- Email: [email protected]
Pharmaceutical and other commercial uses of the dog model
Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 355 – 360 www.elsevier.com/locate/pnpbp
Pharmaceutical and other commercial uses of the dog model Candace J. Ikeda-Douglasa,b, Christina de Riveraa, Norton W. Milgrama,b,T a
b
Department of Pharmacology, 1 King’s College Circle, University of Toronto, Toronto, Canada M5S 1A8 Division of Life Sciences, University of Toronto, 1256 Military Trail, Scarborough, Ontario, Canada M1C 1A4 Accepted 13 December 2004 Available online 24 February 2005
1. Introduction This special journal issue is based on the proceedings of the 10th annual meeting on canine cognition and neuropathology held in Toronto, Ontario, Canada on June 14th and 15th, 2004. The first such meeting, in Toronto, Ontario in 1995, was aimed at providing a forum for researchers from three Labs, Dr. C.W. Cotman of the University of California at Irvine, N.W., Milgram of University of Toronto, and B. Muggenburg of Lovelace Respiratory and Research Institute in Albuquerque, New Mexico, sharing a common interest in the development of a dog model of cognitive aging. We have subsequently met yearly, with progressively increasing attendance and more widespread participation, reflecting an increased scientific awareness and acceptance of the dog model. The articles covered in this issue were based on both oral presentations and posters, given at the meeting. The underlying theme of this year’s meeting was practical applications, which provides an indication of growing maturity of the canine model. The topic issue consists of three sections. The first focuses on recent developments in advancing the canine model. The second deals with other models, including rodent and primate models, and also includes recent comparative neuropsychological data obtained from human subjects. The final section is concerned with practical applications of the canine model, including the assessment of efficacy of interventions.
T Corresponding author. Tel.: +1 416 287 7402; fax: +1 416 287 7642. E-mail address: [email protected] (N.W. Milgram). 0278-5846/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.pnpbp.2004.12.001
2. Recent developments in advancing the canine model of cognitive aging 2.1. Age-related cognitive changes in the dog We have continued to explore the effects of age on visuospatial function. Previous studies have shown that visuospatial memory (Chan et al., 2002) and landmark discrimination learning (Milgram et al., 1999), which involves allocentric spatial memory, show age-dependent cognitive decline. Christie et al. (present issue) have developed a protocol for studying egocentric spatial learning in the dog, in which an animal is required to respond to either the leftmost or rightmost of two otherwise identical objects. This proved to be a very easy task for dogs to learn, and showed no age-sensitivity. Thus, visuospatial function in dogs, like in humans, is not a unitary function and different forms of spatial knowledge appear to be affected differently by aging. Apparently both dogs and humans retain the ability in old age to distinguish where objects are with respect to their own body positions (egocentric spatial knowledge), while the ability to distinguish location with respect to external landmarks (allocentric spatial knowledge) declines with age in both species. Nippak and Milgram (present issue) have developed a new methodology for assessment of canine cognition that is based on analysis of response times (latencies) during performance of cognitive tests. In human studies, response latency is assumed to represent central processing time: the longer the latency, the slower the rate of processing. Thus, elderly humans typically respond more slowly than young humans, particularly on difficult tasks. However, this is not true in dogs tested on a spatial memory task (Nippak et al., 2003). For example, aged dogs respond
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more rapidly than young dogs. Nippak is currently extending the analysis of response latencies to other tasks. First, response latencies of individual dogs are highly correlated when animals are tested on different tasks; some animals respond slowly on all tasks, others respond rapidly. Second, latencies vary inversely with task difficulty: the harder the task, the slower the response rate. These results generally suggest that speed of responding depends on two factors: an individual factor relating to strategy and a more general factor relating to difficulty. 2.2. Sensori-motor correlates of aging in the dog In humans, cognitive impairment also parallels declines in sensory processing, which is an important potential confound. This is in part due to changes in sensory organs. For example, cataracts commonly develop during aging, and will produce general visual impairment. de Rivera et al. have developed a protocol for assessment of visual processing in dogs, which is based on the ability to distinguish contrasts. As with people, the ability to discriminate between two-dimensional shapes deteriorates with reduced contrasts, and this problem is magnified in aged dogs (present issue). 2.3. Neurobiology of age-dependent cognitive decline Recent work has further advanced our knowledge of the neurobiological basis of age-dependent cognitive decline. Head gave an oral presentation at the conference, reporting that beta amyloid protein deposition in some cortical regions of aged dogs is reduced by long-term maintenance on a food enriched with antioxidants and mitochondrial cofactors. This is the first evidence that we know of to show that a nutritional intervention can directly affect rate of development of brain neuropathology. This work may help explain why feeding dogs an antioxidant enriched food can reduce the rate of age-dependent cognitive decline (Milgram et al., 2002a,b, 2005; Milgram, 2003). Imaging technologies provide another means of looking at neuroanatomical changes associated with aging. Su et al. (present issue) reviewed data from structural imaging (MRIs) obtained from a large population of aged beagle dogs over a 4-year period. The most prominent change is in ventricular volume, which shows progressive increases over time. Total cerebral volume, by contrast, did not change significantly, despite the fact that cross-sectional studies do show age-dependent differences in volume of particular brain structures. Structure-specific changes are also notable concomitants of aging in the dog. The frontal lobes also show marked age-dependent changes, decreasing in size with increased age. Other structures, including the hippocampus, show much less dramatic changes (Tapp et al., 2004). These structural changes correlate with age-dependent deficits in executive functions and beta amyloid
pathology (current issue). Volumetric changes are not the only indications of aging. Analysis of MRIs also revealed that age is frequently associated with apparently spontaneously developing brain lesions. It will be important to establish the causes of the lesions and whether the occurrence of these lesions can be linked to cognitive changes. Tapp et al. also measured cerebrovascular volume (CBV), using a procedure called dynamic susceptibility contrast magnetic resonance imaging. CBV varies as a function of age, being larger in young than old dogs, and brain compartment, being larger in gray matter than white matter. CBV is also mediated, at least in part, by acetylcholine (present issue). The neuroanatomical changes associated with aging originate from changes that occur within individual cells. The mitochondria are particularly important. Sullivan and Brown (present issue) discussed the role of mitochondria in oxidative stress and aging. Metabolic dysfunction is a prominent functional change, occurring in localized brain regions during aging and also in Alzheimer’s disease patients. Oxidative stress is one, possibly critical, underlying causal factor, although it is also likely that modified mitochondrial function, in turn, leads to increased oxidative stress. Sullivan (2003) also reported reduced mitochondrial function in aged dogs, and has provided evidence that antioxidant supplementation can partly counteract this reduced function. 2.4. Functional neurobiology of cognition One limitation in our knowledge of the canine model is an absence of information about relationships between brain structure and function. This issue was addressed by Christie et al., who has been studying the structural basis of visuospatial and object recognition memory (2004). Beagle dogs were trained separately on two different tasks, and subsequently were tested daily on both tasks (one in the morning, the other in the afternoon). With this protocol, Christie et al. (2004) showed that lesions to the rhinal cortex impair object recognition memory, but do not affect visuospatial memory. This work helps define the neural cognition circuitry in the canine, which appears to be similar to that of primates. Direct comparisons with primates, however, are difficult to make because of incomplete anatomical data in the dog. Dr. Agnieszka Woz´nicka, who summarized studies performed over a period of several years on the anatomy of the medial temporal lobes, gave an oral presentation specifically focusing on subdivisions of the entorhinal cortex, and is currently correcting this deficiency in knowledge. The dog entorhinal region is made up of an anterior area, which is likely involved in olfaction and a posterior region, which is likely involved in vision. This work provides essential information on the visual circuitry in the canine brain, and helps us to understand the neuro-
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biology of canine vision. The molecular structure of the canine entorhinal cortex is similar to that of the human entorhinal cortex, in that both contain dispersed cellular islands (Woz´nicka and Kosmal, 2003). 2.5. Neurochemical basis of cognitive decline Although, very little is known about neurotransmitter systems and aging in the dog, pharmacological evidence suggests that the dogs’ central cholinergic system is involved in age-dependent memory decline. Araujo et al. (present issue) provided evidence of cholinergic involvement in canine memory based on studies analyzing the cognitive-impairing effects of scopolamine, a muscarinic cholinergic blocking agent. An important aspect of this work was the demonstration that a mild dose of scopolamine (0.15 Ag/kg) selectively disrupts visuospatial memory without producing a notable behavioral response, or affecting performance on a landmark discrimination learning task. Furthermore, scopolamine produced a greater disruptive effect in old dogs than it did in young, which is consistent with the suggestion of age-dependent cholinergic dysfunction. Further work, however, is necessary to quantify the nature of the cholinergic deficit: can it be linked to cellular loss in basal forebrain structures, and to what extent does age-associated cholinergic loss correlate with the development of cognitive dysfunction?
3. Other models 3.1. The rat as a model for studying antioxidants Dr. Barbara Shukitt-Hale provided an overview oral presentation of rodent studies in which polyphenolics have been used as dietary additives. Not only do polyphenolics have beneficial effects on learning, they also provide neuroprotection against brain damage caused by radiation. Polyphenolics can serve as effective antioxidants, but their beneficial effects may also arise from modification produced in neural signaling. This series of studies highlights two benefits of rodent models: short lifespan and the ability to readily utilize invasive techniques. 3.2. Primate models During his oral presentation, Ingram covered certain aspects of primate models of aging. Dr. Ingram discussed an ongoing longitudinal investigation of the effects of restricted food intake on aging in primates. Studies of rodents and other species indicate that a program of caloric restriction can increase maximum lifespan. A long-lifespan is a major disadvantage of using primate models, and this study has been ongoing for several years. Nevertheless, the initial evidence suggests that caloric restriction increases survival, and possibly maximum lifespan. The study has also
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assessed various aspects of cognition, but has not yet found statistically significant effects of diet. Such negative results, however, are not surprising in view of the relatively small number of animals that have completed the entire study. Tinkler and Voytko examined the effects on cognition of estrogen deficiency, which occurs in postmenopausal women. The answer is complex, and depends on both task and age. Estrogen deficient young monkeys show impaired visuospatial attention; older monkeys show more extensive cognitive modification. Furthermore, administration of estrogen can help reverse these impairments. These affects appear linked to actions of estrogen on the brain’s cholinergic system. Importantly, in contrast to rodents, primates have menstrual cycles that are identical in length and hormonal patterns to women and they experience a similar menopause. Thus, this work highlights a uniquely useful aspect of the primate model, namely modeling hormonal and cognitive changes linked to the menstrual cycle and menopause (present issue). 3.3. Comparative neuropsychology The term comparative neuropsychology refers to the application of tests originally developed for assessment in animals to humans. Dr. Julene Johnson’s oral presentation covered a case history report suggesting a practical applicability of comparative neuropsychological approach in diagnosis. The subject appeared to be suffering from fronto-temporal dementia, which accounted for his strange application of language. However, standard neuropsychological testing revealed no obvious abnormalities. The subject was clearly deficient when tested on a reversal learning task. It is difficult, however, to read too much into such case history data based on a single patient, but clearly, this looks like a promising area for further work. Boutet et al. compared human cognitive performance by testing humans on conceptually identical tasks to those used to examine cognitive decline in aged dogs. Relative task difficulty differed between species—humans generally do better on visuoperceptual tasks, which involve object identification or recognition than on visuospatial tasks. Dogs, by contrast, find visuospatial tasks easier to solve. Another difference pertains to behavioral flexibility. Humans do at least as well on reversal learning tasks as they do on the original learning; in many instances human subjects switch their responses after a single non-rewarded trial. Dogs, by contrast, show slower reversal learning when contrasted with the original discrimination learning, and this difference is magnified in aged dogs. The slower reversal learning is a result in part of a perseverative tendency, in which dogs continue to respond to stimulus that is no longer associated with reward. These species differences disappear, however, when AD patients are compared with dogs. AD patients also perform poorly on reversal learning tasks. These findings suggest qualitative similarities between AD patients and aged dogs (present issue).
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Michelle Ryan’s poster presentation showed this neuropsychological test protocol is being studied in children. The results suggest age sensitivity, with children 7 years or younger showing particular difficulty with visuospatial tasks. Finally Nelson et al. has used neuropsychological test protocols to assess cognitive function in Down’s syndrome patients of varying ages in an effort to understand the time course of the onset of dementia. Down’s syndrome patients performed reasonably well on a simple object discrimination learning task. Reversal learning, by contrast, occurred more slowly, which distinguishes Down’s syndrome patients from normal humans (present issue).
4. Practical applications and interventions The last topic area was practical utility: Will the canine model prove useful in developing successful cognitive interventions in humans, and are there other possible applications? Dr. Carl Cotman of the University of California at Irvine raised three important issues pertaining to the use of the dog in development of interventions during his presentation. First, he asked whether the dog is best viewed as a model of AD, or is it more appropriate to view the dog as a model of age-associated memory impairment? The evidence, overall, suggest the latter; dogs do not model AD in one critical characteristic, namely that of the existence of neurofibrillary tangles. Second, Dr. Cotman asked what kinds of pharmacological interventions are likely to be most successful in treating age-related cognitive dysfunction. He answered that the next generation of cognitive-modifying interventions is most likely to be based on cocktails, rather than on single compounds. For example, the longitudinal antioxidant study included both antioxidants and mitochondrial cofactors. Finally, Dr. Cotman summarized a series of studies carried out in his laboratory demonstrating the beneficial effects of exercise, which is apparently linked to an increase in brain synthesis of Brain Derived Neurotrophic Factor (BDNF), a very powerful growth factor (Cotman and Engesser-Cesar, 2002). 4.1. Nutriceutical interventions With respect to development and testing of potential interventions, it is one thing to have an exciting idea and another to put it into action. This was indicated in a presentation given by Zicker from Hill’s Pet Nutrition. Zicker described how Hill’s Pet Nutrition went about developing a test food with high levels of antioxidants and mitochondrial cofactors. Zicker’s presentation highlighted the importance of formulation. Selection of ingredients is only a starting point. The next step is to combine them and administer them appropriately so that they reach their target in sufficient quantities (present issue). The meeting also produced two new reports on the efficacy of an antioxidant-enriched food. Siwak reported
that the food improved performance of aged animals on a concept learning task (present issue), and de Rivera reported that animals initially started on the food at a young age learned a shape discrimination task more rapidly than animals maintained on a control food (present issue). Filburn of Nutramax Laboratories gave an oral presentation on docosahexanoic acid (DHA), an omega three fatty acid that is present in high concentrations in fish oil. DHA is a particularly interesting compound because of evidence, largely epidemiological, that relates higher levels of intake to lower incidence of Alzheimer’s Disease. It is found at high levels in neuronal membrane phospholipids and affects their properties. Filburn also presented new evidence of health benefits in dogs treated with a nutriceutical that includes DHA. 4.2. Pharmaceutical interventions One of the underlying raison d’eˆ tre for studying cognition in aging dogs is to develop interventions for companion animals demonstrating cognitive dysfunction. The first subject drug to be studied, l-deprenyl (Anipryl), has now received regulatory approval and is marketed by Pfizer. Sharon Campbell (Pfizer Animal Health) helped bring us up to date on the status of Anipryl during her oral presentation. In a large trial with pet owners, the overwhelming majority reported improved cognitive function after treatment with Anipryl. The absence of placebo control data, however, was a limiting factor. Landsberg (present issue) presented a wide-ranging discussion on development of pharmaceuticals for use in companion animals to treat behavior disorders. One problem is that of diagnosis, which is largely dependent on observations made by the pet owner. Neuropsychological testing provides a more sensitive means of detecting canine cognitive dysfunction. Anipryl is the only drug currently available for cognitive dysfunction. Several other drugs have been studied, including adrafinil, nicergoline, propentofyline and phenserine. Adrafinil has been found to improve learning (Milgram et al., 2000), but it impairs memory (Siwak et al., 2003). Phenserine is particularly interesting in light of evidence that it improves both learning and memory in aged dogs, as discussed in the article by Araujo et al (current issue). The improvement with phenserine further suggests a decline in cholinergic function with age in the dog similar to that seen in human aging and dementia. The pet food industry has also sought to develop dietary therapies for treatment of cognitive dysfunction, one of which, Hill’s Canine B/D DietR includes a broad spectrum of antioxidants. Studzinski et al. provided an overview of studies in which aged dogs have been used to assess cognitivemodifying effects of pharmaceuticals. In some instances, drugs that proved effective in rodents were ineffective in dogs (current issue). The ampakines are one example. There were several instances in which the results from aged dogs paralleled results from human clinical trials. These findings
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suggest a general utility of the dog in preclinical drug testing—not only to provide evidence of efficacy, but also as a screen for false positives based on studies using rodent models. At this point, however, the human field is still lacking a gold standard that can be used to evaluate efficacy of pharmaceuticals in animal models of Alzheimer’s disease. 4.3. Assessment of satiety Our primary focus of the canine model has been in cognitive assessment. The cognitive assessment protocol, however, can also be used for other purposes, such as in safety studies; cognitive assessment may be able to detect pharmacological cognitive disruption, which may not be obvious in a subject’s behavioral response to the drug. In this volume, Chan et al. describes an additional application of a cognitive assessment protocol in the evaluation of satiating-producing treatments. When dogs are tested on a food-reward based task, overfeeding increases their response latency. It does not, however, affect response accuracy, suggesting dissociation between motivation and performance. This affect also varies as a function of age, with old dogs showing a considerably smaller change than young dogs. These results demonstrate that a cognitive test protocol can be used to provide satiating effects of various treatments, as distinct from nonspecific performance effects.
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The importance of the canine model was also discussed in an oral presentation by Wagster of the National Institute of Aging, who pointed out that the dog model can now fit into the bigger picture of government funded aging research. One aspect emphasized by Wagster was the multidisciplinary nature of the ongoing work, which has enabled us to correlate behavior with both brain structure and cellular indices of pathology. This meeting reviewed several studies in which canines were used to study a variety of interventions. Future research on the canine model is likely to focus in more detail on further mechanistic studies and on the assessment of further interventions. Now that we have a timetable for the development of cognitive decline in the dog, we can search more intently on understanding the underlying processes. Future studies involving interventions will likely continue to focus on combinations of compounds. Conversely, now that we have evidence of the effectiveness of an antioxidant cocktail, it will be important to determine the contributions of the individual components. In addition, it will be important to examine the effect of combining antioxidant therapy with other prescribed interventions, such as anticholinesterases. Finally, further work is clearly warranted on behavioral interventions, such as controlled activity and environmental and cognitive enrichment.
4.4. Assessment of palatability
References
Assessment of palatability in dogs is based typically on two-choice preference tests in which the food considered to be preferred, or more palatable, of two is consumed in greater quantities. It allows food preference to be determined rapidly, but does not control for differences in satiety, or other food effects that may alter the quantity of food consumed. Araujo’s oral presentation summarized the findings using a novel cognitive palatability assessment protocol (CPAP) based on discrimination learning procedures developed for the dog. The CPAP not only provided a reliable and stable instrument for assessing food preference (Araujo et al., 2004), but the results were more consistent than the standard two-pan test when comparing foods that differed only in meat source (Araujo and Milgram, 2004). This suggests that the CPAP provides a more reliable method for assessing food preference in dogs than the standard two-choice tests and that the CPAP avoids confounds of one food affecting the consumption of another, or of little difference between foods.
Araujo, J.A., Milgram, N.W., 2004. A novel cognitive palatability assessment protocol for dogs. J. Anim. Sci. 82, 2200 – 2208. Araujo, J.A., Studzinski, C.M., Larson, B.T., Milgram, N.W., 2004. Palatability of two similar foods: a comparison of the cognitive palatability assessment protocol (CPAP) and the two-pan test. Am. J. Vet. Res. 65, 1490–1496. Chan, A.D.F., Nippak, P., Murphey, H., Ikeda-Douglas, C., Muggenberg, B., Head, E., Cotman, C.W., Milgram, N.W., 2002. Visuospatial impairments in aged canines: the role of cognitive-behavioral flexibility. Behav. Neurosci. 116, 443 – 454. Christie, L., Saunders, R.C., Kowalska, D.M., Head, E., Cotman, C.W., MacKay, W.A., Milgram, N.W., 2004. Rhinal cortex lesions disrupt object recognition while sparing visuospatial memory in dogs. SFN Abstracts No. 82.4. Cotman, C.W., Engesser-Cesar, C., 2002. Exercise enhances and protects brain function. Exerc. Sport Sci. Rev. 30, 75 – 79. Milgram, N.W., 2003. Cognitive experience and its effect on age-dependent cognitive decline in beagle dogs. Neurochem. Res. 28, 1677 – 1682. Milgram, N.W., Adams, B., Callahan, H., Head, E., Mackay, B., Thirlwell, C., Cotman, C.W., 1999. Landmark discrimination learning in the dog. Learn. Mem. 6, 54 – 61. Milgram, N.W., Siwak, C.T., Gruet, P., Atkinson, P., Woehrle´, F., Callahan, H., 2000. Oral administration of adrafinil improves discrimination learning in aged beagle dogs. Pharmacol. Biochem. Behav. 66, 301 – 305. Milgram, N.W., Zicker, S.C., Head, E., Muggenburg, B.A., Murphey, H., Ikeda-Douglas, C., Cotman, C.W., 2002a. Dietary enrichment counteracts age-associated cognitive dysfunction in canines. Neurobiol. Aging 23, 805. Milgram, N.W., Head, E., Muggenburg, B., Holowachuk, D., Murphey, H., Estrada, J., Ikeda-Douglas, C.J., Zicker, S.C., Cotman, C.W., 2002b.
5. Conclusions and future directions In addition to providing a forum for bringing together a collection of data, this issue serves to further validate the aged canine as an animal model of human cognitive aging.
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Landmark discrimination learning in the dog: effects of age, an antioxidant fortified diet, and cognitive strategy. Neurosci. Biobehav. Rev. 26, 679 – 695. Milgram, N.W., Head, E., Zicker, S.C., Ikeda-Douglas, C.J., Murphey, H. Muggenburg, B., Siwak, C., Tapp, D, Cotman, C.W., 2005. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study. Neurobiol. Aging 26, 77–90. Nippak, P.M.D., Chan, A.D.F., Campbell, Z., Muggenburg, B., Head, E., Ikeda-Douglas, C., Murphy, H., Cotman, C., Milgram, N.W., 2003. Response latency in the canine: mental ability or mental strategy? Behav. Neurosci. 117 (5), 1066 – 1075. Siwak, C.T., Tapp, P.D., Woehrle´, F., Milgram, N.W., 2003. Adrafinil disrupts performance on a delayed non-matching to position task in aged beagle dogs. Pharmacol. Biochem. Behav. 76, 161 – 168.
Sullivan, P., 2003. Mitochondrial aging in the canine brain: implications for mitochondrial bioenergetics and homeostasis. Oral Presentation—9th Annual Canine Cognition, Aging and Neuropathology Conference. June 30, Laguna Beach California. Tapp, P.D., Siwak, C.T., Gao, F.Q., Chiou, J.Y., Black, S.E., Head, E., Muggenburg, B.A., Cotman, C.W., Milgram, N.W., Su, M.Y., 2004. Frontal lobe volume, function, and beta-amyloid pathology in a canine model of aging. J. Neurosci. 24 (38), 8205 – 8213. Woz´nicka, A., Kosmal, A., 2003. Cytoarchitecture of the canine perirhinal and postrhinal cortex. Acta Neurobiol. Exp. (Wars) 63 (3), 197 – 209.
Pharmaceutical and other commercial uses of the dog model Candace J. Ikeda-Douglasa,b, Christina de Riveraa, Norton W. Milgrama,b,T a
b
Department of Pharmacology, 1 King’s College Circle, University of Toronto, Toronto, Canada M5S 1A8 Division of Life Sciences, University of Toronto, 1256 Military Trail, Scarborough, Ontario, Canada M1C 1A4 Accepted 13 December 2004 Available online 24 February 2005
1. Introduction This special journal issue is based on the proceedings of the 10th annual meeting on canine cognition and neuropathology held in Toronto, Ontario, Canada on June 14th and 15th, 2004. The first such meeting, in Toronto, Ontario in 1995, was aimed at providing a forum for researchers from three Labs, Dr. C.W. Cotman of the University of California at Irvine, N.W., Milgram of University of Toronto, and B. Muggenburg of Lovelace Respiratory and Research Institute in Albuquerque, New Mexico, sharing a common interest in the development of a dog model of cognitive aging. We have subsequently met yearly, with progressively increasing attendance and more widespread participation, reflecting an increased scientific awareness and acceptance of the dog model. The articles covered in this issue were based on both oral presentations and posters, given at the meeting. The underlying theme of this year’s meeting was practical applications, which provides an indication of growing maturity of the canine model. The topic issue consists of three sections. The first focuses on recent developments in advancing the canine model. The second deals with other models, including rodent and primate models, and also includes recent comparative neuropsychological data obtained from human subjects. The final section is concerned with practical applications of the canine model, including the assessment of efficacy of interventions.
T Corresponding author. Tel.: +1 416 287 7402; fax: +1 416 287 7642. E-mail address: [email protected] (N.W. Milgram). 0278-5846/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.pnpbp.2004.12.001
2. Recent developments in advancing the canine model of cognitive aging 2.1. Age-related cognitive changes in the dog We have continued to explore the effects of age on visuospatial function. Previous studies have shown that visuospatial memory (Chan et al., 2002) and landmark discrimination learning (Milgram et al., 1999), which involves allocentric spatial memory, show age-dependent cognitive decline. Christie et al. (present issue) have developed a protocol for studying egocentric spatial learning in the dog, in which an animal is required to respond to either the leftmost or rightmost of two otherwise identical objects. This proved to be a very easy task for dogs to learn, and showed no age-sensitivity. Thus, visuospatial function in dogs, like in humans, is not a unitary function and different forms of spatial knowledge appear to be affected differently by aging. Apparently both dogs and humans retain the ability in old age to distinguish where objects are with respect to their own body positions (egocentric spatial knowledge), while the ability to distinguish location with respect to external landmarks (allocentric spatial knowledge) declines with age in both species. Nippak and Milgram (present issue) have developed a new methodology for assessment of canine cognition that is based on analysis of response times (latencies) during performance of cognitive tests. In human studies, response latency is assumed to represent central processing time: the longer the latency, the slower the rate of processing. Thus, elderly humans typically respond more slowly than young humans, particularly on difficult tasks. However, this is not true in dogs tested on a spatial memory task (Nippak et al., 2003). For example, aged dogs respond
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more rapidly than young dogs. Nippak is currently extending the analysis of response latencies to other tasks. First, response latencies of individual dogs are highly correlated when animals are tested on different tasks; some animals respond slowly on all tasks, others respond rapidly. Second, latencies vary inversely with task difficulty: the harder the task, the slower the response rate. These results generally suggest that speed of responding depends on two factors: an individual factor relating to strategy and a more general factor relating to difficulty. 2.2. Sensori-motor correlates of aging in the dog In humans, cognitive impairment also parallels declines in sensory processing, which is an important potential confound. This is in part due to changes in sensory organs. For example, cataracts commonly develop during aging, and will produce general visual impairment. de Rivera et al. have developed a protocol for assessment of visual processing in dogs, which is based on the ability to distinguish contrasts. As with people, the ability to discriminate between two-dimensional shapes deteriorates with reduced contrasts, and this problem is magnified in aged dogs (present issue). 2.3. Neurobiology of age-dependent cognitive decline Recent work has further advanced our knowledge of the neurobiological basis of age-dependent cognitive decline. Head gave an oral presentation at the conference, reporting that beta amyloid protein deposition in some cortical regions of aged dogs is reduced by long-term maintenance on a food enriched with antioxidants and mitochondrial cofactors. This is the first evidence that we know of to show that a nutritional intervention can directly affect rate of development of brain neuropathology. This work may help explain why feeding dogs an antioxidant enriched food can reduce the rate of age-dependent cognitive decline (Milgram et al., 2002a,b, 2005; Milgram, 2003). Imaging technologies provide another means of looking at neuroanatomical changes associated with aging. Su et al. (present issue) reviewed data from structural imaging (MRIs) obtained from a large population of aged beagle dogs over a 4-year period. The most prominent change is in ventricular volume, which shows progressive increases over time. Total cerebral volume, by contrast, did not change significantly, despite the fact that cross-sectional studies do show age-dependent differences in volume of particular brain structures. Structure-specific changes are also notable concomitants of aging in the dog. The frontal lobes also show marked age-dependent changes, decreasing in size with increased age. Other structures, including the hippocampus, show much less dramatic changes (Tapp et al., 2004). These structural changes correlate with age-dependent deficits in executive functions and beta amyloid
pathology (current issue). Volumetric changes are not the only indications of aging. Analysis of MRIs also revealed that age is frequently associated with apparently spontaneously developing brain lesions. It will be important to establish the causes of the lesions and whether the occurrence of these lesions can be linked to cognitive changes. Tapp et al. also measured cerebrovascular volume (CBV), using a procedure called dynamic susceptibility contrast magnetic resonance imaging. CBV varies as a function of age, being larger in young than old dogs, and brain compartment, being larger in gray matter than white matter. CBV is also mediated, at least in part, by acetylcholine (present issue). The neuroanatomical changes associated with aging originate from changes that occur within individual cells. The mitochondria are particularly important. Sullivan and Brown (present issue) discussed the role of mitochondria in oxidative stress and aging. Metabolic dysfunction is a prominent functional change, occurring in localized brain regions during aging and also in Alzheimer’s disease patients. Oxidative stress is one, possibly critical, underlying causal factor, although it is also likely that modified mitochondrial function, in turn, leads to increased oxidative stress. Sullivan (2003) also reported reduced mitochondrial function in aged dogs, and has provided evidence that antioxidant supplementation can partly counteract this reduced function. 2.4. Functional neurobiology of cognition One limitation in our knowledge of the canine model is an absence of information about relationships between brain structure and function. This issue was addressed by Christie et al., who has been studying the structural basis of visuospatial and object recognition memory (2004). Beagle dogs were trained separately on two different tasks, and subsequently were tested daily on both tasks (one in the morning, the other in the afternoon). With this protocol, Christie et al. (2004) showed that lesions to the rhinal cortex impair object recognition memory, but do not affect visuospatial memory. This work helps define the neural cognition circuitry in the canine, which appears to be similar to that of primates. Direct comparisons with primates, however, are difficult to make because of incomplete anatomical data in the dog. Dr. Agnieszka Woz´nicka, who summarized studies performed over a period of several years on the anatomy of the medial temporal lobes, gave an oral presentation specifically focusing on subdivisions of the entorhinal cortex, and is currently correcting this deficiency in knowledge. The dog entorhinal region is made up of an anterior area, which is likely involved in olfaction and a posterior region, which is likely involved in vision. This work provides essential information on the visual circuitry in the canine brain, and helps us to understand the neuro-
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biology of canine vision. The molecular structure of the canine entorhinal cortex is similar to that of the human entorhinal cortex, in that both contain dispersed cellular islands (Woz´nicka and Kosmal, 2003). 2.5. Neurochemical basis of cognitive decline Although, very little is known about neurotransmitter systems and aging in the dog, pharmacological evidence suggests that the dogs’ central cholinergic system is involved in age-dependent memory decline. Araujo et al. (present issue) provided evidence of cholinergic involvement in canine memory based on studies analyzing the cognitive-impairing effects of scopolamine, a muscarinic cholinergic blocking agent. An important aspect of this work was the demonstration that a mild dose of scopolamine (0.15 Ag/kg) selectively disrupts visuospatial memory without producing a notable behavioral response, or affecting performance on a landmark discrimination learning task. Furthermore, scopolamine produced a greater disruptive effect in old dogs than it did in young, which is consistent with the suggestion of age-dependent cholinergic dysfunction. Further work, however, is necessary to quantify the nature of the cholinergic deficit: can it be linked to cellular loss in basal forebrain structures, and to what extent does age-associated cholinergic loss correlate with the development of cognitive dysfunction?
3. Other models 3.1. The rat as a model for studying antioxidants Dr. Barbara Shukitt-Hale provided an overview oral presentation of rodent studies in which polyphenolics have been used as dietary additives. Not only do polyphenolics have beneficial effects on learning, they also provide neuroprotection against brain damage caused by radiation. Polyphenolics can serve as effective antioxidants, but their beneficial effects may also arise from modification produced in neural signaling. This series of studies highlights two benefits of rodent models: short lifespan and the ability to readily utilize invasive techniques. 3.2. Primate models During his oral presentation, Ingram covered certain aspects of primate models of aging. Dr. Ingram discussed an ongoing longitudinal investigation of the effects of restricted food intake on aging in primates. Studies of rodents and other species indicate that a program of caloric restriction can increase maximum lifespan. A long-lifespan is a major disadvantage of using primate models, and this study has been ongoing for several years. Nevertheless, the initial evidence suggests that caloric restriction increases survival, and possibly maximum lifespan. The study has also
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assessed various aspects of cognition, but has not yet found statistically significant effects of diet. Such negative results, however, are not surprising in view of the relatively small number of animals that have completed the entire study. Tinkler and Voytko examined the effects on cognition of estrogen deficiency, which occurs in postmenopausal women. The answer is complex, and depends on both task and age. Estrogen deficient young monkeys show impaired visuospatial attention; older monkeys show more extensive cognitive modification. Furthermore, administration of estrogen can help reverse these impairments. These affects appear linked to actions of estrogen on the brain’s cholinergic system. Importantly, in contrast to rodents, primates have menstrual cycles that are identical in length and hormonal patterns to women and they experience a similar menopause. Thus, this work highlights a uniquely useful aspect of the primate model, namely modeling hormonal and cognitive changes linked to the menstrual cycle and menopause (present issue). 3.3. Comparative neuropsychology The term comparative neuropsychology refers to the application of tests originally developed for assessment in animals to humans. Dr. Julene Johnson’s oral presentation covered a case history report suggesting a practical applicability of comparative neuropsychological approach in diagnosis. The subject appeared to be suffering from fronto-temporal dementia, which accounted for his strange application of language. However, standard neuropsychological testing revealed no obvious abnormalities. The subject was clearly deficient when tested on a reversal learning task. It is difficult, however, to read too much into such case history data based on a single patient, but clearly, this looks like a promising area for further work. Boutet et al. compared human cognitive performance by testing humans on conceptually identical tasks to those used to examine cognitive decline in aged dogs. Relative task difficulty differed between species—humans generally do better on visuoperceptual tasks, which involve object identification or recognition than on visuospatial tasks. Dogs, by contrast, find visuospatial tasks easier to solve. Another difference pertains to behavioral flexibility. Humans do at least as well on reversal learning tasks as they do on the original learning; in many instances human subjects switch their responses after a single non-rewarded trial. Dogs, by contrast, show slower reversal learning when contrasted with the original discrimination learning, and this difference is magnified in aged dogs. The slower reversal learning is a result in part of a perseverative tendency, in which dogs continue to respond to stimulus that is no longer associated with reward. These species differences disappear, however, when AD patients are compared with dogs. AD patients also perform poorly on reversal learning tasks. These findings suggest qualitative similarities between AD patients and aged dogs (present issue).
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Michelle Ryan’s poster presentation showed this neuropsychological test protocol is being studied in children. The results suggest age sensitivity, with children 7 years or younger showing particular difficulty with visuospatial tasks. Finally Nelson et al. has used neuropsychological test protocols to assess cognitive function in Down’s syndrome patients of varying ages in an effort to understand the time course of the onset of dementia. Down’s syndrome patients performed reasonably well on a simple object discrimination learning task. Reversal learning, by contrast, occurred more slowly, which distinguishes Down’s syndrome patients from normal humans (present issue).
4. Practical applications and interventions The last topic area was practical utility: Will the canine model prove useful in developing successful cognitive interventions in humans, and are there other possible applications? Dr. Carl Cotman of the University of California at Irvine raised three important issues pertaining to the use of the dog in development of interventions during his presentation. First, he asked whether the dog is best viewed as a model of AD, or is it more appropriate to view the dog as a model of age-associated memory impairment? The evidence, overall, suggest the latter; dogs do not model AD in one critical characteristic, namely that of the existence of neurofibrillary tangles. Second, Dr. Cotman asked what kinds of pharmacological interventions are likely to be most successful in treating age-related cognitive dysfunction. He answered that the next generation of cognitive-modifying interventions is most likely to be based on cocktails, rather than on single compounds. For example, the longitudinal antioxidant study included both antioxidants and mitochondrial cofactors. Finally, Dr. Cotman summarized a series of studies carried out in his laboratory demonstrating the beneficial effects of exercise, which is apparently linked to an increase in brain synthesis of Brain Derived Neurotrophic Factor (BDNF), a very powerful growth factor (Cotman and Engesser-Cesar, 2002). 4.1. Nutriceutical interventions With respect to development and testing of potential interventions, it is one thing to have an exciting idea and another to put it into action. This was indicated in a presentation given by Zicker from Hill’s Pet Nutrition. Zicker described how Hill’s Pet Nutrition went about developing a test food with high levels of antioxidants and mitochondrial cofactors. Zicker’s presentation highlighted the importance of formulation. Selection of ingredients is only a starting point. The next step is to combine them and administer them appropriately so that they reach their target in sufficient quantities (present issue). The meeting also produced two new reports on the efficacy of an antioxidant-enriched food. Siwak reported
that the food improved performance of aged animals on a concept learning task (present issue), and de Rivera reported that animals initially started on the food at a young age learned a shape discrimination task more rapidly than animals maintained on a control food (present issue). Filburn of Nutramax Laboratories gave an oral presentation on docosahexanoic acid (DHA), an omega three fatty acid that is present in high concentrations in fish oil. DHA is a particularly interesting compound because of evidence, largely epidemiological, that relates higher levels of intake to lower incidence of Alzheimer’s Disease. It is found at high levels in neuronal membrane phospholipids and affects their properties. Filburn also presented new evidence of health benefits in dogs treated with a nutriceutical that includes DHA. 4.2. Pharmaceutical interventions One of the underlying raison d’eˆ tre for studying cognition in aging dogs is to develop interventions for companion animals demonstrating cognitive dysfunction. The first subject drug to be studied, l-deprenyl (Anipryl), has now received regulatory approval and is marketed by Pfizer. Sharon Campbell (Pfizer Animal Health) helped bring us up to date on the status of Anipryl during her oral presentation. In a large trial with pet owners, the overwhelming majority reported improved cognitive function after treatment with Anipryl. The absence of placebo control data, however, was a limiting factor. Landsberg (present issue) presented a wide-ranging discussion on development of pharmaceuticals for use in companion animals to treat behavior disorders. One problem is that of diagnosis, which is largely dependent on observations made by the pet owner. Neuropsychological testing provides a more sensitive means of detecting canine cognitive dysfunction. Anipryl is the only drug currently available for cognitive dysfunction. Several other drugs have been studied, including adrafinil, nicergoline, propentofyline and phenserine. Adrafinil has been found to improve learning (Milgram et al., 2000), but it impairs memory (Siwak et al., 2003). Phenserine is particularly interesting in light of evidence that it improves both learning and memory in aged dogs, as discussed in the article by Araujo et al (current issue). The improvement with phenserine further suggests a decline in cholinergic function with age in the dog similar to that seen in human aging and dementia. The pet food industry has also sought to develop dietary therapies for treatment of cognitive dysfunction, one of which, Hill’s Canine B/D DietR includes a broad spectrum of antioxidants. Studzinski et al. provided an overview of studies in which aged dogs have been used to assess cognitivemodifying effects of pharmaceuticals. In some instances, drugs that proved effective in rodents were ineffective in dogs (current issue). The ampakines are one example. There were several instances in which the results from aged dogs paralleled results from human clinical trials. These findings
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suggest a general utility of the dog in preclinical drug testing—not only to provide evidence of efficacy, but also as a screen for false positives based on studies using rodent models. At this point, however, the human field is still lacking a gold standard that can be used to evaluate efficacy of pharmaceuticals in animal models of Alzheimer’s disease. 4.3. Assessment of satiety Our primary focus of the canine model has been in cognitive assessment. The cognitive assessment protocol, however, can also be used for other purposes, such as in safety studies; cognitive assessment may be able to detect pharmacological cognitive disruption, which may not be obvious in a subject’s behavioral response to the drug. In this volume, Chan et al. describes an additional application of a cognitive assessment protocol in the evaluation of satiating-producing treatments. When dogs are tested on a food-reward based task, overfeeding increases their response latency. It does not, however, affect response accuracy, suggesting dissociation between motivation and performance. This affect also varies as a function of age, with old dogs showing a considerably smaller change than young dogs. These results demonstrate that a cognitive test protocol can be used to provide satiating effects of various treatments, as distinct from nonspecific performance effects.
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The importance of the canine model was also discussed in an oral presentation by Wagster of the National Institute of Aging, who pointed out that the dog model can now fit into the bigger picture of government funded aging research. One aspect emphasized by Wagster was the multidisciplinary nature of the ongoing work, which has enabled us to correlate behavior with both brain structure and cellular indices of pathology. This meeting reviewed several studies in which canines were used to study a variety of interventions. Future research on the canine model is likely to focus in more detail on further mechanistic studies and on the assessment of further interventions. Now that we have a timetable for the development of cognitive decline in the dog, we can search more intently on understanding the underlying processes. Future studies involving interventions will likely continue to focus on combinations of compounds. Conversely, now that we have evidence of the effectiveness of an antioxidant cocktail, it will be important to determine the contributions of the individual components. In addition, it will be important to examine the effect of combining antioxidant therapy with other prescribed interventions, such as anticholinesterases. Finally, further work is clearly warranted on behavioral interventions, such as controlled activity and environmental and cognitive enrichment.
4.4. Assessment of palatability
References
Assessment of palatability in dogs is based typically on two-choice preference tests in which the food considered to be preferred, or more palatable, of two is consumed in greater quantities. It allows food preference to be determined rapidly, but does not control for differences in satiety, or other food effects that may alter the quantity of food consumed. Araujo’s oral presentation summarized the findings using a novel cognitive palatability assessment protocol (CPAP) based on discrimination learning procedures developed for the dog. The CPAP not only provided a reliable and stable instrument for assessing food preference (Araujo et al., 2004), but the results were more consistent than the standard two-pan test when comparing foods that differed only in meat source (Araujo and Milgram, 2004). This suggests that the CPAP provides a more reliable method for assessing food preference in dogs than the standard two-choice tests and that the CPAP avoids confounds of one food affecting the consumption of another, or of little difference between foods.
Araujo, J.A., Milgram, N.W., 2004. A novel cognitive palatability assessment protocol for dogs. J. Anim. Sci. 82, 2200 – 2208. Araujo, J.A., Studzinski, C.M., Larson, B.T., Milgram, N.W., 2004. Palatability of two similar foods: a comparison of the cognitive palatability assessment protocol (CPAP) and the two-pan test. Am. J. Vet. Res. 65, 1490–1496. Chan, A.D.F., Nippak, P., Murphey, H., Ikeda-Douglas, C., Muggenberg, B., Head, E., Cotman, C.W., Milgram, N.W., 2002. Visuospatial impairments in aged canines: the role of cognitive-behavioral flexibility. Behav. Neurosci. 116, 443 – 454. Christie, L., Saunders, R.C., Kowalska, D.M., Head, E., Cotman, C.W., MacKay, W.A., Milgram, N.W., 2004. Rhinal cortex lesions disrupt object recognition while sparing visuospatial memory in dogs. SFN Abstracts No. 82.4. Cotman, C.W., Engesser-Cesar, C., 2002. Exercise enhances and protects brain function. Exerc. Sport Sci. Rev. 30, 75 – 79. Milgram, N.W., 2003. Cognitive experience and its effect on age-dependent cognitive decline in beagle dogs. Neurochem. Res. 28, 1677 – 1682. Milgram, N.W., Adams, B., Callahan, H., Head, E., Mackay, B., Thirlwell, C., Cotman, C.W., 1999. Landmark discrimination learning in the dog. Learn. Mem. 6, 54 – 61. Milgram, N.W., Siwak, C.T., Gruet, P., Atkinson, P., Woehrle´, F., Callahan, H., 2000. Oral administration of adrafinil improves discrimination learning in aged beagle dogs. Pharmacol. Biochem. Behav. 66, 301 – 305. Milgram, N.W., Zicker, S.C., Head, E., Muggenburg, B.A., Murphey, H., Ikeda-Douglas, C., Cotman, C.W., 2002a. Dietary enrichment counteracts age-associated cognitive dysfunction in canines. Neurobiol. Aging 23, 805. Milgram, N.W., Head, E., Muggenburg, B., Holowachuk, D., Murphey, H., Estrada, J., Ikeda-Douglas, C.J., Zicker, S.C., Cotman, C.W., 2002b.
5. Conclusions and future directions In addition to providing a forum for bringing together a collection of data, this issue serves to further validate the aged canine as an animal model of human cognitive aging.
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Landmark discrimination learning in the dog: effects of age, an antioxidant fortified diet, and cognitive strategy. Neurosci. Biobehav. Rev. 26, 679 – 695. Milgram, N.W., Head, E., Zicker, S.C., Ikeda-Douglas, C.J., Murphey, H. Muggenburg, B., Siwak, C., Tapp, D, Cotman, C.W., 2005. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study. Neurobiol. Aging 26, 77–90. Nippak, P.M.D., Chan, A.D.F., Campbell, Z., Muggenburg, B., Head, E., Ikeda-Douglas, C., Murphy, H., Cotman, C., Milgram, N.W., 2003. Response latency in the canine: mental ability or mental strategy? Behav. Neurosci. 117 (5), 1066 – 1075. Siwak, C.T., Tapp, P.D., Woehrle´, F., Milgram, N.W., 2003. Adrafinil disrupts performance on a delayed non-matching to position task in aged beagle dogs. Pharmacol. Biochem. Behav. 76, 161 – 168.
Sullivan, P., 2003. Mitochondrial aging in the canine brain: implications for mitochondrial bioenergetics and homeostasis. Oral Presentation—9th Annual Canine Cognition, Aging and Neuropathology Conference. June 30, Laguna Beach California. Tapp, P.D., Siwak, C.T., Gao, F.Q., Chiou, J.Y., Black, S.E., Head, E., Muggenburg, B.A., Cotman, C.W., Milgram, N.W., Su, M.Y., 2004. Frontal lobe volume, function, and beta-amyloid pathology in a canine model of aging. J. Neurosci. 24 (38), 8205 – 8213. Woz´nicka, A., Kosmal, A., 2003. Cytoarchitecture of the canine perirhinal and postrhinal cortex. Acta Neurobiol. Exp. (Wars) 63 (3), 197 – 209.