The prion diseases are neurodegenerative diseases characterized by progressive neurocognitive decline and terminal dementia. In this review, we will discuss the role of neurobehavioral testing in mammalian prion disease model systems, including (1) a review of the clinical phenotype of the major prion diseases in natural disease, (2) an evidence-based summary of the benefits and shortcomings of commonly used behavioral assays, and (3) a review of the neurobehavioral testing in rodent prion models. Based upon this review, and in light of the established importance of model systems in studies of prion pathogenesis and the proven role of behavioral testing in nonprion disease neurodegenerative diseases, it is vital that prion researchers consider the clinical consequences of prion infection so as to maximize the impact of their work.
- prion diseases
- clinical signs
- mouse models
- behavioral testing
- comparative neurosciences
The prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders resulting from the accumulation of a unique, nucleic-acid free/protein-only, infectious agent. Prion diseases affect both humans and nonhumans alike and include diseases that have genetic (familial or sporadic) or infectious causes. The pivotal and unifying event in prion pathogenesis is the posttranslational misfolding of the host-encoded, normal cellular prion protein (denoted PrPC) into a misfolded variant (denoted PrPSc or PrPD). Misfolding is characterized by increased β-sheet content, decreased α-helical content, and by conferred resistance to detergents, alcohol, formalin, proteases, boiling, autoclaving, and radiation . The resulting PrPSc acts as a template for its self-propagation. In addition to their shared mechanism, prion diseases are united by their pathology, which includes amyloid deposition, vacuolization, synaptic dysfunction, glial-mediated neuroinflammation, and neuronal death.
Although the terminal pathologic event in prion disease is neuronal death and the terminal clinical event is neuronal death, the link between these is unclear. Historically, two competing hypotheses have been proposed, namely (1) a loss-of-function hypothesis or (2) a gain-of-function hypothesis. Based upon studies demonstrating that pre- and postnatal knockdown of PrPC expression fails to replicate bona fide prion disease, it seems unlikely loss of function contributes significantly to prion pathogenesis [2–4]. However, while it is increasingly likely that an alternate isoform of PrPC is responsible for prion toxicity, it is unclear whether this species presents a protease-sensitive or resistant form, a monomeric or oligomeric form, or if interactions with additional components are necessary. Lastly, while this model implicates PrPSc as a
In a disease system rife with novelty, one of the most intriguing and clinically relevant aspects of prion disease biology is the existence of strains. Originally recognized in studies of sheep and goats with experimental scrapie, but the best characterized in scrapie-infected mice, the concept of strains reflects clinical, pathologic, and structural variants of prion disease . Prion strains are unique isolates that demonstrate different phenotypical and biochemical differences when transmitted into identical hosts. Classically, these differences include pattern of PrPSc distribution (both within and outside of the CNS), PrPSc plaque morphology, vacuolar profile, incubation period, susceptibility to PK digestion, glycosylation profile, incubation period, and, most important for this article, clinical disease phenotype [12–14]. The biologic basis for strains is not entirely clear, but it is hypothesized that unique PrPSc confirmations and polymorphisms are significant contributors [15–17].
In a review of neurobehavioral testing in prion diseases, it is worth noting that there is not always a clear or proportional relationship between disease neuropathology (i.e., PrPSc accumulation, gliosis, and neuronal loss) and clinical phenotype. This is most dramatically represented in subclinical prion disease (i.e., measurable CNS PrPSc without clinical disease) and in prion-infected animals demonstrating significant clinical disease but lacking detectable PrPSc [7, 18, 19]. This lack of correlation between patterns of brain PrPSc deposition and clinical disease is well documented in many natural and experimentally infected TSE affected animals, including TSE-infected cattle, goats, and mice [18, 20–22]. In addition, a discordant relationship between neuronal loss and clinical signs is reported in BSE-infected cattle and between neuroinflammation and clinical signs in scrapie-infected sheep [23–27]. The cause(s) of this disparate relationship between PrPSc and prion disease are not completely clear, but the limited sensitivity of traditional PrPSc detection tools, the increasing recognition of the toxicity of protease-sensitive forms of misfolded PrP, and the complexity of the tissue response to misfolded prion protein likely contribute . Finally, it is likely that shortcomings in behavioral testing have contributed to historical inabilities to document clinical disease in prion-infected animals, particularly those in which neurobehavioral deficits may be subtle. This is particularly likely in large animals, in which the vague and imprecise early clinical signs of TSE infection can mimic a number of nonprion infectious conditions.
2. Clinical phenotype of prion diseases
Despite their unifying cause, individual prion diseases demonstrate unique clinical presentations. This clinical heterogeneity not only applies between differing diseases (i.e., CJD vs. FFI) but also within a particular disease. The following section summarizes the major clinical features of the most common prion diseases of humans and domestic animals.
2.1. Creutzfeld-Jakob disease (CJD)
Creutzfeld-Jakob disease (CJD) is the most common form of human prion disease and can be divided into sporadic, hereditary (i.e., familial), iatrogenic, or variant forms. The hereditary form can be further subdivided into three distinct phenotypic subtypes, namely (1) Gerstmann-Straussler-Scheinker (GSS) disease, (2) fatal familial insomnia (FFI), and familial CJD (fCJD). Although the following section will review the unique clinical features of each of these forms, all variants of CJD are generally characterized by a rapid, progressive onset of dementia of unknown origin .
Sporadic CJD (sCJD) is the most common form of CJD, representing approximately 85% of cases . Although six major variants of sCJD are recognized according to differences in molecular, genetic, and biochemical features, most CJD variants present a similar clinical phenotype [30, 31]. The common features of CJD are represented by progressive dementia with some combination of myoclonus, visual deficits, cerebellar disturbances, pyramidal or extrapyramidal symptoms (spasticity, hyperactive reflexes, muscle contractions, alterations in movement, tremor) or akinetic mutism (alertness with a lack of motor functions, including speech, gestures, and facial expression) . However, notable clinically unique CJD subtypes include cerebellar (or ataxic subtypes), myoclonic CJD, thalamic CJD, and the Heidenhain variant (which manifests significant visual deficits) [33–36]. In addition to these variants, 41 distinct forms of inherited TSEs have been described in humans, each demonstrates unique clinical phenotypes unique point mutations or octapeptide insertion mutations .
Fatal familial insomnia (FFI) is a clinicopathologic variant of human prion disease considered to be a familiar variant of CJD. Genetically, FFI is characterized by a mutation at codon 178 of the prion protein gene (aspartic acid to asparagine) coupled to a methionine polymorphism at codon 129 on the corresponding abnormal allele. As the name indicates, FFI patients chiefly suffer from sleep disturbances—principally insomnia, but also including hypersomnia, restless sleep, and sleep attacks . Beyond these, FFI patients demonstrate a range of clinical signs that are both similar to, and unique from classic CJD. Overlapping signs include cognitive deficits, spatial disorientation, ataxia, and hallucinations whereas clinical signs unique to FFI include weight loss, hyperhidrosis, and husky voice . However, even among FFI patients, there are unique clinical syndromes that depend upon the codon 129 genotype. For example, it has been reported that hallucinations and myoclonus are more common in patients that are methionine homozygous (i.e., MM) at codon 129, whereas vegetative disturbances and nystagmus are more common in methionine heterozygous patients . Interestingly, although the diagnosis of unique variants of prion disease based on clinical phenotype only is considered difficult, an algorithm of FFI specific and sensitive clinical signs has been developed which correctly identified 81% of patients during early disease stages .
Like FFI, Gerstmann-Straussler-Scheinker (GSS) is a mutational variant of CJD in which a number of differing prion protein gene point mutations have been identified, the most common of which is the P102L/129M variant . There are two typical clinical phenotypes of P102L GSS, namely (1) a typical type with cerebellar ataxia and slow onset dementia and (2) a CJD-like form with acute dementia and myoclonus [29, 39].
Like other TSEs, scrapie is a clinically progressive disease that is most classically characterized by pruritus, altered behavior, and locomotion deficits . However, like other prion diseases, the clinical phenotype of sheep scrapie varies somewhat according to strain and host characteristics. Accordingly, three profiles of clinical disease have been described, namely (1) a pruritic form, (2) a paralytic form (which lack pruritus), and (3) an atypical cerebellar (Nor98) form . The neurologic signs of scrapie are wide-ranging, and include mentation abnormalities (e.g., hyperresponsiveness), motor deficits (e.g., incoordination, exaggerated gait, hypermetria, ataxia, tremors), visual deficits (including nystagmus and blindness), loss of the menace response, dysphagia, and dysphonia [42, 43]. Although not always the case, deficits in locomotion, including hypotonia, proprioceptive deficits, reduced withdrawal reflex, and ataxia, are reported to occur later in disease [27, 43]. Terminal sheep scrapie is characterized by depression, recumbency, and/or seizure activity. In addition to the classical form of the disease, an alternate strain of scrapie, denoted atypical or Nor98, has been described and is characterized clinically by motor deficits, including progressive ataxia and incoordination whereas pruritus is very uncommon . Scrapie-infected goats demonstrate many of the same clinical signs as seen in sheep, including pruritus, restlessness, and terminal ataxia/recumbency . Similar to sheep, discrete clinical phenotypes have been identified in goat scrapie, namely a “scratching syndrome” characterized principally by pruritus and a “drowsy syndrome” characterized by decreased activity and depression absent pruritus [21, 45, 46]. However additional features have been reported, including teeth grinding, irritability, and heightened alertness . Additional noted differences between scrapie-infected sheep and goats include hyperesthesia in goats (as opposed to hypoesthesia in sheep) and nibbling of the body in goats (as opposed to rubbing of the body in sheep) .
2.3. Bovine spongiform encephalopathy (BSE)
In contrast to the prion diseases of nondomestic species, the clinical features of BSE-infected cattle are well described. Like other prion diseases, BSE infection in cattle is principally associated with progressive changes in behavior and locomotion. Early disease is dominated by changes in behavior, including increased alertness, nervousness, excitability, nervous ear/eye movements, and hypersensitivity to touch, sound, and visual stimuli, head shyness, panic-stricken response, reluctance to enter the milking parlor, and change in temperament [20, 47–49]. During this early phase, specific tests used to elicit hyperesthesia include: (1) the “flash test” (reactivity to a camera flash), (2) the “clipboard test” (reactivity to waving a clipboard towards the animal, (3) the “hand clap” (reactivity to clapping hands), and (4) the “stick test” (reactivity to a light touch of the hindlimbs with a flexible stick) . As disease progresses, BSE-infected cattle develop deficits in locomotion include tremors, hypermetria, hindlimb and generalized ataxia, difficulty rising, spastic gait, and thermal recumbency . Terminally, cattle may enter into a “dull” form of the disease characterized by loss of previous hyperesthesia and disinterest in surroundings . Previous studies have shown that at least one, either apprehension, hyper-reactivity, or ataxia, is found in 97% of cattle with BSE .
Outside of cattle, there is sparse information on BSE infection in other species. In BSE-infected goats, hyperesthesia, pruritus, head tossing, or shaking, overreactivity to touch of the hindlimbs, and hypermetria are reported . There are conflicting reports on the clinical phenotype of BSE-infected sheep, which may reflect route of inoculation, age of infected sheep, or intensity of clinical monitoring. In one report, BSE-infected sheep demonstrate a uniform clinical disease characterized by early pruritus with late locomotion deficits . Whereas, other studies suggest that sudden-onset ataxia is common in BSE-infected sheep .
In addition to classical BSE (C-BSE), two unique strains of BSE have been described. These strains denoted by H-BSE and L-BSE according to their biochemical characteristics and migration profile of the proteinase-resistant fragments on Western blot, demonstrate some clinical features unique from C-BSE. Similar to C-BSE, cattle experimentally infected with either H-BSE or L-BSE demonstrate both hyperesthesia and dullness, however the magnitude of hyperresponsiveness is reported to be higher in C-BSE . While no consistent differences were noted when the clinical phenotype of H- and L-type BSE were compared, cattle with either of these two forms of atypical BSE did not progress to permanent recumbency and failed to demonstrate tremors, which contrasts with C-BSE .
2.4. Chronic wasting disease (CWD)
Chronic wasting disease (CWD) is an endemic prion disease of cervids, affecting white-tailed deer, mule deer, elk, moose, red deer, sika deer, muntjac, and reindeer. The two most recognized clinical signs of natural CWD are behavioral changes and loss of body mass. Not surprisingly, the behavioral phenotype of CWD in wild, naturally infected animals is not well-described, but work with captive (both naturally and experimentally infected) animals has provided some descriptive insights. Like other ruminant TSEs, CWD is a progressive disease. Early in the progression of CWD, the behavioral abnormalities in CWD are considered subtle and best appreciated by those who are in repeated contact with infected animals. Early clinical signs include alterations in patterns of interaction with humans (either increased or decreased contact), fixed gaze, repetitive behaviors (head tossing, exaggerated lifting of the legs), diminished alertness, prolonged periods of somnolence, and aggressive behavior which, late in disease, progresses to motor deficits (incoordination, trembling, and stumbling) [42, 53, 54]. Although distinct strains of CWD have been identified, as reflected by incubation period and neuropathologic differences, their neurobehavioral characteristics have not been reported [55, 56].
3. The basic toolkit of behavioral phenotyping
Behavioral research in laboratory rodent species has progressed for decades, largely with the aim of understanding the biological basis of normal behavior and brain function. When properly utilized, behavioral analysis has the potential to be both explanatory of the
3.1. Neuromotor function
One of the first classes of behaviors that is often looked at in phenotyping studies, is the effect of the manipulation on neuromotor function, e.g., general activity, coordination, strength. A wide array of assays is available to assess the diverse aspects of neuromotor function. All of these assays are very approachable and several are amenable to automated scoring systems (for further review see Pierce and Kalivas and Wahlsten ) [58, 59]. The main differences to note in the assessment of these tests are the aspect of motor function being examined, the context of the test environment, and the motivational drive for movement.
3.2. Learning and memory
Another broad category of behavior that is regularly looked at is learning and memory (cognitive function). Assessing cognitive function can take many forms as there are multiple domains of cognitive function. Some of the basic domains include spatial navigation learning, working memory, and conditioning can be readily studied in mouse models without complicated and prolonged training. Additionally, each of these tests measures very different functions that involve different neural circuitry.
3.3. Anxiety and depression-related behavior
This is an area of research typified by some very approachable tests that are useful in their own right to study the impact of manipulations on anxiety and depression-related behavior [67, 68]. These assays are also important tools to use as controls for altered motivation in cognitive assays . These assays are often fairly easy to employ, but can be easily impacted by uncontrolled external variabilities, and many times subject to misinterpretation/overinterpretation of data. Critical to effectively using these behavioral tests is an understanding their test validity, be it construct, face, or predictive . Also, as there is some inherent fallibility in interpreting these behaviors as they relate to affective and mood disorders, it is important to utilize multiple tests in combination for a thorough evaluation.
This discussion of behavioral testing has mostly focused on individual tests, what they are, how they work, what is the utility and what the confounds are to their use. However, at this point it is important to discuss the use of combinations of tests into so-called “test batteries.” The idea of a broad-based analysis of behavior is at the heart of behavioral phenotyping efforts that have grown in response to advances in murine genetics and increasing emphasis on disease modeling research (for review see Crawley) [74, 75]. The construction of a proper test battery is not a trivial or even standardized operation. Test batteries can be designed to be intentionally broad with an emphasis on observation and characterization as is often done with gene knockout studies. Such designs tend to take a relatively agnostic approach to hypotheses about phenotype and may use an initial screen to suggest more detailed behavioral analysis or follow-up mechanistic studies. Another way to design a screen is with investigation of a very specific endpoint in mind (e.g., cognitive deficit). In this case supplemental tests may be chosen to satisfy controls for confounding behavioral deficits (motor dysfunction, sensory deficit, or changes in motivation). In all situations it is advisable to at least consider the use of multiple tests within the same behavioral domain that utilize different outputs or behavioral abilities to complete the test.
4. Behavior assays used in mouse models of prion disease
The adjective insidious is commonly used to describe the prion diseases because there are no obvious outward symptoms to alert the public to infection and progression. This presents a problem to those seeking to provide a therapeutic intervention. A common theme in medicine is the idea that early intervention in disease progression is more likely to lead to a better prognosis. Thus, the conundrum with prion diseases is that since this disease progresses silently, how are we to be alerted to its progression in order to intervene? Luckily for us, prion diseases are neurological diseases and there is an expansive literature on brain—behavior relationships. Thus, behavioral testing using experimental animal model systems allows for sufficient control of variables to rigorously test specific hypotheses about the impact of prion disease progression on behavior. As such, there have been a number of studies that have attempted to use behavior assays to document the progression of prion diseases.
Although this chapter focuses on the utility of behavior analysis for understanding prion diseases, it is interesting that early studies used scrapie to understand brain-behavior relationships. Savage and Field used the open field test to measure emotionality (at various dpi) in mice that were intracerebrally (IC) inoculated with scrapie (third passage from sheep) . Their data indicate that disease progression is correlated with a decline in emotionality, but not ambulation. A subsequent study used 263 K scrapie inoculum to unilaterally ablate the striatum in golden hamsters . Striatal destruction was verified using the apomorphine stimulated rotation task. The authors suggest that scrapie might be a useful tool for studying other brain regions such as the basal ganglia.
Clinical signs of disease progression in IC inoculated scrapie mouse model systems are observed around 23 weeks or 161 days post inoculation [78, 79]. By this time, the disease has progressed to the point where no therapeutic intervention will succeed. Mice at this stage of the disease show reduced mobility, hunched posture and lack of grooming [78, 79]. Heitzman and Corp wanted to determine if they could detect behavioral symptoms of scrapie prior to the then current standard of 16 weeks post inoculation . They tested mice that were IC inoculated with scrapie using the open field test and the emergence test. Although they did not observe any effect of early disease progression on the open field test, they did observe a statistically significant effect of scrapie on the emergence test at 6 weeks post inoculation. This data suggests that scrapie inoculated mice show reduced exploratory behavior or increased anxiety. More importantly, this data also indicates that it is possible to observe changes in behavior 9 weeks prior to the onset of clinical symptoms in scrapie-inoculated mice.
Outram put forth several “to be met” criteria required for scrapie-behavior correlations . (1) The behavior change must be a consequence of scrapie. (2) One should determine whether the change in behavior is correlated with altered central or peripheral nervous system activity. (3) The behavioral assay itself should not modify disease progression. (4) The behavior assayed and its neural bases should be well characterized. With these criteria in mind Outram demonstrated that drinking behavior is altered in IC inoculated scrapie mice . Declines in drinking behavior were observed approximately 7 weeks post inoculation using a number of fluids, including sucrose, water, and glucose + saline. This finding was seen in mice that were IC or IP inoculated with several scrapie strains including ME7, 22A, 79A. This effect was also observed in several mouse strains, including C57BL, A2G, VL, and VM mice.
Subsequent work by McFarland et al. found that both mouse strain and scrapie strain affected the open field and Y maze performance . In Nya:NYLAR, C57/10J, and ICR mice that were IC inoculated with Chandler scrapie, only ICR mice showed a statistically significant reduction in spontaneous alternation in the y-maze task. Moreover, Y-maze performance was diminished in the Nya:NYLAR and ICR mice, but not C57 mice. In the second experiment Nya:NYLAR mice were IC inoculated with one of three scrapie strains: 22C, ME7, and 79-A and tested at 95–103 dpi. The 22-C inoculated mice exhibited a statistically significant decrease in activity, but 79-A mice exhibited a statistically significant increase in activity. Moreover, only the ME-7 and 79-A strains resulted in a reduced entry into the center field. Although there was no effect of scrapie strain on y-maze spontaneous alternation, 79-A inoculated mice exhibited an increased number of arm entries. The strain specificity of prion clinical phenotype was further demonstrated by a study examining behavioral effects on C57BL/6 mice IC inoculated with either the scrapie strains 139A or ME7 or the mouse adapted BSE strain 301C . Mice inoculated with 301C were generally less active during the dark phase of the light-dark cycle than control or scrapie inoculated mice. In contrast, ME7 inoculated mice also showed a decline in activity during the dark phase, although not to the same extent as 301C inoculated mice. Statistically significant scrapie strain effects were observed in measures of the duration of several open field behaviors including, rearing, wall rearing, sniffing, grooming, and walking . Scrapie inoculated mice did show a decline in water consumption around 10 weeks post inoculation, consistent with data published by Outram . All mice exhibited similar scrapie induced neuropathological changes . Taken together, these studies indicate that scrapie strain and mouse strain may impact the outcome of behavioral assays.
More recently, a battery of behavioral tests has been successfully used to visualize the progression of prion disease across several scrapie strains [61, 78, 79, 84–86]. Based on their work over the years, the aforementioned authors have elucidated the timing of behaviors that are affected. Nesting and affective behaviors (glucose consumption and burrowing) are first to be affected. Motor, strength, and coordination deficits appear subsequently. Finally, mice show decreased activity and prototypical clinical signs of scrapie. Betmouni et al. took advantage of evidence that the ME7 scrapie strain apparently targets the hippocampus, in order to determine if behavioral testing is useful for detecting early, subtle, hippocampal deficits in scrapie inoculated mice [78, 79]. Hippocampal deficits have been associated with hyperactivity and deficits at passive avoidance tasks. The authors observed increased locomotor activity and impaired retention of a multitrial passive avoidance task in scrapie inoculated mice around 12–14 weeks post inoculation. The authors also observed motor function impairments on the inverted screen and horizontal bar tests before the onset of known clinical signs of scrapie. A subsequent study examined the behavioral correlates of scrapie progression using a similar battery of tests . Burrowing of food in the home cage was found to be inversely proportional to disease progression in scrapie inoculated mice. Consistent with other studies there was a decline in spontaneous alternation, beginning around 10 weeks post inoculation and there was a statistically significant reduction in glucose consumption in scrapie inoculated mice during weeks 15–19. A statistically significant effect of group was also observed in the horizontal bar test, which tests motor coordination . The authors did not observe any statistically significant differences between groups in the rotarod or the inverted screen test. In sum, the development of a battery of behavioral assays is a boon for science in that it facilitates the comparison of experimental findings across investigators.
As previously discussed, early studies provide evidence that both scrapie and mouse strain may impact on the outcome of behavior assays. Cunningham et al. examined the behavioral progression of scrapie in C57BL/6J mice inoculated with one of the following strains: ME7, 79A, 22L, and 22A . All mice were intrahippocampally inoculated with one of the aforementioned scrapie strains or normal brain homogenate. After recovery mice were subjected to the battery of behavioral tests described above. A similar disease progression was observed in all scrapie inoculated mice, except those that were inoculated with 22A. These mice exhibited a delayed disease progression. ME7 inoculated mice were the first to show decreased glucose consumption around 10 weeks post inoculation, followed by 79A and 22L at 12 weeks post inoculation. In these mice, although the progression of scrapie was generally similar, there were differences in end stage neuropathology. Although all scrapie inoculated mice showed microglial activation, the degree of activation appeared to be less in the 22L inoculated mice. There were strain differences in vacuolation in the hippocampus, septum, and thalamus. Although all scrapie inoculated mice showed widespread PrPSC staining, there were also strain-dependent differences in the density of scrapie with some strains showing more diffuse immunoreactivity and others show plaques or punctate immunoreactivity. Neuron loss was fairly similar in all scrapie inoculated mice. One striking finding was that there was a lack of hippocampal cell death in 22L or 22A inoculated mice, despite the fact that all scrapie inoculated mice received an intrahippocampal injection. The authors note that this is consistent with the idea that variables other than site of exposure contribute to PrPSC spread and neuropathology.
Taken together this brief review of the literature indicates that it is possible to use behavioral testing as a proxy to monitor the progression of prion disease in mouse model systems. An important caveat, however, is that investigators must carefully consider scrapie strain effects, mouse strain effects or interactions between the two. Although this is an important variable to consider, there are exceptions to this generalization. For instance, Asuni et al. noted that their previous studies used C57BL/6J mice from Harlan laboratories, a mouse strain that was subsequently shown to have a spontaneous deletion of alpha synuclein . The authors were concerned that the absence of alpha synuclein represented a potential confound with data that correlate synaptic loss with prion disease progression. A comparison of C57 mice with and without alpha synuclein revealed no impact of alpha synuclein on the progression of scrapie as assayed by behavioral testing.
4.1. Behavioral studies in transgenic mouse models
4.1.1. Behavior assays have been used to validate prion knockout mice
As mentioned earlier, our current understanding of prion disease is that it is a consequence of misfolded PrPC. However, the function of PrPC is not wholly known. To further understanding of its function, a number of groups have developed PrPC knockout (PrPKO) mice. As part of these studies, behavior assays have been used to assess the impact of PrP ablation. The first PrPKO mouse, also known as the
However, other researchers have found that
Meotti et al. used a number of thermal and chemical nociception tests, to determine whether PrPC has a role in pain detection .
5. Future directions
This review of the behavioral effects of prion disease has attempted to demonstrate the dramatic, host, agent, and disease-specific heterogeneity in natural and experimental systems. While these differences are recognized, the reasons underlying them are not known. As much as this unknown reflects uncertainties regarding the mechanisms of prion neurotoxicity, it also demonstrates the limited body of work that has systematically cataloged and characterized the clinical deficits these systems. Due to this knowledge gap, in concert with a growing understanding of the scientific importance of behavioral testing, it is important that prion researchers continue to consider clinical phenotype in future
Pan KM, Baldwin M, Nguyen J, et al. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA1993;90:10962–10966.
Bueler H, Fischer M, Lang Y, et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature1992;356:577–582.
Bueler H, Aguzzi A, Sailer A, et al. Mice devoid of PrP are resistant to scrapie. Cell1993;73:1339–1347.
Mallucci GR, Ratte S, Asante EA, et al. Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. EMBO J2002;21:202–210.
Haley NJ, Mathiason CK, Zabel MD, et al. Detection of sub-clinical CWD infection in conventional test-negative deer long after oral exposure to urine and feces from CWD+ deer. PLoS One2009;4:e7990.
Castilla J, Gutierrez-Adan A, Brun A, et al. Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein. J Neurosci2004;24:5063–5069.
Hill AF, Collinge J. Subclinical prion infection. Trends Microbiol2003;11:578–584.
Ersdal C, Ulvund MJ, Benestad SL, et al. Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie. Vet Pathol2003;40:164–174.
Thackray AM, Klein MA, Aguzzi A, et al. Chronic subclinical prion disease induced by low-dose inoculum. J Virol2002;76:2510–2517.
Race R, Meade-White K, Raines A, et al. Subclinical scrapie infection in a resistant species: persistence, replication, and adaptation of infectivity during four passages. J Infect Dis2002;186 Suppl 2:S166–170.
Fraser H, Dickinson AG. Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation. J Comp Pathol1973;83:29–40.
Langevin C, Andreoletti O, Le Dur A, et al. Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model. Neurobiol Dis2011;41:219–225.
Aguzzi A, Sigurdson C, Heikenwaelder M. Molecular mechanisms of prion pathogenesis. Annu Rev Pathol2008;3:11–40.
Collinge J, Clarke AR. A general model of prion strains and their pathogenicity. Science2007;318:930–936.
Safar J, Wille H, Itri V, et al. Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med1998;4:1157–1165.
Goldfarb LG, Petersen RB, Tabaton M, et al. Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science1992;258:806–808.
Dickinson AG, Meikle VM. Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent. Mol Gen Genet1971;112:73–79.
Lasmezas CI, Deslys JP, Robain O, et al. Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science1997;275:402–405.
Collinge J, Owen F, Poulter M, et al. Prion dementia without characteristic pathology. Lancet1990;336:7–9.
Konold T, Bone GE, Clifford D, et al. Experimental H-type and L-type bovine spongiform encephalopathy in cattle: observation of two clinical syndromes and diagnostic challenges. BMC Vet Res2012;8:22.
Konold T, Bone GE, Phelan LJ, et al. Monitoring of clinical signs in goats with transmissible spongiform encephalopathies. BMC Vet Res2010;6:13.
Konold T, Lee YH, Stack MJ, et al. Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain. BMC Vet Res2006;2:31.
Jeffrey M, Halliday WG. Numbers of neurons in vacuolated and non-vacuolated neuroanatomical nuclei in bovine spongiform encephalopathy-affected brains. J Comp Pathol1994;110:287–293.
Jeffrey M, Halliday WG, Goodsir CM. A morphometric and immunohistochemical study of the vestibular nuclear complex in bovine spongiform encephalopathy. Acta Neuropathol1992;84:651–657.
Austin AR, Meek S, Webster S, et al. Heart rate variability in BSE. Vet Rec1996;139:631.
Mackenzie A. Immunohistochemical demonstration of glial fibrillary acidic protein in scrapie. J Comp Pathol1983;93:251–259.
Jeffrey M, Gonzalez L. Classical sheep transmissible spongiform encephalopathies: pathogenesis, pathological phenotypes and clinical disease. Neuropathol Appl Neurobiol2007;33:373–394.
Manix M, Kalakoti P, Henry M, et al. Creutzfeldt-Jakob disease: updated diagnostic criteria, treatment algorithm, and the utility of brain biopsy. Neurosurg Focus2015;39:E2.
Ironside JW, Ghetti B, Head MW, et al. Prion diseases In: Love S, Louis DN,Ellison DW, eds. Greenfield's Neuropathology. 8th ed. London: Hodder Arnold, 2008;1197–1273.
Parchi P, Saverioni D. Molecular pathology, classification, and diagnosis of sporadic human prion disease variants. Folia Neuropathol2012;50:20–45.
Parchi P, Giese A, Capellari S, et al. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol1999;46:224–233.
Brown P, Brunk C, Budka H, et al. WHO Manual for surveillance of human transmissible spongiform encephalopathies including variant Cretuzfeldt-Jakob disease. Geneva: World Health Organization; 2003.
Cali I, Castellani R, Yuan J, et al. Classification of sporadic Creutzfeldt-Jakob disease revisited. Brain2006;129:2266–2277.
Baiardi S, Capellari S, Ladogana A, et al. Revisiting the heidenhain variant of Creutzfeldt-Jakob disease: evidence for prion type variability influencing clinical course and laboratory findings. J Alzheimers Dis2015;50:465–476.
Grant MP, Cohen M, Petersen RB, et al. Abnormal eye movements in Creutzfeldt-Jakob disease. Ann Neurol1993;34:192–197.
Alema G, Bignami A. Subacute degenerative presenile polioencephalopathy with akinetic stupor and decorticate rigidity with myoclonus (“myoclonic” variety of the Jakob-Creutzfeld disease). Riv Sper Freniatr Med Leg Alien Ment1959;83 Suppl 4:1485–1623.
Krasnianski A, Bartl M, Sanchez Juan PJ, et al. Fatal familial insomnia: Clinical features and early identification. Ann Neurol2008;63:658–661.
Krasnianski A, Sanchez Juan P, Ponto C, et al. A proposal of new diagnostic pathway for fatal familial insomnia. J Neurol Neurosurg Psychiatry2014;85:654–659.
Iwasaki Y, Mori K, Ito M, et al. Gerstmann-Straeussler-Scheinker disease with P102L prion protein gene mutation presenting with rapidly progressive clinical course. Clin Neuropathol2014;33:344–353.
Parry HB, Oppenheimer DR. Scrapie disease in sheep: historical, clinical, epidemiological, pathological, and practical aspects of the natural disease. London; New York: Academic Press; 1983.
Konold T, Bone G, Vidal-Diez A, et al. Pruritus is a common feature in sheep infected with the BSE agent. BMC Vet Res2008;4:16.
Imran M, Mahmood S. An overview of animal prion diseases. Virol J2011;8:493.
Healy AM, Weavers E, McElroy M, et al. The clinical neurology of scrapie in Irish sheep. J Vet Intern Med2003;17:908–916.
Benestad SL, Sarradin P, Thu B, et al. Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec2003;153:202–208.
Pattison IH, Millson GC. Scrapie produced experimentally in goats with special reference to the clinical syndrome. J Comp Pathol1961;71:101–109.
Pattison IH, Millson GC. Further observations on the experimental production of scrapie in goats and sheep. J Comp Pathol1960;70:182–193.
Saegerman C, Speybroeck N, Roels S, et al. Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy. J Clin Microbiol2004;42:172–178.
Braun U, Schicker E, Hornlimann B. Diagnostic reliability of clinical signs in cows with suspected bovine spongiform encephalopathy. Vet Rec1998;143:101–105.
Konold T, Bone G, Ryder S, et al. Clinical findings in 78 suspected cases of bovine spongiform encephalopathy in Great Britain. Vet Rec2004;155:659–666.
Konold T, Sivam SK, Ryan J, et al. Analysis of clinical signs associated with bovine spongiform encephalopathy in casualty slaughter cattle. Vet J2006;171:438–444.
Wilesmith JW, Hoinville LJ, Ryan JB, et al. Bovine spongiform encephalopathy: aspects of the clinical picture and analyses of possible changes 1986–1990. Vet Rec1992;130:197–201.
Houston EF, Gravenor MB. Clinical signs in sheep experimentally infected with scrapie and BSE. Vet Rec2003;152:333–334.
Mathiason CK, Hays SA, Powers J, et al. Infectious prions in pre-clinical deer and transmission of chronic wasting disease solely by environmental exposure. PLoS One2009;4:e5916.
Williams ES, Miller MW, Kreeger TJ, et al. Chronic wasting disease of deer and elk: a review with recommendations for management. Journal of Wildlife Management2002;66:551–563.
Angers RC, Kang HE, Napier D, et al. Prion strain mutation determined by prion protein conformational compatibility and primary structure. Science2010;328:1154–1158.
Raymond GJ, Raymond LD, Meade-White KD, et al. Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains. J Virol2007;81:4305–4314.
Tecott LH, Nestler EJ. Neurobehavioral assessment in the information age. Nat Neurosci2004;7:462–466.
Pierce RC, Kalivas PW. Locomotor behavior. Curr Protoc Neurosci2007;Chapter 8:Unit 8 1.
Wahlsten D. Mouse behavioral testing: how to use mice in behavioral neuroscience. 1st ed. London; Burlington, VT: Academic, 2011.
Luong TN, Carlisle HJ, Southwell A, et al. Assessment of motor balance and coordination in mice using the balance beam. J Vis Exp2011;49:1–3.
Deacon RM. Measuring the strength of mice. J Vis Exp2013;76:1–4.
Pompl PN, Mullan MJ, Bjugstad K, et al. Adaptation of the circular platform spatial memory task for mice: use in detecting cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer's disease. J Neurosci Methods1999;87:87–95.
Mowrer OH, Lamoreaux RR. Fear as an intervening variable in avoidance conditioning. J Comp Psychol1946;39:29–50.
Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci1992;106:274–285.
Fanselow MS. Contextual fear, gestalt memories, and the hippocampus. Behav Brain Res2000;110:73–81.
Raybuck JD, Lattal KM. Bridging the interval: theory and neurobiology of trace conditioning. Behav Processes2014;101:103–111.
File SE, Lippa AS, Beer B, et al. Animal tests of anxiety. Curr Protoc Neurosci2004;Chapter 8:Unit 8 3.
Cryan JF, Holmes A. The ascent of mouse: advances in modelling human depression and anxiety. Nat Rev Drug Discov2005;4:775–790.
Hunsaker MR. The importance of considering all attributes of memory in behavioral endophenotyping of mouse models of genetic disease. Behav Neurosci2012;126:371–380.
Davis KL. American College of Neuropsychopharmacology. Neuropsychopharmacology: the fifth generation of progress: an official publication of the American College of Neuropsychopharmacology. Philadelphia: Lippincott Williams & Wilkins, 2002.
Griebel G, Belzung C, Perrault G, et al. Differences in anxiety-related behaviours and in sensitivity to diazepam in inbred and outbred strains of mice. Psychopharmacology (Berl)2000;148:164–170.
Mathiasen LS, Mirza NR, Rodgers RJ. Strain- and model-dependent effects of chlordiazepoxide, L-838,417 and zolpidem on anxiety-like behaviours in laboratory mice. Pharmacol Biochem Behav2008;90:19–36.
Groenink L, Vinkers C, van Oorschot R, et al. Models of anxiety: stress-induced hyperthermia (SIH) in singly housed mice. Curr Protoc Pharmacol2009;Chapter 5:Unit 5 16.
Crawley JN. Behavioral phenotyping strategies for mutant mice. Neuron2008;57:809–818.
Crawley JN. What's wrong with my mouse? Behavioral phenotyping of transgenic and knockout mice. 2nd ed. Hoboken, NJ: Wiley-Interscience, 2007.
Savage RD, Field EJ. Brain damage and emotional behaviour: the effects of scrapie on the emotional responses of mice. Anim Behav1965;13:443–446.
Gorde JM, Bert J, Gambarelli D, et al. Apomorphine-induced circling behaviour in hamsters following unilateral injection of scrapie gent in the striatum. Neurosci Lett1981;22:201–204.
Betmouni S, Perry VH. The acute inflammatory response in CNS following injection of prion brain homogenate or normal brain homogenate. Neuropathol Appl Neurobiol1999;25:20–28.
Betmouni S, Clements J, Perry VH. Vacuolation in murine prion disease: an informative artifact. Curr Biol1999;9:R677–679.
Heitzman RJ, Corp CR. Behaviour in emergence and open-field tests of normal and scrapie mice. Res Vet Sci1968;9:600–601.
Outram GW. Early reduction of drinking in mice with scrapie. Lancet1971;1:397.
McFarland DJ, Baker FD, Hotchin J. Host and viral genetic determinants of the behavioral effects of scrapie encephalopathy. Physiol Behav1980;24:911–914.
Dell'Omo G, Vannoni E, Vyssotski AL, et al. Early behavioural changes in mice infected with BSE and scrapie: automated home cage monitoring reveals prion strain differences. Eur J Neurosci2002;16:735–742.
Deacon RM. Measuring motor coordination in mice. J Vis Exp2013:e2609;75:1–8.
Guenther K, Deacon RM, Perry VH, et al. Early behavioural changes in scrapie-affected mice and the influence of dapsone. Eur J Neurosci2001;14:401–409.
Cunningham C, Deacon RM, Chan K, et al. Neuropathologically distinct prion strains give rise to similar temporal profiles of behavioral deficits. Neurobiol Dis2005;18:258–269.
Deacon RM, Raley JM, Perry VH, et al. Burrowing into prion disease. Neuroreport2001;12:2053–2057.
Asuni AA, Hilton K, Siskova Z, et al. Alpha-synuclein deficiency in the C57BL/6JOlaHsd strain does not modify disease progression in the ME7-model of prion disease. Neuroscience2010;165:662–674.
Lipp HP, Stagliar-Bozicevic M, Fischer M, et al. A 2-year longitudinal study of swimming navigation in mice devoid of the prion protein: no evidence for neurological anomalies or spatial learning impairments. Behav Brain Res1998;95:47–54.
Coitinho AS, Freitas AR, Lopes MH, et al. The interaction between prion protein and laminin modulates memory consolidation. Eur J Neurosci2006;24:3255–3264.
Tobler I, Gaus SE, Deboer T, et al. Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature1996;380:639–642.
Roesler R, Walz R, Quevedo J, et al. Normal inhibitory avoidance learning and anxiety, but increased locomotor activity in mice devoid of PrP(C). Brain Res Mol Brain Res1999;71:349–353.
Nico PB, Lobao-Soares B, Landemberger MC, et al. Impaired exercise capacity, but unaltered mitochondrial respiration in skeletal or cardiac muscle of mice lacking cellular prion protein. Neurosci Lett2005;388:21–26.
Meotti FC, Carqueja CL, Gadotti Vde M, et al. Involvement of cellular prion protein in the nociceptive response in mice. Brain Res2007;1151:84–90.
Le Pichon CE, Valley MT, Polymenidou M, et al. Olfactory behavior and physiology are disrupted in prion protein knockout mice. Nat Neurosci2009;12:60–69.
Budefeld T, Majer A, Jerin A, et al. Deletion of the prion gene Prnp affects offensive aggression in mice. Behav Brain Res2014;266:216–221.
Koolhaas JM, Coppens CM, de Boer SF, et al. The resident-intruder paradigm: a standardized test for aggression, violence and social stress. J Vis Exp2013:e4367;77:1–7.
Lobao-Soares B, Walz R, Carlotti CG, Jr., et al. Cellular prion protein regulates the motor behaviour performance and anxiety-induced responses in genetically modified mice. Behav Brain Res2007;183:87–94.