutic intervention.
Genetic Dissection of a Behavioral Quantitative Trait Locus Shows That Rgs2 Modulates Anxiety in Mice
Here we present a strategy to determine the genetic basis of variance in complex phenotypes that arise from natural, as opposed to induced, genetic variation in mice. We show that a commercially available strain of outbred mice, MF1, can be treated as an ultrafine mosaic of standard inbred strains and accordingly used to dissect a known quantitative trait locus influencing anxiety. We also show that this locus can be subdivided into three regions, one of which contains Rgs2, which encodes a regulator of G protein signaling. We then use quantitative complementation to show that Rgs2 is a quantitative trait gene. This combined genetic and functional approach should be applicable to the analysis of any quantitative trait.
Hippocampal Lesions, Species-typical Behaviours and Anxiety in Mice
The hippocampus is believed to play an important role in spatial cognition and anxiety. Much of the supporting evidence is derived from rat studies. Recent reports on hippocampal lesioned mice also showed impairments in spatial function, but anxiety was not uniformly diminished. There were, however, striking impairments in several "species typical" behaviours; lesioned mice made poorer nests, and hoarded and burrowed less. In the present experiments, mice with excitotoxic hippocampal lesions were tested in a well-established anxiety paradigm, the light-dark box. As in previous anxiety tests, the results were mixed; some measures (reduced dark time) suggested lesioned mice were less anxious; others (fewer light-dark transits) suggested greater anxiety. However, lesioned mice only made fewer transits when the door was small. This suggested that the tendency to enter small holes, so characteristic of small rodents, was reduced; subsequent tests showed lesioned mice preferred to explore in an alley rather than enter its attached tunnels. Further tests of "species typical" behaviours revealed that lesioned mice spent less time digging and climbing, and made less use of cardboard shelters in their cages. This was not due to inactivity; lesions did not reduce grooming or locomotion. Finally, tests of hyponeophagia showed hippocampal lesions reduced this measure of anxiety, so long as the control baseline was sufficiently high. Overall, the results suggest that the hippocampus is important in many species-typical behaviours, potentially influencing performance in a range of behavioural tests. However, species-typical behaviours offer easy and economical ways to test for hippocampal dysfunction, for example, in genetically modified mice.
Hippocampal Scrapie Infection Impairs Operant DRL Performance in Mice
In differential reinforcement of low rates of responding (DRL) tasks, animals are trained to respond for rewards that become available only after some set time has elapsed since the animal's previous response. DRL performance is impaired by hippocampal lesions regardless of their precise location, and can be measured using automated operant equipment, whereas spatial tasks are selectively impaired by dorsal, but not ventral hippocampal lesions, and are typically conducted by hand. Earlier studies of prion infection following dorsal hippocampal micro-injections of scrapie have shown clear impairments of spatial alternation, but these occurred significantly later than dysfunction in hippocampus-dependent 'domestic' tasks such as nesting or burrowing. In the present experiment, mice were trained to respond on an automated DRL schedule prior to dorsal hippocampal ME7 scrapie injection. Post-operative DRL performance was monitored, along with performance on 'domestic' and other tests, which provided additional measures of disease progression. Animals with scrapie developed a clear DRL deficit at approximately the same time as their deficits on the other tests became apparent, and long before clinical signs were detectable. DRL deficits thus appeared earlier in the sequence of disease progression than previously reported for spatial alternation, suggesting that early signs of scrapie infection are caused in part by neuronal dysfunction extending beyond the dorsal hippocampal region of initial infection.
A Protocol for High-throughput Phenotyping, Suitable for Quantitative Trait Analysis in Mice
Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.
Increased Expression of the 5-HT Transporter Confers a Low-anxiety Phenotype Linked to Decreased 5-HT Transmission
A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.
T-maze Alternation in the Rodent
This protocol details a method for using a T-maze to assess the cognitive ability of rodents. The T-maze is an elevated or enclosed apparatus in the form of a T placed horizontally. Animals are started from the base of the T and allowed to choose one of the goal arms abutting the other end of the stem. If two trials are given in quick succession, on the second trial the rodent tends to choose the arm not visited before, reflecting memory of the first choice. This is called 'spontaneous alternation'. This tendency can be reinforced by making the animal hungry and rewarding it with a preferred food if it alternates. Both spontaneous and rewarded alternation are very sensitive to dysfunction of the hippocampus, but other brain structures are also involved. Each trial should be completed in under 2 min, but the total number of trials required will vary according to statistical and scientific requirements.
Appetitive Position Discrimination in the T-maze
This protocol details a method to perform appetitively motivated tasks in rodents to test cognitive ability. When testing cognition in animals, the simplest paradigms can potentially yield quick results with minimal investment from the experimenter. Although appetitively motivated tasks are generally learnt more slowly than aversively motivated ones, they may be essential for distinguishing the effects of a treatment on learning from its effects on aversive motivation per se. For example, if a treatment improves learning in both types of paradigm, this is better evidence that it affects cognition rather than sensorimotor processes. Rats and mice easily learn position discriminations in a T-maze, especially if multiple cues, such as different objects and floor textures in the goal arms, are provided. To start, the rodent is placed in the maze and it chooses an arm. This Trial 1, however, is the only one on which this arm will be rewarded. From now on, it must always choose the other arm. The rule is simple: for example, always turn left into the arm with diagonal black stripes on the walls and gravel glued to the floor. High levels of correct responding can be achieved within 20-40 trials. The test may therefore be particularly useful with animals of low cognitive ability, such as transgenic mice derived from some 129 or SJL strains. Once the animals are habituated, each trial should take approximately 1 min. Thus, to test ten animals for 40 trials would take around 7 h.
Burrowing in Rodents: a Sensitive Method for Detecting Behavioral Dysfunction
Virtually all rodents display burrowing behavior, yet measurement of this behavior has not yet been standardized or formalized. Previously, parameters such as the latency to burrow and the complexity of the burrow systems in substrate-filled boxes in the laboratory or naturalistic outdoor environments have been assessed. We describe here a simple protocol that can quantitatively measure burrowing in laboratory rodents, using a simple apparatus that can be placed in the home cage. The test is very cheap to run and requires minimal experimenter training, yet seems sensitive to a variety of treatments, such as the early stages of prion disease in mice, mouse strain differences, lesions of the hippocampus and prefrontal cortex in mice, also effects of lipopolysaccharide and IL-1beta in rats. Other species such as hamsters, gerbils and Egyptian spiny mice also burrow in this apparatus, and with suitable size modification probably almost any burrowing animal could be tested in it. The simplicity, sensitivity and robustness of burrowing make it ideal for assessing genetically modified animals, which in most cases would be mice. The test is run from late afternoon until the next morning, but only two measurements need to be taken.
Digging and Marble Burying in Mice: Simple Methods for in Vivo Identification of Biological Impacts
Mice exhibit various species-typical behaviors such as digging and burrowing. They dig in the ground to find food, to hoard food, to create a refuge from predators or cold and to make a safe nursery area for the young. In the laboratory, mice dig vigorously in deep bedding such as wood chips. This behavior is sensitive to strain differences and drugs. For example, the effects of anxiolytics and 5-HT-active compounds, including those used clinically for obsessive-compulsive disorder (OCD), can be detected. Digging can be quantified by manual timing. Alternatively, the bedding can be covered with glass marbles and the number buried can be counted after a set time. These behaviors can be assessed using very little specialized equipment, and results can be obtained from ten animals in about an hour. Species-typical behaviors may be sensitive to a wide variety of treatments, and their simplicity and ability to yield robust quantitative data might be particularly useful in assessing genetically modified mice, even in laboratories not primarily oriented to behavioral work.
Housing, Husbandry and Handling of Rodents for Behavioral Experiments
Most animals used in research are rodents, mainly mice because of their predominance in genetics and molecular biology. This article attempts to provide an introduction to mice and rats: health considerations (of the experimenter); choice of species, age, strain and sex; housing and environmental enrichment; and animal identification, handling and dosing. These considerations apply to animal work in general; the rest of the article focuses on the preliminary aspects of behavioral testing, including a protocol for an open field test. This procedure is traditionally associated with activity measurements, and although automated versions are readily available these days, the latter are expensive and may be unavailable in many non-behavioral departments. Moreover, particularly when testing novel genetically modified animals or pharmacological agents, there is no substitute for direct visual observation to detect abnormal signs in the animals: for example, ptosis, piloerection, tremor, ataxia or exophthalmos. The open field test can be adapted in several ways: to assess general behavior and activity (similar to a primary screen in the pharmaceutical industry) or to measure memory (habituation) or anxiety.
Assessing Nest Building in Mice
For small rodents, nests are important in heat conservation as well as reproduction and shelter. Nesting is easily measured in the home cages of mice, particularly with the advent of pressed cotton materials. The mice first shred the tightly packed material, then arrange it into a nest. Published studies have often used materials such as hay, twine or tissues, sometimes preshredded, and have assigned scores of the quality of the resulting nest with rather rudimentary rating scales; e.g., 0, no nest; 1, flat nest; 2, nest covering the mouse. The protocol described here uses pressed cotton squares and a definitive 5-point nest-rating scale. Any unshredded material left after a bout of nesting can also be weighed, providing a semi-independent objective assay of nesting ability. Nesting has been shown to be sensitive to brain lesions, pharmacological agents and genetic mutations. This is a simple, cheap and easily done test that, along with other tests of species-typical behavior, is a sensitive assay for identifying previously unknown behavioral phenotypes. The test needs to be done overnight, but it should take no more than 5 minutes to set up plus 1 minute to assess one nest and weigh the untorn residue.
Assessing Hoarding in Mice
Hoarding is a species-typical behavior shown by rodents, as well as other animals. By hoarding, the rodent secures a food supply for times of emergency (for example, when threatened by a predator) or for times of seasonal adversity such as winter. Scatter hoarding, as seen typically in squirrels and birds, involves placing small caches of food in hidden places, generally underground. Most rodents, however, hoard a supply of food in or near the home base--for example, in 'larders' near the sleeping quarters in a burrow. In the laboratory, measurement of hoarding involves simply weighing the food transported into the home cage from an external source, but the route to that source must be secure and animal-proof; for example, there should be no holes large enough to permit escape of a mouse, and no weak points that could be enlarged by gnawing. A suitable and easily constructed apparatus is described in the protocol. Hoarding has been shown to be sensitive to brain lesions and pharmacological agents, and is a suitable test for species-typical behavior in genetically modified mice.
Impaired Synaptic Plasticity and Motor Learning in Mice with a Point Mutation Implicated in Human Speech Deficits
The most well-described example of an inherited speech and language disorder is that observed in the multigenerational KE family, caused by a heterozygous missense mutation in the FOXP2 gene. Affected individuals are characterized by deficits in the learning and production of complex orofacial motor sequences underlying fluent speech and display impaired linguistic processing for both spoken and written language. The FOXP2 transcription factor is highly similar in many vertebrate species, with conserved expression in neural circuits related to sensorimotor integration and motor learning. In this study, we generated mice carrying an identical point mutation to that of the KE family, yielding the equivalent arginine-to-histidine substitution in the Foxp2 DNA-binding domain. Homozygous R552H mice show severe reductions in cerebellar growth and postnatal weight gain but are able to produce complex innate ultrasonic vocalizations. Heterozygous R552H mice are overtly normal in brain structure and development. Crucially, although their baseline motor abilities appear to be identical to wild-type littermates, R552H heterozygotes display significant deficits in species-typical motor-skill learning, accompanied by abnormal synaptic plasticity in striatal and cerebellar neural circuits.
Increased Striatal Dopamine Release and Hyperdopaminergic-like Behaviour in Mice Lacking Both Alpha-synuclein and Gamma-synuclein
Alpha-synuclein is intimately involved in the pathogenesis of Parkinson's disease, and has been implicated in the regulation of synthesis, release and reuptake of dopamine (DA). However, mice lacking members of the synuclein family have been reported to display no overt behavioural phenotype. This may be a result of compensatory upregulation of other synucleins during development. Here we report on behaviour and DA synapse function of alpha-synuclein null, gamma-synuclein null, and alpha-gamma-synuclein double-null knockout mice. Double-null mice were hyperactive in a novel environment and alternated at a lower rate in a T-maze spontaneous alternation task, a phenotype reminiscent of mice expressing reduced levels of the DA transporter. To investigate a possible hyperdopaminergic phenotype in alpha-gamma-synuclein double-null mice, we used fast-scan cyclic voltammetry at carbon-fibre microelectrodes to assess DA release and reuptake in striatal slices from wild-type, alpha-null, gamma-null and double-null mice in real time. Double-null mice were found to have a twofold increase in the extracellular concentration of DA detected after discrete electrical stimuli in the striatum. By measuring the rate of reuptake of DA and tissue DA content in these animals, we showed that the observed increase in size of striatal DA transients was not attributable to a decrease in reuptake of DA via the DA transporter, and can not be attributed to an increase in tissue DA levels in the striatum. Rather, we propose that loss of both alpha- and gamma-synuclein causes an increase in release probability from dopaminergic synapses.
Marked for Life? Effects of Early Cage-cleaning Frequency, Delivery Batch, and Identification Tail-marking on Rat Anxiety Profiles
Daily handling of preweanling rats reduces their adult anxiety. Even routine cage-cleaning, involving handling, reduces adult anxiety compared with controls. Cage-cleaning regimes differ between animal breeders, potentially affecting rodent anxiety and experimental results. Here, 92 adult male rats given different cage-cleaning rates as pups, were compared on plus-maze, hyponeophagia, corticosterone, and handling tests. They were pair-housed and half were tail-marked for identification. Anxiety/stress profiles were unaffected by cage-cleaning frequency, suggesting that commercial-typical differences in husbandry contribute little variance to adult rat behavior. However, delivery batch affected some elevated plus-maze measures. Also, tail-marked rats spent three times longer on the plus-maze open arms than their unmarked cagemates, suggesting reduced anxiety, yet paradoxically they showed greater chromodacryorrhoea responses to handling, implying increased aversion to human contact. A follow-up study showed that rats avoided the odor released from the marker pen used. Thus, apparently trivial aspects of procedure can greatly affect experimental results.
Systemic Inflammation Induces Acute Behavioral and Cognitive Changes and Accelerates Neurodegenerative Disease
Chronic neurodegeneration results in microglial activation, but the contribution of inflammation to the progress of neurodegeneration remains unclear. We have shown that microglia express low levels of proinflammatory cytokines during chronic neurodegeneration but are "primed" to produce a more proinflammatory profile after systemic challenge with bacterial endotoxin (lipopolysaccharide [LPS]).
A Double Dissociation Between the Effects of Sub-pyrogenic Systemic Inflammation and Hippocampal Lesions on Learning
Immune system activation has been found to affect the function of the hippocampus. Sub-pyrogenic systemic inflammation impairs performance of species-typical behaviours that are also disrupted by hippocampal lesions in rodents. In a series of experiments the effect of a low, sub-pyrogenic dose of lipopolysaccharide (LPS) on hippocampus-dependent learning and memory was tested. LPS failed to impair hippocampus-dependent spatial reference memory and working memory. However, LPS affected learning a simple side-discrimination task in which an arm of a T-maze was rewarded (correct arm), and the other arm was never rewarded (incorrect arm). Whereas LPS actually enhanced performance when reward was available on every trial in the correct arm, LPS impaired learning when the correct arm was rewarded on 50% of trials. Hippocampal lesions did not impair either the continuous or partial reinforcement versions of the task. These results demonstrate that a low, sub-pyrogenic dose of LPS can impair cognitive function, but can, depending on the demands of the task, also facilitate learning. However, the double dissociation between the effects of LPS and hippocampal lesions demonstrate that sub-pyrogenic inflammation does not affect learning by disrupting hippocampal function.
Deterioration of Physical Performance and Cognitive Function in Rats with Short-term High-fat Feeding
Efficiency, defined as the amount of work produced for a given amount of oxygen consumed, is a key determinant of endurance capacity, and can be altered by metabolic substrate supply, in that fatty acid oxidation is less efficient than glucose oxidation. It is unclear, however, whether consumption of a high-fat diet would be detrimental or beneficial for endurance capacity, due to purported glycogen-sparing properties. In addition, a high-fat diet over several months leads to cognitive impairment. Here, we tested the hypothesis that short-term ingestion of a high-fat diet (55% kcal from fat) would impair exercise capacity and cognitive function in rats, compared with a control chow diet (7.5% kcal from fat) via mitochondrial uncoupling and energy deprivation. We found that rats ran 35% less far on a treadmill and showed cognitive impairment in a maze test with 9 d of high-fat feeding, with respiratory uncoupling in skeletal muscle mitochondria, associated with increased uncoupling protein (UCP3) levels. Our results suggest that high-fat feeding, even over short periods of time, alters skeletal muscle UCP3 expression, affecting energy production and physical performance. Optimization of nutrition to maximize the efficiency of mitochondrial ATP production could improve energetics in athletes and patients with metabolic abnormalities.
A Mouse Model for the Metabolic Effects of the Human Fat Mass and Obesity Associated FTO Gene
Human FTO gene variants are associated with body mass index and type 2 diabetes. Because the obesity-associated SNPs are intronic, it is unclear whether changes in FTO expression or splicing are the cause of obesity or if regulatory elements within intron 1 influence upstream or downstream genes. We tested the idea that FTO itself is involved in obesity. We show that a dominant point mutation in the mouse Fto gene results in reduced fat mass, increased energy expenditure, and unchanged physical activity. Exposure to a high-fat diet enhances lean mass and lowers fat mass relative to control mice. Biochemical studies suggest the mutation occurs in a structurally novel domain and modifies FTO function, possibly by altering its dimerisation state. Gene expression profiling revealed increased expression of some fat and carbohydrate metabolism genes and an improved inflammatory profile in white adipose tissue of mutant mice. These data provide direct functional evidence that FTO is a causal gene underlying obesity. Compared to the reported mouse FTO knockout, our model more accurately reflects the effect of human FTO variants; we observe a heterozygous as well as homozygous phenotype, a smaller difference in weight and adiposity, and our mice do not show perinatal lethality or an age-related reduction in size and length. Our model suggests that a search for human coding mutations in FTO may be informative and that inhibition of FTO activity is a possible target for the treatment of morbid obesity.
The Puzzle Box As a Simple and Efficient Behavioral Test for Exploring Impairments of General Cognition and Executive Functions in Mouse Models of Schizophrenia
Deficits in executive functions are key features of schizophrenia. Rodent behavioral paradigms used so far to find animal correlates of such deficits require extensive effort and time. The puzzle box is a problem-solving test in which mice are required to complete escape tasks of increasing difficulty within a limited amount of time. Previous data have indicated that it is a quick but highly reliable test of higher-order cognitive functioning. We evaluated the use of the puzzle box to explore executive functioning in five different mouse models of schizophrenia: mice with prefrontal cortex and hippocampus lesions, mice treated sub-chronically with the NMDA-receptor antagonist MK-801, mice constitutively lacking the GluA1 subunit of AMPA-receptors, and mice over-expressing dopamine D2 receptors in the striatum. All mice displayed altered executive functions in the puzzle box, although the nature and extent of the deficits varied between the different models. Deficits were strongest in hippocampus-lesioned and GluA1 knockout mice, while more subtle deficits but specific to problem solving were found in the medial prefrontal-lesioned mice, MK-801-treated mice, and in mice with striatal overexpression of D2 receptors. Data from this study demonstrate the utility of the puzzle box as an effective screening tool for executive functions in general and for schizophrenia mouse models in particular.
Opposing Alterations in Anxiety and Species-typical Behaviours in Serotonin Transporter Overexpressor and Knockout Mice
Human gene association studies have produced conflicting findings regarding the relationship between the 5-HT transporter (5-HTT) and anxiety. In the present study genetically modified mice were utilised to examine the effects of changes in 5-HTT expression on anxiety. In addition, the influence of 5-HTT expression on two innate "species-typical" behaviours (burrowing and marble burying) and body weight was explored. Across a range of models, 5-HTT overexpressing mice displayed reduced anxiety-like behaviour whilst 5-HTT knockout mice showed increased anxiety-like behaviour, compared to wildtype controls. In tests of species-typical behaviour 5-HTT overexpressing mice showed some facilitation whilst 5-HTT knockout mice were impaired. Reciprocal effects were also seen on body weight, as 5-HTT overexpressors were lighter and 5-HTT knockouts were heavier than wildtype controls. These findings show that variation in 5-HTT gene expression produces robust changes in anxiety and species-typical behaviour. Furthermore, the data add further support to findings that variation of 5-HTT expression in the human population is linked to changes in anxiety-related personality traits.
The Passive Transfer of Immunoglobulin G Serum Antibodies from Patients with Longstanding Complex Regional Pain Syndrome
The aetiology of Complex Regional Pain Syndrome (CRPS) is unknown. Recent evidence suggests that there may be autoantibodies directed against peripheral nerves, but it is unclear whether such autoantibodies are merely biomarkers or whether they cause or contribute to the underlying pathology. The transfer of disease after injection of a patient's serum or IgG fraction into mice ('passive transfer') is the classic way to demonstrate a functional role of autoantibodies.
Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse
The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.
Functional Alterations to the Nigrostriatal System in Mice Lacking All Three Members of the Synuclein Family
The synucleins (α, β, and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.
Hemisphere-specific Optogenetic Stimulation Reveals Left-right Asymmetry of Hippocampal Plasticity
Postsynaptic spines at CA3-CA1 synapses differ in glutamate receptor composition according to the hemispheric origin of CA3 afferents. To study the functional consequences of this asymmetry, we used optogenetic tools to selectively stimulate axons of CA3 pyramidal cells originating in either left or right mouse hippocampus. We found that left CA3 input produced more long-term potentiation at CA1 synapses than right CA3 input as a result of differential expression of GluN2B subunit-containing NMDA receptors.
Hippocampal Lesions Can Enhance Discrimination Learning Despite Normal Sensitivity to Interference from Incidental Information
Spatial properties of stimuli are sometimes encoded even when incidental to the demands of a particular learning task. Incidental encoding of spatial information may interfere with learning by (i) causing a failure to generalize learning between trials in which a cue is presented in different spatial locations and (ii) adding common spatial features to stimuli that predict different outcomes. Hippocampal lesions have been found to facilitate acquisition of certain tasks. This facilitation may occur because hippocampal lesions impair incidental encoding of spatial information that interferes with learning. To test this prediction mice with lesions of the hippocampus were trained on appetitive simple simultaneous discrimination tasks using inserts in the goal arms of a T-maze. It was found that hippocampal lesioned mice were facilitated at learning the discriminations, but they were sensitive to changes in spatial information in a manner that was similar to control mice. In a second experiment it was found that both control and hippocampal lesioned mice showed equivalent incidental encoding of egocentric spatial properties of the inserts, but both groups did not encode the allocentric information. These results demonstrate that mice show incidental encoding of egocentric spatial information that decreases the ability to solve simultaneous discrimination tasks. The normal egocentric spatial encoding in hippocampal lesioned mice contradicts theories of hippocampal function that suggest that the hippocampus is necessary for incidental learning per se, or is required for modulating stimulus representations based on the relevancy of information. The facilitated learning suggests that the hippocampal lesions can enhance learning of the same qualitative information as acquired by control mice. © 2011 Wiley Periodicals, Inc.
Systemic Inflammation Induces Acute Working Memory Deficits in the Primed Brain: Relevance for Delirium
Delirium is an acute, severe neuropsychiatric syndrome, characterized by cognitive deficits, that is highly prevalent in aging and dementia and is frequently precipitated by peripheral infections. Delirium is poorly understood and the lack of biologically relevant animal models has limited basic research. Here we hypothesized that synaptic loss and accompanying microglial priming during chronic neurodegeneration in the ME7 mouse model of prion disease predisposes these animals to acute dysfunction in the region of prior pathology upon systemic inflammatory activation. Lipopolysaccharide (LPS; 100 μg/kg) induced acute and transient working memory deficits in ME7 animals on a novel T-maze task, but did not do so in normal animals. LPS-treated ME7 animals showed heightened and prolonged transcription of inflammatory mediators in the central nervous system (CNS), compared with LPS-treated normal animals, despite having equivalent levels of circulating cytokines. The demonstration that prior synaptic loss and microglial priming are predisposing factors for acute cognitive impairments induced by systemic inflammation suggests an important animal model with which to study aspects of delirium during dementia.
