Molecular correlates of emotional learning using genetically selected rat lines.

The genetic contributions to active avoidance learning in rodents have been well established, yet the molecular basis for genetically selected line differences remains poorly understood. To identify candidate genes influencing this active avoidance paradigm, we utilized the bidirectionally selected Syracuse high- and low-avoidance (SHA and SLA) rat lines that markedly differ in their two-way active avoidance behavior. Rats were phenotyped, rested to allow recovery from testing stress and then hippocampi were dissected for gene expression profiling (Affymetrix U34A chips; approximately 7000 known genes), comparing SLA to SHA. Next, a subset of differentially expressed genes was confirmed by real-time PCR (RT-PCR) in hippocampi. Additional studies at the protein level were performed for some genes. Using triplicate arrays on pooled hippocampal samples, differentially expressed genes were identified by microarray suite 5.0 and robust multi-array average analyses. By RT-PCR analysis in hippocampi, eight genes were nominated as potential candidate genes consistent with the differential expression from the microarray data. Four genes, Veli1 (mlin-7B), SLC3a1, Ptpro and Ykt6p, showed higher expression in SHA hippocampi than SLA. Four genes, SLC6A4, Aldh1a4, Id3a and Cd74, showed higher expression in SLA hippocampi than SHA. The active avoidance behavioral difference between lines probably emerges from 'many small things'. These potential candidate genes generate hypotheses for future testing in human association and rodent studies. Differences in levels of a pleiotropic gene like Ptpro and SLC6A4 suggest that small differences over a lifespan may contribute to large behavioral differences.

A selective genetic analysis of the Syracuse high- and low-avoidance (SHA/Bru and SLA/Bru) strains of rats (Rattus norvegicus).

Selective breeding of Long-Evans rats for good and poor avoidance learning in a two-way shuttle box resulted in the Syracuse strains that differ markedly in the selected phenotypes. These phenotypes have many associated traits, five of which are studied here: emotionality (open-field defecation), Pavlovian fear conditioning (CER suppression), passive avoidance training (punishment), size (weight) of the adrenal glands and adrenal concentration of corticosterone. Specifically, animals of the low-avoidance strain are more emotional, show greater fear conditioning, exhibit faster passive avoidance learning, and have larger adrenal glands in which adrenal corticosterone levels are lower than those of the high-avoidance strain. A reciprocal dihybrid cross of the two strains produced F1 hybrids, which were used to produce the segregating second filial and high and low backcross generations from which animals displaying the extreme high- and low-avoidance phenotypes were selected for study of the associated traits. Measurement of the five traits in these high and low phenotypic animals indicated that all five remain significantly associated with the avoidance phenotypes, in the expected direction, and comparably in all three segregating generations. The results indicate that the hypothesis of a major gene controlling avoidance learning must be rejected and that the few (2-3) genetic units thought to be involved may be closely linked to those that mediate these five associated characters, or express all five pleiotropically.

How do rats cope with the two-way escape problem in a homogeneous shuttle box?

The behavior of 25 rats trained in a homogeneous shuttle box to escape unsignalled grid-shock was analyzed. Three categories of escape were distinguished: (1) species-specific fly away from the charged grid, (2) long-latency crossing preceded and accompanied by other behaviors that compete with the escape response, and (3) short-latency escape which followed an anticipatory postural pose. The animals displayed species-specific fly away only during the initial trials of a session. Subsequently long-latency crossings develops, reflecting a resistance to enter the opposite compartment. A measure based on a comparison of escape latency distributions in the two halves of the 1st session discriminates between good and poor learners. Subgroups of good and poor learners differed in performance efficiency in all five training sessions. Good learners were able to overcome the resistance to enter the opposite compartment and recall the learned short-latency escape.

Differential behavioral and endocrinological effects of corticotropin-releasing hormone (CRH) in the Syracuse high- and low-avoidance rats.

The Syracuse high- and low-avoidance rats, which have been selectively bred for good (SHA/Bru) or poor (SLA/Bru) avoidance learning in a two-way shuttle box, differ in emotionality. This experiment investigated the effect of corticotropin-releasing hormone (CRH), administered centrally (0, 0.1, 0.5, and 1.0 microg), on conditioned suppression and on the hypothalamic-pituitary-adrenocortical system. Three groups of animals were used: SHA/Bru rats conditioned at 0.21 or 0.43 mA and SLA/Bru rats conditioned at 0.21 mA. The results confirm those of previous studies which found that SLA/Bru rats show greater conditioned suppression than the SHA/Bru rats at the low shock intensity and that at 0.43 mA, the SHA/Bru animals acquire a level of conditioning comparable to that of the SLA/Bru animals at 0.21 mA. The results show that the nonlinear behavioral effect of CRH is independent of strain and produces comparable effects in animals of both strains, but only when level of conditioning is equated. Adrenal and plasma concentrations of corticosterone increased in all three groups of animals as a direct linear function of dose of CRH. Both greater levels of conditioning and larger amounts of CRH increase the synthesis of corticosterone more in SHA/Bru animals than in the SLA/Bru animals. Thus, genetic variation, which differentiates the behavioral and endocrinological characteristics of these animals, shows that these effects of CRH can be independent of each other and suggests that some minimal level of conditioned fear is necessary for CRH to exert its anxiogenic effect.

The effects of extended training and acute administration of an anxiolytic on avoidance learning and intertrial responding in the Syracuse strains of rats.

Male and female animals of the SHA/Bru and SLA/Bru strains of rats were given extended two-way avoidance training in the shuttle box at the rate of 30 trials per day for 11 days. SLA/Bru animals increased their avoidance responses (AVRs) from approximately 10 to roughly 25%, whereas animals of the SHA/Bru strain remained unchanged at approximately 100% AVRs. SHA/Bru animals made a number of intertrial responses (ITRs) early in the experiment; these declined after about 3 days to the low level made by SLA/Bru animals. Chlordiazepoxide (CDP) had no effect on AVRs in animals of either strain, and had no effect on ITRs made by animals of the SHA/Bru strain, but increased ITRs, in a dose-dependent way, in animals of the SLA/Bru strain. These results are interpreted in terms of the well-established genetic difference in emotional reactivity between animals of the two strains and in terms of genetically determined differences in sensitivity to anxiolytic drugs such as CDP.

Conditioned taste and taste-potentiated odor aversions in the Syracuse high- and low-avoidance (SHA/Bru and SLA/Bru) strains of rats (Rattus norvegicus).

Syracuse high- and low-avoidance Long-Evans rats (Rattus norvegicus; SHA/Bru and SLA/Bru) were selectively bred for good and poor active-avoidance learning. However, SLA/Bru animals are superior to SHA/Bru rats in conditioned suppression and passive avoidance learning. In this experiment, saccharin taste and almond odor were the components of a compound conditioned stimulus (flavor) in an illness-induced aversive conditioning paradigm. SLA/Bru rats (n = 17) showed stronger conditioned flavor, taste, and odor aversion than did SHA/Bru animals (n = 18). Unselected Long-Evans rats (n = 18) were intermediate between the selected strains. SLA/Bru and Long-Evans rats showed taste-potentiated odor aversions in this experiment, whereas SHA/Bru animals did not. The results provide evidence that genetic factors, as exemplified by the different strains, are importantly involved in the mechanisms underlying interoceptive and exteroceptive aversive conditioning.

Opposite effects of naltrexone on ETOH intake by Syracuse high and low avoidance rats.

Rats which were selectively bred for good (Syracuse High Avoidance: SHA) and poor (Syracuse Low Avoidance: SLA) shuttle-box avoidance learning were used to assess the effects of naltrexone on ethanol ingestion. Male rats from both strains were offered a free choice of water and ethanol (10%, v/v) for two 8-day periods between which was inserted a 4-day period of forced ethanol consumption. The net ethanol consumption and ethanol preference ratio were significantly greater in control SHA rats than in control SLA rats in the first choice period, but they did not differ in the forced and the second choice periods. Chronic naltrexone administration from an implanted 30-mg pellet showed bidirectional effects, i.e., suppression of ethanol consumption in SLA animals and enhancement in SHA rats.

Genetic determinants of individual differences in avoidance learning: behavioral and endocrine characteristics.

Bidirectional genetic selection for good and poor active avoidance learning in a shuttle box has been carried out in three independent laboratories using remarkably similar discrete-trial training procedures. The resulting strains are known as the Roman High and Low Avoidance (RHA and RLA), the Syracuse High and Low Avoidance (SHA and SLA) and the Australian High and Low Avoidance (AHA and ALA) strains, respectively. An additional unidirectionally selected strain, known as the Tokai High Avoider (THA) strain was developed in Japan using a free-operant Sidman avoidance procedure in a Skinner box. This paper reviews the selection of the Syracuse strains, enumerates the various behavioral and endocrine characteristics of the strains, and compares them to the other similarly selected strains. The behavioral work suggests that genetic selection from diverse breeding stocks has resulted in common characteristics that differentiate the strains in the emotional, not learning, domain. The endocrine data, however, are somewhat at odds. The Syracuse strains differentiate one way with respect to endocrine function, and the Roman strains differentiate in the opposite way. We suggest, therefore, that the endocrine correlates are not tightly linked to the avoidance genotype. Genetic analysis of all of the selected strains for both the avoidance phenotype and the endocrine correlates will be needed to test this hypothesis.

Characteristics of the pituitary-adrenal system in the Syracuse high- and low-avoidance strains of rats (Rattus norvegicus).

After many generations of selective breeding, rats of the high-avoidance strain (SHA) average 67% avoidance responses in a two-way shuttle box, whereas those of the low-avoidance strain (SLA) average 0%. Adrenal gland weights, both absolutely and relative to body weight, are 40-50% greater in adult SLA than SHA rats of both sexes. Females of both strains have larger adrenal glands than males. Morphometry revealed that the difference in adrenal size in adults is entirely in the three cortical zones. The strain difference occurs as early as 21 days of age, whereas the sex difference appears only after puberty. A 2-min exposure to either vapor induced an elevation in adrenal concentration of corticosterone which was significantly greater in SHA than SLA animals of both sexes at some time periods following the ether stress. Despite having smaller glands, older previously stressed SHA rats have higher basal adrenal concentrations of corticosterone than do SLA animals. The reduced steroidogenesis in the large adrenals of SLA rats suggests that there may be an enzymatic defect of genetic origin in those animals.

Adrenal morphometry in unilateral and sham adrenalectomized Syracuse high and low avoidance rats.

Syracuse high (SHA) and low (SLA) Long-Evans rats, bred for differences in avoidance performance, exhibit dramatic differences in adrenal gland weight. Here we examined adrenal weight and composition (i.e., the size of the medulla, zonae fasciculata/reticularis and glomerulosa) following unilateral adrenalectomy and sham surgery in these strains. Adrenals of SLA animals, regardless of treatment, were heavier and contained larger medullas and cortices than did adrenals of SHA animals. When individual regions were expressed as a percent of total adrenal area, SHA glands (age 31-45 days), although smaller in weight, contained a larger percentage of glomerulosa than did adrenals of SLA animals. Unilateral adrenalectomy produced significant compensatory growth in SHA and SLA animals as indexed by increases in adrenal weight as soon as 7 days after surgery. The adrenal enlargement was the net result of an increase in absolute size of the fasciculata/reticularis (significant 14 days following surgery) and a decrease in the absolute size of glomerulosa (significant 7 days following surgery). These results suggest that SHA and SLA adrenal differences may be the result of genetically determined differential pituitary-adrenal activity.

CER suppression, passive-avoidance learning, and stress-induced suppression of drinking in the Syracuse high- and low-avoidance strains of rats (Rattus norvegicus).

The Syracuse strains of Long-Evans rats were selectively bred for good (SHA) or poor (SLA) avoidance learning in a two-way shuttle box, which resulted in a phenotypic difference that is correlated with behavior patterns indicative of emotional reactivity, SLA animals showing evidence of greater emotional reactivity than SHA animals. The first three experiments examined conditioned suppression of bar pressing and compared paired and unpaired conditioned- and unconditioned-stimulus presentations to evaluate the influence of conditioning versus primary aversive stimulation on baseline responding. SLA animals acquired conditioned suppression faster than SHA animals and also showed greater suppression of baseline responding than SHA animals. In Experiment 4, SLA animals learned a passive-avoidance task faster than SHA animals. In Experiment 5, SLA animals showed greater stress-induced suppression of drinking a weak quinine solution than SHA animals. These data are consistent with the hypothesis that SLA animals are more emotionally reactive than SHA animals.

Running-wheel avoidance learning in rats (Rattus norvegicus): effects of contingencies and comparisons of different strains.

In Experiment 1, we showed that active- and passive-avoidance responding in a running wheel was learned because of the avoidance contingency. In Experiment 2, strain differences among four commercially bred rats were assessed in an active-avoidance paradigm. Wistar, Donryu, and Fischer rats learned faster than Sprague-Dawleys. In Experiment 3, learning in a multiple active/passive avoidance schedule was examined, and both components of this task were learned. This multiple schedule was used to investigate strain differences in selectively bred rats in Experiments 4 and 5. Tsukuba low-emotional (TLE) rats responded more than Tsukuba high-emotional (THE) rats in both components. However, discrimination of passive components was better in THE than in TLE rats. Syracuse high-avoidance rats were superior in the active component, whereas Syracuse low-avoidance rats showed superior performance in the passive component.

Genetic differences in avoidance learning by Rattus norvegicus: escape/avoidance responding, sensitivity to electric shock, discrimination learning, and open-field behavior.

The behaviors of rats selectively bred for either good or poor shuttle box avoidance learning were studied. The results of Experiment 1 indicated that the phenotypic difference in avoidance learning is not associated with differences in speed of escape or avoidance responding. Differences between the lines in frequency of intertrial responses (ITRs), which appear during training but not during pretest, suggest that ITRs in animals of the low-avoidance (SLA) line are more suppressed by electric shock than in animals of the high-avoidance (SHA) line. This result suggests that SLA animals may be more emotionally responsive than SHA animals. Experiment 2 demonstrated that the animals of the two lines do not differ in absolute sensitivity to electric shock, and Experiment 3 showed that the poor performance of the SLA line is not due to an inability to learn. Experiment 3 also provided evidence which suggests that the poor avoidance learning by SLA animals is due to their emotional reactivity. Observations of open-field behavior in Experiment 4 are consistent with this hypothesis. The major consistent correlate of the phenotypic difference in avoidance learning is greater emotionality or emotional reactivity in SLA than in SHA animals.

Genetic differences in avoidance behavior: cardiovascular activity, pain sensitivity and stress-induced analgesia.

Four experiments are reported which examine cardiovascular activity, pain sensitivity and stress-induced analgesia in rats selectively bred for differences in shuttlebox avoidance behavior. The results of Experiments 1 and 2 indicate that the two genetic lines differ in basal pain sensitivity, as measured by the hot-plate test. This difference in pain sensitivity appears not to be mediated by endogenous opioids, because it was not altered by pretreatment with a large dose of naloxone. In Experiments 1, 3 and 4 tail-flick tests of basal pain sensitivity failed to reveal line differences. Basal and stress levels of cardiovascular activity also showed no differences between the lines. In Experiment 3, LA but not HA animals showed profound stress-induced analgesia which was not blocked by a large dose of naltrexone. In Experiment 4, both LA and HA animals showed stress-induced analgesia, perhaps because the procedure of this experiment permitted conditioning mechanisms to contribute to the analgesia. Differential genetic selection for avoidance behavior also selected for differential pain sensitivity and some forms of non-opioid stress-induced analgesia but without concomitant selection for differential cardiovascular activity.

Effect of ACTH and vasopressin on extinction: evidence of opiate mediation.

These experiments demonstrated that both ACTH and arginine vasopressin (AVP) delay the extinction of a shuttle box avoidance response in intact rats. Furthermore, the effect of ACTH (2.5 IU) and AVP (0.2 IU) on extinction was the same when these hormones were administered daily throughout the course of extinction testing. Doses of naloxone, which did not affect this behavior, differentially blocked the effects of these hormones, ACTH being more easily blocked than AVP. The results are interpreted in terms of opiate mediation of the effects of the hormones and do not support the assertion that ACTH works through motivation whereas vasopressin works through memory.

Genetic selection for avoidance behavior in the rat.

Long-Evans rats were selectively bred for over 20 generations using two behavioral criteria: (1) limited responding to the warning signal during ten pretest trials and (2) either good or poor avoidance behavior during 60 avoidance training trials in a two-way shuttlebox. The results suggest that avoidance behavior in the rat is a heritable characteristic that may be selected independently of activity level.

Central mediation of hormonal influences on instrumental avoidance conditioning.

Two behaviorally active hormones of the pituitary-adrenal system are adrenocorticotropic hormones (ACTH) and corticosterone, and their behavioral effects are facilitation and inhibition of performance of previously learned avoidance responses, respectively. Their uptake, distribution and effects on central nervous system are reviewed. Hypothalamic neurotransmitter control of corticotropin releasing hormone (CRH) is described together with hypothalamic and extra-hypothalamic (hippocampal) regulation of pituitary-adrenal activity. Extra-hypothalamic mediation of the behavioral effects of ACTH is evaluated. Recent isotopic mappings of the efferents of the hippocampal formation have identified pathways from hippocampal subiculum to hypothalamus and posterior lateral and anterior thalamic nuclei. The evidence reviewed suggests a complex circuit involving hippocampal subiculum, thalamus and hypothalamus may be involved both in regulating pituitary-adrenal responses to stress and in mediating the effects of ACTH on avoidance behavior.