Abstract

Section 1

Section 2

Section 3

Section 4

Section 5

Discussion
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Development of two new inbred rat strains, WKHA and WKHT, started in the late 1970’s from progenitors, SHR (spontaneously hypertensive rats) and normotensive WKY (Wistar-Kyoto) rats. The need for developing WKHA and WKHT arose from the findings by us and others that although the SHR had been selectively inbred for the hypertensive trait, a feature which makes it the most widely used animal model of human essential hypertension (1), SHRs have also been fortuitously endowed with a number of behavioral abnormalities as an unintended consequence of inbreeding.

Most prominent among the behavioral abnormalities is that SHRs are hyperactive in a novel environment and hyperreactive to stress, when compared with WKY controls (2-4). SHRs also habituate more poorly to novel stimuli than WKY, they are more aggressive, more exploratory, and they respond to d-amphetamine by a decrease in activity, in contrast to WKY rats who increased their activity (5). Taken together, these behavioral patterns suggested to us that the SHR exhibited some of the characteristics of human hyperactivity disorders, and should be considered potentially as a naturalistic animal model of ADHD.

A major disadvantage in considering the SHR as a model of hyperactive disorders, however, is that hypertension co-exists with their behavioral abnormalities. The hypertension is of neurogenic origin, as is also the behavior, thus any attempts to study the neurological or genetic basis of either trait will be compromised by the co-segregation of both traits in the same genome.

In the late 1970s we applied a recombinant inbreeding strategy starting from a crossbreeding of SHR with WKY. From the hybrids (F1) we bred a large population of F2’s in which the genes for hypertension and hyperactivity were maximally segregated, allowing us to examine the inheritance patterns of each trait as well as search for evidence for a possible genetic linkage between the two (6). We found that the inheritance of each of the traits was additive rather than Mendelian in distribution, indicating a polygenic basis for both hypertension and hyperactivity.

Furthermore, there was no correlation between blood pressure and activity score among individual F2 rats, indicating the two traits were not genetically linked. This provided the feasibility for proceeding further to separate the two traits genetically using successive, selective, recombinant inbreeding (brother/sister), starting from the F2 generation, to eventually produce two new homozygous strains: WKHA with the characteristics of hyperactivity and normal blood pressure, and WKHT with high blood pressure and normal activity (7, 8). Both WKHA and WKHT strains became fully homozygous (surpassing 20 successive inbreedings) in 1990 and 1992, respectively, and they are currently in the F36 generation of WKHA rats and F33 generation of WKHT rats. There was a fixation of the desired phenotypes as early as the F5 generation (7), allowing us early on to make use of the two new strains in experimental protocols designed to search for co-segregation of biological or behavioral changes with either hypertension or hyperactivity. We were able to do this by utilizing WKHT, WKHA and the parental SHR and WKY in a 4-strain comparison of genetically related inbred rats, where hypertension and hyperactivity are expressed in all possible combinations: SHR have both traits, WKY have neither, and WKHT and WKHA express each trait separately. Thus, if a given biological change were related to hyperactivity, then it should be observed in the two hyperactive strains (WKHA and SHR) and be absent in the non-hyperactive strains (WKY and WKHT). Similarly, if a biological change were related to hypertension, then it should be observed in the two hypertensive strains (WKHT and SHR) and be absent in the two normotensive strains (WKY and WKHA).

Using this strategy, we have accumulated a body of data, over more than a decade, which allows us to present here a limited profile of the characteristics of the WKHA rat, by which to judge its suitability as a model of hyperactivity and hyperreactivity to stress.

HYPERACTIVITY AND HYPERREACTIVITY TO STRESS ARE CLOSELY ASSOCIATED IN WKHA RATS

WKHA, like the SHR, consistently exhibit increased activity levels when exposed to a novel environment, whether tested in the open field (9, 10) or in an activity cage equipped with light beam/photocell detectors for following movements of the rat (7). This hyperactivity is a lifelong trait, and it is more pronounced in females than males (8). When re-exposed to the activity cage at hourly intervals for four repeated tests of spontaneous activity, WKHA rats decrease activity scores with each successive trial, unlike the SHR who habituate poorly and continue to show high activity scores despite repeated exposure to an originally novel environment (8).

When all four strains were examined in a free-exploration, open-field paradigm, devoid of the aversive features of a novel environment, (9), WKHA rats were not as exploratory as the SHR, but rather the WKHT appear to have inherited this trait from the SHR. This characteristic of less exploratory behavior in WKHA rats was also noted in their tendency to avoid entering the inner portions of the open field (10).

Chiueh and McCarty (11) made the observation that footshock stress resulted in a marked increase in plasma catecholamines in SHR compared with WKY rats. In 1988 we revisited this paradigm in R. McCarty’s laboratory (12), this time adding WKHA and WKHT strains to the comparison. The hypothesis that was most intuitively appealing to us at the time was that the sympatho-adrenomedullary hyperresponsiveness to stress in SHR was related to the hypertension. Instead, we found that WKHA rats increased plasma norepinephrine and epinephrine levels significantly above those of WKY, as did the SHR, whereas WKHT rats did not. Similar findings were made using a different paradigm, air-jet stress, a form of "mental stress". With S. Knardahl (13) we reported that air-jet stress resulted in a greater increase in blood pressure, heart rate and peripheral vascular resistance in WKHA and SHR rats than in WKY and WKHT rats; i.e. a co-segregation of cardiovascular hyperreactivity to stress with the hyperactivity trait and not the hypertension.

In hindsight, the association of hyperactivity with heightened reactivity to stress in WKHA rats should have been predictable; we had selected breeders of the WKHA strain for their high activity scores when exposed to novelty stress, the strange test cage . The same consideration can be made for why SHRs have higher cardiovascular reactivity to stress than WKYs; SHRs were selected for high systolic blood pressure under conditions for determining blood pressure non-invasively, which entails the use of restraint and heat stress (1).

THE HPA AXIS SHOWS CHANGES ASSOCIATED WITH BOTH HYPERACTIVITY AND HYPERTENSION

Together with our colleagues in Bordeaux, France, (10) we examined the hypothalamic pituitary adrenal (HPA) axis and stress (open-field exposure) in the four strains. We found that the hyperactive strains, WKHA and SHR, both exhibited a blunted response to injection of CRF, a hypothalamic releasing facter which stimulates anterior pituitary ACTH secretion and subsequently secretion of adrenal glucocorticoids. Other neuroendocrine and behavioral responses of the WKHA rat were found to resemble those seen by my colleagues in Bordeaux in another strain of hyperreactive rats, the Roman High-Avoidance strain, particularly a decreased release of anterior pituitary prolactin during open field exposure.

With colleagues at the University of Vermont we made other interesting observations of changes in the pituitary gland among the four strains. Using radioimmunoassay, immunocytochemistry and in situ hybridization we reported that the expression of pituitary proopiomelanocortin (POMC) and its peptide products was significantly altered in both hypertension and hyperactivity. In the anterior pituitary lobe POMC and its peptide products, ACTH and beta-endorphin, were all markedly decreased in content, as were also POMC mRNA and the number of corticotrope cells which secrete POMC and its peptides (14). Interestingly, the number of corticotropes was significantly increased in the WKHA strain compared with WKY rats. One may hypothesize that by having selected for low blood pressure in developing the WKHA rat, we had selected for maintaining the anterior lobe corticotropes and their expression of POMC peptides. Alternatively, the higher number of corticotropes may reflect an adaptive remodeling of the HPA axis in WKHA rats in the face of a diminished responsivity of corticotropes to a CRF challenge (10).

In line with an increase in anterior lobe corticotropes, the melanotropes of the intermediate pituitary lobe were also increased in number in WKHA and SHR rats (15). Like the corticotropes, the melanotropes secrete the prepropeptide, POMC, however its peptide products are alpha-MSH and beta-endorphin. We found that POMC and its peptide products were all markedly elevated in the hyperactive strains, WKHA and SHR, as well as expression of POMC mRNA (Braas, Hendley and May, unpublished findings). These interesting neuroendocrine changes, together with the blunted prolactin response to stress reported by Castanon et al. (10) in WKHA rats point to a possible change in hypothalamic dopamine neuronal function in WKHA rats, considering that both prolactin secretion and melanotrope POMC secretion are under hypothalamic dopaminergic control.

BRAIN CATECHOLAMINERGIC FUNCTION IN WKHA RATS

In 1981 we reported that the high-affinity uptake of norepinephrine was increased in SHR compared with WKY during early postnatal development, in all brain areas tested (16). At the same time, dopamine uptake was decreased, but only in the frontal cortical region of the brain. Assuming that high-affinity uptake is a measure of neuronal activity, we concluded that in the prehypertensive phase of development there was a hypernoradrenergic and hypodopaminergic innervation of the frontal cortex of SHR. We revisited the issue of catecholamine uptake when the four strains were available, although the ages of the rats ranged from young to old adults rather than the early postnatal weeks (17). We found that increased norepinephrine uptake was significant for the hypertensive trait in several brain regions, confirming the earlier prediction from SHR and WKY. However, the most interesting changes were seen in the frontal cortex in WKHA female rats. They had the lowest uptake rates of norepinephrine and the highest uptake rates of dopamine among all four strains. This imbalance of catecholaminergic input to the frontal cortex in WKHA rats suggests a hypoinnervation by locus coeruleus noradrenergic neurons, and a hyperinnervation by mesolimbic/mesocortical dopaminergic neurons in this major brain area for regulating motivated behaviors.

OTHER BEHAVIORAL CHARACTERISTICS OF WKHA RATS

1). Sugar and Its Effects on Activity and on Distractibility/Attention

We searched for a possible exacerbation of hyperactivity when sugar is ingested by examining the behavioral and metabolic changes following ad lib sucrose-supplemented chow feeding (18). We found that the hyperactive strains had the same activity scores regardless of whether sugar was eaten or not, or whether sugar had been eaten acutely or over a two week period. We also used a distractibility/attention test to examine the effects of sugar in WKHA vs WKY rats. In this test the rats were given the open-field test on day 1, then a repeated test was given on day 2 in which the features of the open field had been modified to provide a distracting stimulus. The greater the distractibility, the less the tendency to decrease activity scores on day 2. Two interesting findings were made: a) among chow-fed controls WKHA male rats were more distractible than either WKY rats or female WKHA rats; and b) after chronic sucrose ingestion WKHA females as well as males were more significantly distracted by the stimulus change in the field.

2) Aggression

We examined one particular form of interstrain aggression in the four strains by observing dominance and attack behaviors when individuals were paired with another rat of a different strain but the same sex, in a neutral arena. We scored the number of species-specific aggressive acts in 15 min, using a checklist of aggressive acts ranging from mildly aggressive allogrooming to biting attacks (19). The results indicated that WKHT were the most aggressive strain, and that allogrooming was by far the most common expression of dominance behaviors. Interestingly, although total aggression score was low in WKHA rats, the females, when they did attack, used the most severe forms of aggression rather than the milder allogrooming.

In another aggression study with the late Jim Henry and his colleagues (20) WKHA males were housed with Long-Evans and Sprague Dawley rats for over four months under conditions of severe psychosocial stress. The WKHAs were observed to be the least aggressive among the strains, and it was interesting to note that all strains experienced an increase in blood pressure after months of crowding and unstable housing conditions, with the exception of the WKHA rats, whose pressures remained stable.

SUMMARY

In this brief review, we have presented a limited behavioral, neuroendocrine and neurochemical profile of the WKHA strain, which allows us to propose that it is a valuable naturalistic model of the kind of hyperactivity that is associated with hyperreactivity to stress. Recently, molecular genetic studies by our colleagues in Montreal (21) affirmed that WKHA and WKHT strains are truly inbred, and our colleagues in Bordeaux (22) identified a quantitative trait locus on chromosome 8 that promises to reveal a major gene or set of genes associated with hyperactivity and hyperreactivity in WKHA rats (see this symposium).

REFERENCES

1. Yamori, Y (1984) Development of the spontaneously hypertensive rat (SHR) and of various spontaneous rat models, and their implications. In: DeJong, W (Ed) Handbook of Hypertension, Elsevier, New York, 4: 224-239.
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8. Hendley, ED and Ohlsson, WG (1991) Two new inbred rat strains derived from SHR: WKHA, hyperactive, and WKHT, hypertensive, rats. Amer. J. Physiol., 261: H583-H589.
9. Sagvolden, T, Hendley, ED and Knardahl, S (1992) Behavior of hypertensive and hyperactive rat strains: hyperactivity is not unitarily determined. Physiol. and Behav., 52: 49-57.
10. Castanon, N, Hendley, ED, Fan, X-M and Mormede, P (1993) Psychoneuroendocrine profile associated with hypertension or hyperactivity in spontaneously hypertensive rats. Amer. J. Physiol., 265: R1304-R1310.
11. Chiueh, CC and McCarty, R (1981) Sympatho-adrenal hyperreactivity to footshock stress but not to cold exposure in spontaneously hypertensive rats. Physiol. and Behav., 26: 85-89.
12. Hendley, ED, Cierpial, MA and McCarty, R (1988) Sympathetic-adrenal medullary response to stress in hyperactive and hypertensive rats. Physiol. and Behav., 44: 47-51.
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14. Braas, KM, Hendley, ED and May, V (1994) Anterior pituitary proopiomelanocortin expression is decreased in hypertensive rat strains. Endocrinology, 134: 196-205.
15. Braas, KM, Hendley, ED and May, V (1993) Regulation of pituitary gland pro-opiomelanocortin in hypertensive and hyperactive rats. Soc. for Neurosci. Absts., 19: 1185.
16. Myers, MM, Whittemore, SR and Hendley, ED (1981) Changes in catecholamine neuronal uptake and receptor binding in the brains of spontaneously hypertensive rats (SHR). Brain Research, 220: 325-338.
17. Hendley, ED and Fan, X-M (1992) Regional differences in brain norepinephrine and dopamine uptake kinetics in inbred rat strains with hypertension and/or hyperactivity. Brain Research, 586: 44-52.
18. Hendley, ED, Conti, LH, Wessel, DJ, Horton, ES and Musty, RE (1987) Behavioral and metabolic effects of sucrose-supplemented feeding in hyperactive rats. Amer. J. Physiol., 253: R434-R443.
19. Hendley, ED, Ohlsson, WG and Musty, RE (1992) Interstrain aggression in hypertensive and/or hyperactive rats: SHR, WKY, WKHA, WKHT. Physiol. and Behav., 51: 1041-1046.
20. Henry, JP, Liu, Y-Y, Nadra, WE, Qian, C-G, Mormede P, Lemaire, V, Ely, D and Hendley, ED (1993) Psychosocial stress can induce chronic hypertension in normotensive strains of rats. Hypertension, 21: 714-723.
21. Deschepper, CF, Prescott, G, Hendley, ED and Reudelhuber, TL (1997) Genetic characterization of novel strains of rats derived from crosses between Wistar-Kyoto and spontaneously hypertensive rats, and comparisons with their parental strains. Lab. Animal Science, 47: 638-646.
22. Moisan, M-P, Courvoisier, H, Bihoreau, M-T, Gauguier, D, Hendley, ED, Lathrop, M, James, MR and Mormede, P (1996) A major quantitative trait locus influences hyperactivity in the WKHA rat. Nature Genetics, 14: 471-473.

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