De Wied and Colleagues II: Further Clarification of the Roles of Vasopressin and Oxytocin in Memory Processing

Barbara B. McEwen

De Wied and Colleagues II: Further Clarification of the Roles of Vasopressin and Oxytocin in Memory Processing

I. Chapter Overview

________________________________________________________________________________________________

The research presented in Chapter 2 led De Wied and colleagues to conclude that vasopressin (VP) and oxytocin (OT) have important roles in modulating the formation of long-term memory, but are not importantly involved in the early learning phase of memory processing. Tested in both aversive and appetitive paradigms, peripherally administered VP consistently facilitated memory consolidation. The influence of OT was examined only in aversive paradigms in which, in general, it produced an amnestic effect. However, dose level and route of administration (central versus peripheral) were important parameters determining the specific type of memory modulation produced by this peptide. Although this early phase of their research launched the view herein referred to as the ‘‘VP/OT Central Memory Theory,’’ the heavy reliance on the peripheral route of administration opened the door for a number of alternative explanations of the means by

Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

103

104

Barbara B. McEwen

which these injected neuropeptides exerted their behavioral effects on learning paradigms. The evidence presented in this chapter pertains to four major questions: (1) Are VP and OT physiologically involved in memory processing, or could the findings presented in Chapter 2 be due to some nonphysiological pharmacological effect of the peptides? (2) If endogenous VP and OT influence memory processing, is this influence exerted by their peripheral (hormonal) and/or central (neurogenic) activities? (3) How important is a VP interaction with the central arousal system for the learning and memory effects induced by peripheral administration of the peptide? (4) Do peripherally administered VP and OT affect memory storage by an action exerted at peripheral and/or central receptor sites? As in Chapter 2, propositions formulated to define the major components of the ‘‘VP/OT Central Memory Theory’’ are presented at the end of this chapter along with relevant experimental support.

II. Establishing the Roles of Endogenous VP in Memory Processing: The Brattleboro Rat Model

_____________________________________________________

A. Introductory Comments De Wied and colleagues used three experimental models to study the physiological involvement of VP and/or OT in memory processing: (1) posterior pituitary lobectomized subjects are deprived of VP, OT, and other hormones present in the posterior/intermediate lobes of the pituitary gland, but not of central VP-ergic and OT-ergic circuitry. In an early series of experiments, De Wied (1965) compared these subjects with sham operates on learning and memory tasks before and after hormonal replacement therapy. These experiments were discussed in Chapter 2; (2) the Brattleboro rat, derived from the Long-Evans strain of hooded rats, is an inbred strain of rat that exhibits a genetic mutation at the single-gene locus encoding synthesis of the VP precursor (Schmale and Richter, 1984). Rats homozygous for the mutation (HODI rats) lack VP and suffer from diabetes insipidus (a disorder of water metabolism characterized by excessive ingestion of water, and urination). Rats heterozygous at the gene site (HEDI rats) are partially deprived of VP and do not have diabetes insipidus (Van Wimersma Greidanus and De Wied, 1977). Numerous studies have compared HODI, HEDI, and Long-Evans normal (LENO) rats on learning and memory tasks and the results of these studies and their controversial interpretations are discussed below; and (3) selective neutralization of peripheral and central endogenous VP or OT by antisera allows comparison of the relative contribution of peripheral and central stores of the peptide(s) to memory processing; this research is discussed in a subsequent section of this chapter.

The Roles of Vasopressin and Oxytocin in Memory Processing

105

B. Early Research Studies by De Wied and Colleagues with the Brattleboro Rat 1. Selected Studies a. De Wied et al. (1975) De Wied et al. (1975) compared HODI and HEDI rats on learning and retention of a passive avoidance (PA) response, using a single-trial step-through passive avoidance task. HODI rats were differentiated from HEDI rats by measuring water intake over a 2-day period before experimental testing. Examination of VP in the posterior pituitary lobe indicated nondetectable levels in HODI rats, and the level was significantly lower (about one-half the amount) in HEDI rats than in Wistar rats. In experiment 1, subjects were randomly assigned to the nonshock group or to shock groups that received a 3-s footshock (FS) at one of three levels of intensity (0.25, 0.50, or 1.0 mA). The reentry latencies of these subjects were tested 24, 48, and 120 h after the single PA training trial. For the HODI rats, none of the FS intensity levels produced significant PA behavior relative to the nonshock controls (i.e., none of the FS groups differed from the nonshock controls in median reentry latency on any of the three retention tests). Although the reentry latency for the highest FS level was slightly above preshock levels, the difference was not statistically significant. For the HEDI rats, PA behavior was observed for the 0.5- and 1.0-mA FS levels (reentry latencies significantly longer than the nonshock controls for the 0.5-mA FS in the 48- and 120-h retention test and in all three retention tests for the 1.0-mA FS level). Moreover, the 1.0-mA FS produced maximum PA behavior (median reentry latency of 300 s) in the HEDI rats for all three retention tests. When the unconditioned stimulus (UCS) was made more aversive (i.e., the 1.0-mA FS was given for 10 s rather than then 3 s), the HODI rats still failed to exhibit PA behavior. However, when they were exposed to a 3-s FS of 1.0 mA, and injected with either arginine vasopressin (AVP) or desglycinamide-lysine vasopressin (DG-LVP; 1 g/rat) immediately after the FS trial, they did show normal PA behavior on all three retention tests (i.e., their reentry latencies were significantly longer than those of the nonshocked controls, and highly similar to the PA behavior of HEDI rats assessed under identical test conditions). These latter observations indicated that the effect of the parent peptide on PA behavior was not due to its ability to normalize water metabolism in these VP-deficient rats, because the DG-LVP analog lacks endocrinological effects. Experiment 2 used a 3-s FS of 1.0-mA intensity, and tested reentry latency either immediately or 3 h after the FS training trial. At both test times the median reentry latency for the HEDI rats was at the maximum value (300 s). For the HODI rats, reentry latency tested immediately after the FS trial was near the maximal value, and although the reentry latency was markedly reduced at the 3-h test, it was still significantly different from

106

Barbara B. McEwen

preshock values. These findings suggest that, unlike retention, PA learning (reentry latency at zero delay) is normal in HODI rats. b. Bohus et al. (1975) Bohus et al. (1975) compared Brattleboro HODI, HEDI, and Wistar normal (WNO) rats for acquisition and extinction on two multitrial active avoidance-learning tasks, an open field test of exploratory and emotional reactivity, and a threshold reactivity test for various FS intensities. HODI and HEDI rats were also tested for acquisition/retention, and for pituitary–adrenal reactivity during retention testing in a single-trial passive avoidance task. Independent groups of subjects were used for each of the avoidance tasks, the open field test, and for assessing sensitivity to FS. Shuttlebox avoidance training lasted 12 days (10 trials/day) and was followed by 7 sessions of extinction (10 trials/session). The results are diagrammed in Fig. 1, and were as follows: (1) HODI rats reached the learning criterion (80% avoidance responses in 12 days), although their rate of acquisition was slower, and their total number of avoidance responses was significantly lower, than that for HEDI and WNO rats; acquisition performance was similar for the latter two groups; and (2) avoidance responding extinguished more rapidly in HODI rats than in either HEDI or WNO rats. Acquisition training in the pole-jump avoidance task occurred over a 6-day period (10 trials/day) and was followed by 4 days of extinction (10 trials/day). The results, given in Fig. 2, were as follows: (1) HODI and HEDI rats were similar in learning performance (both groups reached an approximately 75% performance level at the end of the 6-day acquisition period), and they were both deficient (slower in reaching the avoidance criterion and

FIGURE 1 Acquisition and extinction of a shuttlebox avoidance response in homozygous (Ho-DI) and heterozygous (He-DI) diabetes insipidus rats and in Wistar (Wi) rats. Source: Bohus et al., 1975 (Fig. 1, p. 611). Copyright ß 1975 by Brain Research Publications Inc. Reprinted by courtesy of Elsevier Science, present publisher of this journal. CAR, conditioned avoidance response.

The Roles of Vasopressin and Oxytocin in Memory Processing

107

FIGURE 2 Acquisition and extinction of a conditioned avoidance response in a pole-jumping situation in homozygous (Ho-DI) and heterozygous (He-DI) diabetes insipidus rats and in Wister (Wi) rats. Source: Bohus et al., 1975 (Fig. 2, p. 611). Copyright ß 1975 by Brain Research Publications Inc. Reprinted by courtesy of Elsevier Science, present publisher of this journal.

made significantly fewer correct avoidance responses) relative to WNO rats; and (2) WNO rats were significantly more resistant to response extinction than HEDI rats, which, in turn, made significantly more avoidance responses than their HODI counterparts. Independent groups of HODI and HEDI rats were randomly assigned to shock (3 s, 1.0-mA FS) and nonshock conditions, and after a single learning trial were tested for PA retention at 1 min (0-h delay), 3 h, and 24 h. Pituitary–adrenal activity (plasma corticosterone levels) was assessed 15 min after the beginning of each retention test. The behavioral results indicated the following: (1) full PA behavior (maximum reentry latency of 300-s duration) occurred in both groups of Brattleboro rats when tested 0 h after the learning trial; and (2) in contrast to HEDI rats, which continued to exhibit full avoidance behavior in the retention tests 3 and 24 h after the learning trial, HODI rats showed partial passive avoidance at 3 h (much reduced but significantly different from nonshock controls) and none at 24 h (no difference from nonshock controls). Thus HODI rats were strongly and significantly impaired in PA retention tested 24 h after the learning trial. Measures of endocrine activity indicated that the plasma corticosterone level during retention was positively related to PA behavior. On each retention test, HEDI rats showed full PA behavior and corticosterone levels that were consistently significantly higher than those of nonshocked controls. Plasma corticosterone levels in HODI rats decreased over the longer retention intervals, as did PA behavior. At 0-h retention, when full PA behavior occurred in HODI rats, plasma corticosterone levels did not differ from HEDI levels. At the 3-h retention test, when HODI rats showed partial PA behavior, plasma corticosterone levels were drastically reduced relative to the

108

Barbara B. McEwen

HEDI level, although still higher than that of nonshocked controls. At the 24-h retention test, when there was no evidence of PA behavior in HODI rats, plasma corticosterone levels remained significantly lower than those of HEDI rats and did not differ from nonshocked control levels. These observations indicate that in the absence of VP there is an impairment of the psychological mechanisms underlying PA behavior and a correlated deficiency in the pituitary–adrenal endocrine response to the fear-provoking environment. Painted lines marked off a number of central and peripheral squares on the floor of the circular open field. Subjects were placed in the center of the arena at the beginning of each daily 3-min session over four consecutive days and observed for ambulation (number of squares entered), rearing (both in the center of the field and near the walls), grooming (face washing, etc.), and defecation (number of boluses deposited). Results of the open field test were as follows: (1) ambulation (number of squares entered) was higher for both Brattleboro rat groups relative to WNO rats in both sessions 3 and 4 because of the failure of the former groups to show exploratory behavior habituation over the course of testing; (2) relative to WNO rats, Brattleboro rats showed a greater incidence of rearing in the middle of the arena in session 1, and near the wall in sessions 2 and 3 (HODI rats) or in sessions 3 and 4 (HEDI rats); and (3) except for session 1, during which defecation was performed more frequently by Brattleboro rats, and sessions 3 or 4, during which both HODI and HEDI rats groomed more frequently than did WNO rats, the groups were similar in these measures of open field behavior. Taken together, these findings suggest that, relative to WNO rats, Brattleboro rats evidenced more exploratory activity (ambulation), an initially higher level of autonomic reactivity (defecation), and a slower rate of habituation to the novel environment of the open field test. The test for sensitivity to FS pain used 2 sets of 12 FS intensities varying between 33 and 300 A. The index of threshold responsiveness was defined as the lowest intensity of FS that elicited a reaction such as flinch and jerk and/or jump and run. Pituitary–adrenal responsiveness was also determined in this test. The results of these tests indicated that the threshold currents for behavioral reactivity were similar in magnitude for HODI and HEDI rats but less for WNO rats. Pituitary–adrenal activity, indicated by plasma corticosterone levels after presentation of an FS series, did not differ between HODI and HEDI or between HODI and WNO rats, although HEDI rats exhibited larger increases in plasma corticosterone than did WNO rats. In summary, the results indicated that (1) learning may take place in the partial (HEDI) or total (HODI) absence of endogenous VP, because both groups learned both pole-jump and shuttlebox avoidance responses and exhibited full PA behavior when tested 1 min after the FS learning trial. Nevertheless, learning deficits were observed in HODI rats on both multitrial avoidance tasks. Relative to WNO rats, they were slower to acquire successful avoidance responses, and made fewer correct avoidance responses

The Roles of Vasopressin and Oxytocin in Memory Processing

109

during the learning period; (2) a consistent retention deficit occurred in both HODI and HEDI rats, compared with WNO rats, on both active avoidance tasks. Of the two, HODI rats were more profoundly impaired in all three active and passive avoidance behavior retention tasks; (3) among the Brattleboro rats, pituitary–adrenal endocrine reactivity appears to be positively related to retention performance in the passive avoidance task; this is consistent with the suggestion that the memory of the FS mediates the pituitary–adrenal reaction elicited during the PA retention test. This is further supported by the lack of difference between HODI and WNO rats in pituitary–adrenal endocrine reactivity in response to a pain stimulus (FS); (4) relative to WNO rats, both Brattleboro groups showed more exploratory activity (ambulation, rearing) and autonomic reactivity (defecation) and slower habituation when placed in a novel environment (open field test results); and (5) because HODI and HEDI rats did not differ in open field behavior or pain sensitivity, yet did differ in both active and passive avoidance retention behavior, it was concluded that the former factors were not causal influences on the differences in retention between the two groups.

C. Inconsistencies in the Research Literature Regarding the Putative Brattleboro Diabetes Insipidus Retention Deficit Because De Wied and colleagues propose that vasopressin has an important role in memory storage and retrieval but not in the processes underpinning learning per se, this discussion has been limited to evidence relating the genetically associated VP deficiency to performance on tasks of retention. Subsequent to the early studies by De Wied and colleagues (Bohus et al., 1975; De Wied et al., 1975), a number of investigators reported that Brattleboro rats were equal to, or in some cases superior to, Long-Evans normal (LENO) rats in various tests of retention. Brito (1983) and Williams et al. (1983a) observed that HODI rats were equivalent to LENO rats in PA retention in a step-through PA task, and Miller et al. (1976) observed no significant differences among HODI, HEDI, and LENO rats in extinction performance in a shuttlebox shock avoidance task. Although Bailey and Weiss (1979) observed that HODI rats were poorer in PA retention than HEDI rats, both groups did exhibit memory for the shock experience because latencies to enter the dark chamber were sharply increased in postshock relative to preshock trials; moreover, reentry latencies in both Brattleboro groups were higher than those observed in LENO rats tested in a separate experiment. Bailey and Weiss (1978) found that PA retention was equivalent for HODI and HEDI rats and was superior to LENO rats throughout the 4-day postshock observation period. Failure to observe a retention deficit in Brattleboro VP-deficient rats has also been reported by researchers using other types of retention tests. Brito

110

Barbara B. McEwen

(1983) observed no impairment in working memory in HODI relative to LENO rats in a T-maze alternation task tested with a short (8 s) delay between the forced-choice (information) and the free-choice run. Moreover, Brito and colleagues (Brito et al., 1981, 1982) observed that HODI rats retained a punishment-induced inhibition of a food-approach response longer than did LENO rats. On the other hand, several investigators have obtained evidence suggesting a retention deficit in HODI rats. Ambrogi Lorenzini et al. (1985) and Drago and Bohus (1986) reported that Brattleboro DI rats extinguished a conditioned one-way shock avoidance response more rapidly than did LENO rats. Stoehr et al. (1993) tested the effect of vasopressin on conditioned freezing behavior to aversive shock treatment. HODI, HEDI, and LENO rats received three footshocks in a sound-attenuated box on the training day. The following day, and for 3 days thereafter, the rats were returned to the box without further shock treatment and evaluated for spontaneous freezing behavior. HODI rats showed significantly less freezing behavior than did HEDI or LENO rats on each of the four test days. The authors interpreted these data in support of the notion that vasopressin has an important role in mediating appropriate autonomic and emotional responsivity in fear-conditioning paradigms. However, the reduced fear could also have been mediated, in part, by poorer retention in the HODI rats. Colombo et al. (1992) used a delayed alternation T-maze task, with varying intertrial intervals, to assess ability to retain spatial information in memory using a recently developed VP-deficient strain of rat. Relative to M520/NO rats, M520/DI rats exhibited diabetes insipidus and were significantly impaired in memory on this task. M520/HZ rats did not display diabetes insipidus and their task performance was intermediate between that of M520/DI and M520/NO rats.

D. Colony-Specific Heritable Traits and Inconsistent Findings Concerning a Retention Deficit in the Brattleboro DI Rat These contradictory findings may have been due, in part, to heritable behavioral characteristics, other than VP deficiency, that distinguish different laboratory colonies of Brattleboro rats. Although various groups of HODI rats share the VP gene mutation, differences between them in genetic background may be a more critical determinant of their behavior abnormalities (Brito, 1983; Williams et al., 1983a). For example, Gash et al. (1982) have pointed out that because small numbers of Brattleboro rats have been dispersed to found the various established colonies, it is possible that colony behavioral differences, independent of AVP deficiency, are due to differences in characteristics of the various colony founders.

The Roles of Vasopressin and Oxytocin in Memory Processing

111

1. Selected Studies a. Herman et al. (1986a) Herman et al. (1986a) subsequently tested this hypothesis. Brattleboro DI (DI) and normal Long-Evans (NO) rats were obtained from different laboratory colonies and tested for timidity in an open field emergence task, and for learning and memory in a simple runway approach/avoidance task and in a delayed nonmatching-to-sample (DNMS) task. DI and NO rats were obtained from colonies maintained at Charing Cross Hospital in London (CC/DI and CC/NO) and colonies of American suppliers in Rochester, New York (i.e., RO/DI and RO/NO). These researchers found that (1) DI rats from both colonies exhibited a high degree of neophobia (only 3 of the 10 subjects left the home cage to enter the open field arena over the 4-day test period). In contrast, both NO groups showed a strong tendency to enter the open field with decreasing emergence latency as a function of testing experience; (2) acquisition of the food-rewarded runway response was equivalent for the CC/DI, RO/DI, and RO/NO groups but slower for the CC/NO group; (3) the mouthshocks given on the last day of training severely disrupted the food-approach habit in all groups in the first postshock trial, indicating memory for the shock in all the subjects; (4) recovery from the punishment-induced response inhibition was quite variable among the groups, with the RO/DI group showing accelerated recovery of approach compared with the CC/DI group and both NO groups; (5) all four groups evidenced dispositional memory (acquired the nonmatching-to-sample contingency in the DNMS task), but RO/DI rats learned the contingency more quickly than either the CC/DI group or the CC/NO group; and (6) RO/DI rats also outperformed CC/DI rats on representational memory as indicated by the greater accuracy of their responses, which depended on trial-specific information, and by a lesser tendency to adopt a position habit, which does not require representational memory. The results were interpreted as indicating that the VP deficiency accounted for the ‘‘fearfulness’’ observed in the open field emergence test, but that colony-specific genetic and/or early experiential factors, rather than a VP deficiency per se, contributed to the differences observed in long-term memory in the approach/avoidance task (i.e., rate of recovery from the mouthshock experience) and in dispositional and representational memory in the DNMS task. Confirmation of the Herman et al. hypothesis about colony differences in emotional and cognitive behavior led these researchers to develop an inbred strain of rats homozygous for the VP deficiency (Roman high avoidance [RHA]:di/di) but otherwise identical to a normal Roman high avoidance (RHA:þ/þ) inbred strain of rats in heritable behavioral traits. The RHA:di/di strain was derived from an original mating between Brattleboro HODI rats and RHA:þ/þ rats and subsequent backcrosses between F2 generations of offspring homozygous for the VP deficiency and the RHA:þ/þ parent strain.

112

Barbara B. McEwen

b. Herman et al. (1986b) Herman et al. (1986b) compared RHA:di/di rats with RHA:þ/þ rats in the same tasks used by Herman et al. (1986a), and included a test of PA behavior and additional tests of open field emotional behaviors. The results indicated that (1) in the approach/avoidance task the groups were equivalent in adapting to the maze (eating in the goal box) and in acquiring the runway response, and both showed memory for the shock experience, although di/di rats showed shorter postshock latency to approach the goal box. The main difference between the groups concerned performance recovery from the shock experience: di/di rats gradually recovered their preshock goal approach speed during successive postshock sessions whereas þ/þ rats showed no strong tendency to enter the goal box during any of the postshock sessions; (2) in the DNMS task, both groups were able to use representational and dispositional memory to solve the problem, but relative to þ/þ rats, di/di rats were less accurate over all sessions and required significantly more trials to acquire the DNMS contingency, suggesting impaired dispositional memory; and (3) in the PA task, the two groups did not differ on latency to enter the dark (shock) compartment, either before or 24 h after the shock trial. Reentry latencies tended to be distributed in a bimodal fashion for both groups. The open field test results indicated that di/di rats habituated more slowly to the open field than did þ/þ rats. The two groups did not differ in ambulation scores on trial 1, but did so on subsequent trials. In comparison with þ/þ rats, di/di rats showed higher rates and slower habituation of rearing, higher defecation scores, and a greater tendency to ambulate in the central squares; there were no differences in freezing at the center of the open field or in grooming behavior. These findings indicated that di/di rats were able to learn the simple runway goal-approach response in a normal fashion and evidenced no absolute memory impairment because they were able to use dispositional and representational memory to solve the DNMS problem, showed inhibited goal box approach 24 h after the shock experience, and PA retention equivalent to that of þ/þ rats. Nevertheless, poorer performance in the cognitive tasks indicated by a significantly shorter postshock approach latency, greater recovery of the goal-approach response in the runway, more trials required to master the DNMS contingency, and reduced accuracy in performing this memory task could suggest relative memory impairment of di/di rats. An alternative interpretation is suggested by the open field behavior of di/di rats; subsequent to day 1 in the open field arena, di/di rats showed greater frequency in ambulation and rearing and slower habituation of this behavior than did þ/þ rats. Moreover, the heightened autonomic reactivity (defecation) of di/di rats relative to þ/þ rats was present on all test days. This difference in open field behavior suggests a heightened emotional/ arousal level among þ/þ subjects that, in turn, could have influenced

The Roles of Vasopressin and Oxytocin in Memory Processing

113

performance in the cognitive tasks. Herman et al. (1986b) were inclined to favor this latter interpretation. They hypothesized that VP deficiency may, directly or indirectly, result in greater than normal increases in arousal and/or arousability in certain situations (e.g., under food deprivation), which can then affect attentional selectivity and other cognitive functions necessary for normal performance on behavioral tasks, thereby indirectly influencing memory processes.

E. Brattleboro Rat Retention Deficit I: A Primary or Secondary Effect of VP Deficiency? Bailey and Weiss (1981) suggest that the HODI retention deficits that have been observed in avoidance tasks may represent secondary rather than primary effects of chronic VP deficiency. These include increased plasma levels of OT (released by the chronic state of mild dehydration), deficiency in growth hormone (may be responsible for the smaller size of HODI rats), and decreased responsiveness of the pituitary–adrenocortical system to some stressors [associated with decreased release of corticotropin-releasing factor (CRF) and reduced anterior pituitary responsiveness to CRF, both of which are reversed by VP treatment]. For example, a PA retention deficit could be due to a reduced pituitary–adrenal response to FS in the learning trial (Weiss et al., 1969, 1970). However, the finding that a single injection of DG-AVP promptly reversed the PA retention deficit without correcting the diabetes insipidus disorder suggested that the VP deficiency had a primary effect on retention (De Wied et al., 1975).

F. Brattleboro Rat Retention Deficit II: An Arousal-Mediated Phenomenon? The study of Brattleboro rats is also pertinent to determining the mechanism by which exogenous vasopressin may influence retention in avoidance tasks. For example, arousal level has been suggested as an indirect mechanism by which vasopressin alters retention behavior in avoidance tasks (discussed in Chapters 6 and 7). Apropos to this idea, several investigators have suggested that differences in retention between Brattleboro and LENO rats may be due to alterations in emotionality, temperament, or arousal level produced directly or indirectly by VP deficiency. As previously discussed, Herman et al. (1986a,b) share this viewpoint. Several researchers have suggested that Brattleboro rats are more fearful than LENO rats. This view has been supported by the following findings: (1) Bailey and Weiss (1981) observed that compared with LENO rats, HODI and HEDI rats showed less exploratory behavior (crossings) in the open field arena and remained frozen or inactive rather than running from the experimenter’s approach at the end of the test. This behavior of the Brattleboro

114

Barbara B. McEwen

rats was interpreted as indicating greater fear, which in turn was considered to be consistent with the longer PA reentry latencies observed in these rats (Bailey and Weiss, 1978, 1979); and (2) Brito et al. (1981) and Brito (1983) judged HODI rats to be more timid than LENO rats as indicated by (a) their slower emergence from the home cage into an open field, (b) the smaller amount eaten when fed in a novel environment, and (c) their slower adaptation to a T-maze apparatus used for various cognitive tests (Brito, 1983; Brito et al., 1981). Some researchers, although failing to observe evidence of greater fear and timidity, nevertheless have obtained data suggestive of an altered arousal level in HODI and HEDI rats. Williams et al. (1983a) observed that compared with normal LE rats, HODI rats (both males and females) exhibited significantly higher activity scores (crossing and rearing activity) during the initial 15 trials in an open field test. Williams et al. (1983b) showed that peripherally administered AVP (5 g/rat, subcutaneous) counteracted the early-trial increase in activity level exhibited in HODI rats. Moreover, AVP decreased open field activity to a lesser degree in normal rats, indicating their lower sensitivity to the influence of the hormone on this aspect of behavior. The finding that the activity level of the VP-deficient rat is elevated during an early observational period and that exogenous vasopressin can counteract this activity was interpreted as indicating that AVP has a direct effect on the processes that underlie this increased activity (presumably the altered emotional, motivational, and/or attentional state of the animal). Still other researchers have reported no evidence of increased timidity in novel environments associated with inherited VP deficiency. Laycock and Gartside (1985) reported that Brattleboro DI rats showed less fear and performed significantly better than LENO rats in an operant task when first placed in the Skinner box and Colombo et al. (1992) reported no significant difference among M520/HO, M520/HE, and M520/NO rats in rate of adapting to a novel test environment (the T-maze alternation task), a measure used by Brito et al. (1981) to operationally define ‘‘timidity.’’

G. Brattleboro Rat Model Revisited: Recent Findings by De Wied and Colleagues 1. Selected Study: De Wied et al. (1988) De Wied et al. (1988) reported a reinvestigation of retention behavior in the Brattleboro rat. HODI, HEDI, Long-Evans normal (LENO), and Wistar normal (WNO) rats were tested for emotional behavior in large and small open fields, and for acquisition and/or retention in three avoidance tasks (polejump avoidance, step-through PA, and shock-avoidant discrimination in a T-maze) and two appetitive tasks (food-rewarded visual discrimination and the hole board search task).

The Roles of Vasopressin and Oxytocin in Memory Processing

115

The open field test results verified earlier reports of an altered state of emotionality or motivation in HODI rats (Bohus et al., 1975). In both the small and large open fields, HODI rats exhibited a higher level of activity and slower habituation than the other groups. With the exception of normal learning in the original and reversed discrimination of the aversive T-maze task, HODI and the HEDI rats exhibited learning and retention deficits in all the remaining behavioral tests. Thus, in comparison with LENO and WNO rats, both HODI and HEDI rats were significantly impaired in acquisition of the black–white discrimination in the food-rewarded Y-maze, learning, working and reference memory in the hole board food search task, acquisition and maintenance of the pole-jump shock avoidance response, and retention in the PA task. Moreover, the HEDI rats tested in this study were more impaired in performing active and passive avoidance tasks than was observed earlier (Bohus et al., 1975) and did not differ from HODI rats in the other tests of learning and retention. The observation that the DI rats in these more recent studies appeared to be even more impaired in learning and retention than those used earlier supported the hypothesis that genetic differences exist for various colonies of this inbred strain. On the other hand, the authors noted that while there were differences in degree, the VP-deficient rats were consistently impaired in avoidance task retention in both their earlier and more recent investigations.

H. Section Summary and Concluding Remarks The Brattleboro DI rat model would seem to be a highly valuable asset to the investigation of the putative role of vasopressin in memory processing. Theoretically, if endogenous vasopressin has an important role in memory storage and retrieval, even if limited to stressful learning conditions (e.g., avoidance paradigms), VP-depleted subjects should exhibit poorer memory than their VP-repleted counterparts in such learning environments. Early findings by De Wied and colleagues (Bohus et al., 1975; De Wied et al., 1975) did indicate that HODI rats were significantly impaired in memory in active, and especially passive, avoidance tasks. Subsequent research from other laboratories, however, questioned such a memory impairment (e.g., Bailey and Weiss, 1978, 1979; Brito, 1983; Miller et al., 1976; Williams et al., 1983a). The research of Herman and colleagues (1986a) suggested that variation in genetic backgrounds of different laboratory colonies of Brattleboro rats could produce inherited variations in behavior tendencies that affect performance in many studies. This could contribute to the inconsistent retention effects observed in various studies and also obscure which of the behavioral results were due to the VP deficiency rather than to colony-specific genetic background influences.

116

Barbara B. McEwen

De Wied and colleagues (1988) acknowledged the influence of colonyspecific genetic differences on performance when comparing the results of their earlier and later studies with the Brattleboro rat model, but noted that retention impairment was a consistent finding in both sets of studies. Aside from the inconsistent results regarding a Brattleboro-associated retention deficit, there is the problem of interpreting the cause of this deficit when it does occur. A number of investigators disagree with De Wied and colleagues’ proposal that the absence of vasopressin at memory-processing sites is the major cause of the retention impairment observed in Brattleboro DI rats. It has been pointed out that any or some combination of hormonal and/or metabolic secondary effects of diabetes insipidus may influence cognitive performance in these animals (Bailey and Weiss, 1981). In addition, alterations in emotional, temperamental, and/or arousal levels, directly or indirectly caused by the VP deficiency, are viewed by many researchers as chief mediators of the cognitive effects observed in these Brattleboro rats (e.g., Bailey and Weiss, 1981; Brito et al., 1981; Herman et al., 1986b; Williams et al., 1983a,b). When considering the secondary effects associated with the genetic VP deficiency, together with heritable trait differences among localized colonies of Brattleboro or other inbred strains of VP-deficient rats, it seems an inescapable conclusion that this experimental model is of limited value in settling issues raised by alternative theories regarding the nature of the contribution of vasopressin to memory processing.

III. Further Study of the Role of Endogenous VP and OT in Memory Processing: Peripheral and/or Central Mechanisms?

_______________________________________________________________________________________________

A. Introductory Comments Because VP and OT are present in the body as both hormones and neural transmitters, and are released centrally and peripherally by a variety of stress stimuli (Chapter 1), it is important to learn the sources of these peptides that have putative effects on retention. De Wied and colleagues theorize that it is the central, and not the peripheral, systems that physiologically modulate memory consolidation and retrieval, and mediate the retention effects observed after peripheral injection of these peptides. Participation of central VP and OT circuitry in memory processing is quite feasible given the presence of VP-ergic and OT-ergic fiber terminals and binding sites within brain structures implicated in memory processing (Buijs, 1987; Buijs et al., 1978; Caffe et al., 1987; De Vries and Buijs, 1983; Sofroniew, 1983; Sofroniew and Weindl, 1978; Van Leeuwen and Caffe, 1983). De Wied and colleagues have used experimental and correlational techniques in their studies of physiological involvement of peripheral and central

The Roles of Vasopressin and Oxytocin in Memory Processing

117

VP and OT systems in memory storage, in both active and passive avoidance paradigms. The experimental technique discussed below temporarily removed the influence of a centrally or peripherally localized neuropeptide by neutralizing it through selective injection of antiserum to the peptide. Their correlational studies examined levels of VP or OT within the plasma, cerebrospinal fluid (CSF), or specific brain sites at designated times during learning or retention testing and correlated these levels with measures of learning and/or retention.

B. Neutralizing Peripheral or Centrally Circulating VP or OT by Antiserum Treatment: Effect on Memory Processing 1. Selected Study: Van Wimersma Greidanus et al. (1975a) Van Wimersma Greidanus et al. (1975a) neutralized central or peripheral levels of vasopressin by injection of VP antiserum, and studied the effect on passive avoidance (PA) retention in male Wistar rats. They were tested in a single-trial step-through PA task during which they received either no FS or a 3-s FS of 0.75-mA intensity. Retention testing occurred either 2 min, 1 h, 4 h, 24 h, or 48 h after the learning trial, with 300 s allowed for maximal reentry latency. One group of rats was injected intracerebroventricularly with 1 l of anti-VP serum or control rabbit serum. This procedure neutralized central but not hormonal levels of VP because it had no effect on urine production, water consumption, or urine vasopressin levels. Another group was injected intravenously with 100 l of anti-VP serum or control rabbit serum. Injections were given either 0.5 h before, or immediately after, the PA learning trial. Neutralization of central VP levels did not affect PA learning because reentry latencies were maximal (300 s) in the rats injected intracerebroventricularly with anti-VP serum, as they were in the controls, when tested 2 min and 1 h after the learning trial. However, PA retention was severely retarded in the anti-VP-treated subjects, as indicated by reentry latencies at 4 h that were significantly reduced, relative to those of the controls, and even more markedly reduced at 24 and 48 h after the learning trial. Retention, however, was not affected when peripherally circulating AVP was neutralized by an intravenous injection of 100 times the amount of antiserum used for intracerebroventricular injection. For these peripheral VP-neutralized rats, median reentry latency was maximal, as it was for the rats given control rabbit serum at both the 24- and 48-h retention tests. A role for limbic VP-ergic systems in PA retention has been indicated by the observation that anti-VP serum more efficiently neutralizes central vasopressin when it is injected into limbic system structures rather than into a lateral ventricle. Thus, PA retention impairment can be achieved by a more

118

Barbara B. McEwen

diluted antiserum solution if injected into the various limbic system structures rather than into the lateral ventricle (reported in Van Wimersma Greidanus et al., 1986). A role for central OT systems in memory modulation is suggested by the findings that neutralizing central levels of OT by intracerebroventricularly injected anti-OT serum improved PA retention (Bohus et al., 1978b; and see Chapter 2).

C. Correlational Studies 1: Avoidance Retention and AVP Levels in the Blood Correlational studies have been conducted to further investigate the role of endogenous vasopressin in memory storage and retrieval. Because it is possible that vasopressin released from the brain into peripheral circulation during learning contributes to memory processing, studies have been conducted to relate plasma levels of vasopressin to active and passive avoidance behavior. 1. Selected Studies a. Thompson and De Wied (1973) Thompson and De Wied (1973) tested the proposition that VP is released by environmental cues previously associated with an aversive experience. Antidiuretic (AD) activity was bioassayed from eye plexus blood collected from anesthetized male rats of an inbred Wistar strain. The blood was collected from rats anesthetized with ether for 45 s, immediately after the first (24 h), or in some experiments after the second (48 h), PA retention trial. The assay was accomplished by injecting the collected serum into female rats of the same inbred Wistar strain, which were then bioassayed for AD activity by assessing the effect of the injected serum on urine flow rate in water-loaded alcohol-anesthetized rats over consecutive 10-min periods. Eye plexus blood instead of trunk blood was used because of its higher level of AD activity. In addition, AD activity was measured in rats whose PA behavior had been modified by treatment with either adrenocorticotropic hormone peptide [ACTH(1–10); 30 g/rat, subcutaneous] or DG-LVP (0.5 g/rat, subcutaneous), which lacks the pressor and antidiuretic effects of the parent peptide (De Wied et al., 1972). In the experiment that studied AD activity under nonpeptide treatment conditions, the subjects were assigned to either the nonshocked (NS) control group or received a 3-s FS at an intensity of 0.25, 0.50, or 1.00 mA in the PA learning trial, and were tested for retention 24 h later. In the two experiments that used peptide-treated subjects, PA training and testing was similar except that the shocked groups received an FS of only 0.25-mA intensity and were tested for retention at both 24 and 48 h after the FS trial. The peptides, either ACTH(1–10) (30 g/rat, subcutaneous) or DG-LVP (0.5 g/rat, subcutaneous), or placebo solutions were injected 1 h before the 24-h retention test trial (retrieval design).

The Roles of Vasopressin and Oxytocin in Memory Processing

119

The results were as follows: (1) although the numbers of subjects in the nonpeptide-treated FS groups were too small for statistical analysis, the median reentry latencies in the 24-h retention trial increased in correspondence with increased shock intensities, consistent with previous observations (Ader and De Wied, 1972); (2) AD activity level in the nonpeptide-treated NS group, sampled immediately after the 24-h retention trial, was significantly lower than that in each of the FS groups, and AD levels increased incrementally in correspondence with increased levels of shock intensity; (3) relative to placebo treatment, ACTH(4–10) did not influence AD activity in the NS controls but significantly increased it in the shocked subjects. Median reentry latency was also significantly higher in the ACTH-treated group than in the placebo-treated FS group. However, neither the increased reentry latency nor the increased AD activity was maintained in the second retention test, 24 h later; (4) DG-LVP, devoid of endocrine activity, unexplainably increased AD activity in the nonshocked controls after the 24-h retention trial; nevertheless, AD activity in the DG-LVP-treated shocked subjects was significantly higher than that for the DG-LVP-treated nonshocked controls; and (5) DG-LVP treatment markedly increased reentry latency and significantly increased AD activity in the shocked subjects relative to the placebotreated controls, and these measures remained largely unchanged in the second retention test trial. These results led to the following conclusions: (1) the proposal that ACTH-like peptides and vasopressin influence memory retrieval by different mechanisms was again supported by the results indicating a short-term effect of ACTH(4–10) versus a long-term effect of DG-LVP on PA behavior; (2) conditioned cues, which can elicit memory retrieval for the footshock experience, can also cause a release of VP into the peripheral circulation, and these effects are related to severity of the aversive experience; and (3) when reentry latency was increased by either DG-LVP or ACTH(1–10) the release of AVP into the peripheral circulation also increased. Although this could imply that the VP released into the circulation may have fed back to influence the behavioral response, the authors pointed out the need for further experiments on this question. Information relevant to this issue is provided by experimental findings discussed below. b. Van Wimersma Greidanus et al. (1979a) Van Wimersma Greidanus et al. (1979a) examined AVP levels in the peripheral circulation (trunk blood) in male inbred Wistar rats tested in active (pole-jump) and passive (single-trial, step-through) avoidance tasks. AVP levels in trunk blood were measured in independent groups of rats killed at various points in time during the course of active or passive avoidance testing. For the active avoidance task, the animals received acquisition training (10 trials/day) on days 1–3 and extinction testing (10 trials) on day 4. Plasma levels of AVP were assessed from trunk blood collected either before (i.e.,

120

Barbara B. McEwen

after a 3-min exposure to the pole-jump apparatus) or immediately after each day’s session of acquisition and extinction. The results were as follows: (1) successful avoidance responding increased over the 3-day acquisition period, reached the criterion performance level on day 3, and, as expected, decreased below asymptotic performance level during extinction testing on day 4; and (2) there was no significant change in plasma AVP level measured before or after the behavioral test session on any day of acquisition or during extinction. Moreover, neither pre- nor postsession AVP levels during extinction differed significantly from any values observed during acquisition training. Thus plasma AVP levels were not related to behavioral changes that occurred during acquisition or extinction testing in this active avoidance task. In the passive avoidance task, depending on the group, the rats were not shocked (NS group) or were given a 3-s FS of low intensity (0.25 mA; LS group) or high intensity (1.0 mA; HS group). Trunk blood was collected either 5, 60, or 300 s (maximal duration for observing reentry latency) after the onset of the 24-h retention test. In addition, trunk blood was collected 300 s after the learning trial for one group of subjects tested with the highintensity FS. The results indicated that (1) reentry latency increased with increasing FS intensity, with all subjects in the HS group exhibiting a maximal PA response (300-s reentry latency); (2) when trunk blood was collected 5, 60, or 300 s after the onset of the retention test trial, there was no significant difference between the LS group and its NS control group in plasma level of AVP; (3) the HS group did not differ from its NS control group in plasma AVP when blood was collected 5 or 60 s after the onset of the retention test. However, when blood was collected at the end of the test trial, plasma AVP level in the HS group (median reentry latency, 300 s) was significantly higher than that of either the NS or LS group; and (4) plasma AVP levels did not differ from basal levels in rats killed 300 s after receiving the 1.00-mA FS, in accordance with a report by Husain et al. (1976) that noxious stimuli do not necessarily cause a rise in plasma AVP levels. The lack of relation between learning performance and plasma AVP levels (assessed either during the 3 days of multitrial pole-jump avoidance learning, or during the single PA learning trial with the 1.00-mA FS) indicated that plasma AVP is an unlikely route by which endogenous vasopressin influences retention in these tasks. The findings that plasma AVP levels in the HS group increased after 300 s, but not after 5 or 60 s in the postshock test environment, suggests that the longer the exposure to this environment, the greater the hormonal AVP released by the conditioned fear stimuli. c. Mens et al. (1982a) Mens et al. (1982a) determined plasma levels of oxytocin (OT) and vasopressin (VP) during acquisition and retention of passive avoidance (PA) behavior in male inbred Wistar rats. Rats in the OT assessment groups were given either no shock (NS group) or a 3-s FS

The Roles of Vasopressin and Oxytocin in Memory Processing

121

of low intensity (0.25 mA) or high intensity (0.75 mA). Blood was collected in these subjects either before the behavior test (basal level assessment), 20 min after the learning trial, or 180 s (maximal duration for retention trial) after the 24-h retention test. Rats in the VP assessment groups received either no shock or a 3-s FS of 0.50-mA intensity. In these rats, blood was collected after the 2-min adaptation trial on day 1 of PA training, 20 min after the learning trial, or 180 s after the 24-h retention test. Plasma levels of the peptides were measured by a radioimmunoassay (RIA) procedure. These tests permitted a measure of peptide release into peripheral circulation by the noxious unconditioned stimulus (FS) during the PA learning trial, and by the conditioned stimuli present during the PA retention trial. The results for subjects in the OT assessment groups were as follows: (1) reentry latencies were positively related to shock intensity, with a maximal median reentry latency (180 s) for the rats given the highest FS level; (2) when measured 20 min after the learning trial, mean plasma OT in the nonshocked group did not differ significantly from the basal value, nor did the nonshocked group significantly differ from the 0.75-mA shock group in this measure. However, the 0.25-mA shock group unexpectedly exhibited a significant decrease in plasma OT relative to the nonshocked group; and (3) after the retention trial, there were no significant differences between the nonshocked controls and either of the shocked groups in mean plasma OT levels, nor was there any relationship between plasma OT levels and reentry latencies. The results for the VP assessment groups were as follows: (1) reentry latencies were bimodally distributed and subjects were classified as avoiders (median reentry latency, 180 s) or nonavoiders (median reentry latency, 68 s); (2) plasma levels of AVP assessed 20 min after the FS trial or 3 min after onset of the retention trial did not differ from levels obtained during adaptation to the task; and (3) there was no relationship between reentry latency and plasma levels of AVP. Summarizing, barring the exception of the plasma OT result, which needs replication, this study indicates, for the FS levels used, no change in plasma levels of VP or OT during PA learning or retention; nor were these levels related to reentry latencies. These results further support the view that hormonal VP or OT feedback during PA acquisition does not contribute to memory processing in this task. d. Laczi et al. (1983c) Laczi et al. (1983c) investigated vasopressin levels in eye plexus blood in relation to passive avoidance (PA) acquisition and retention in normal Wistar (WNO) rats, Brattleboro HODI and HEDI rats, and Brattleboro HONO variant rats, normal for the gene locus encoding the VP precursor. Because only female HONO rats were available, their data were compared with data obtained from female WNO rats. Male rats were used in all other experimental tests. Depending on the experiment, eye plexus blood was collected from anesthetized rats at various times after the

122

Barbara B. McEwen

PA learning trial and/or the 24-h retention trial. The collected blood samples were used to assess the plasma level of antidiuretic (AD) activity and/or immunoreactive AVP (irAVP). Experiment 1 was designed to replicate and extend the experimental study of Thompson and De Wied (1973), and assessed both AD activity and irAVP in eye plexus blood withdrawn either 30 s after the PA learning trial or 30 s after the 24-h retention trial. The WNO subjects were assigned to either a nonshock (NS) group or to one of three FS groups (a 3-s FS at an intensity of 0.25, 0.50, or 1.0 mA). The results of experiment 1 were as follows: (1) each of the FS groups displayed significantly longer reentry latencies than did the NS controls; (2) reentry latencies increased with FS intensity, but maximum latency (300 s) was already present in rats given the 0.50-mA FS; (3) reentry latency was not affected by the procedure of collecting eye plexus blood after the training trial because there was no significant difference in this measure between subjects from which the blood was taken after the learning trial and those from which it was taken after the retention trial; (4) plasma AD activity and irAVP levels were high in nonshocked as well as in shocked rats assessed after the training trial, and these levels were not significantly correlated with FS intensity. This finding suggested that handling and the test environment itself were sufficient to release VP into the circulation and FS stress failed to add to this effect; (5) when assessed after the retention trial, AD activity was slightly but significantly higher in the 0.25-mA FS than in the nonshocked group, and marked and significant differences in this measure occurred between the subjects given higher FS levels and NS controls; and (6) AD activity after the retention trial was positively and significantly correlated with FS intensity used in the learning trial, indicating that the noxious conditioned stimuli caused a release of VP into the peripheral circulation during the retention trial. The results of this experiment corroborated the observations of Thompson and De Wied (1973), which showed that PA performance is related to AD activity in eye plexus blood (i.e., the higher the reentry latencies the higher the levels of AD activity in eye plexus blood collected immediately after the 24-h retention trial). Experiment 2 was designed to replicate the unexpected finding in experiment 1 that FS did not further increase the release of VP into the peripheral circulation. Plasma VP was collected for RIA assessment 30 s (0 min), 10 min, and 30 min after the PA learning trial to compare WNO groups receiving no shock, a low FS (0.25 mA), or a high FS (1.00 mA). The results indicated that (1) no significant differences among the three FS groups in irAVP levels were observed for any of the three collection times; (2) nevertheless, reentry latency in the 24-h retention trial was significantly related to FS intensity; and (3) irAVP levels 30 min after the PA learning trial were significantly reduced compared with those determined 0 min after the learning trial for the NS and shocked groups.

The Roles of Vasopressin and Oxytocin in Memory Processing

123

In experiment 3, male HEDI and HODI subjects and female HONO and WNO rats received either no FS or a 1.0-mA FS during the PA learning trial, and were tested for retention 24 h later. Eye plexus blood, collected 30 s after the learning trial, was tested by RIA, and samples obtained 30 s after the 24-h retention test were measured by both AD assay and RIA. The results were as follows: (1) for the HEDI subjects: reentry latencies were maximal (300 s) for the 1.0-mA FS group, irAVP levels did not significantly differ between NS and FS groups whether sampled after the learning or the retention trial, and irAVP was lower in comparison with NS WNO rats; (2) none of the HODI rats showed PA behavior, and irAVP levels were under the limit of detection in both NS controls and shocked rats; and (3) comparisons between HONO and WNO female rats indicated significantly longer reentry latencies for the FS relative to the NS groups for both strains of rat; for blood collected after the learning trial there were no differences in AD and irAVP activity between the FS and NS groups of either strain. When sampled after the retention trial, however, AD and irAVP activity were significantly higher in the FS than in the NS groups in both strains of rat. In experiment 4, VP levels in eye plexus blood and PA behavior were tested in WNO rats pretreated with intracerebroventricularly injected antiVP serum (1 l of undiluted serum) or control (normal rabbit serum) serum. Injections were given 0.5 h before the PA learning trial (consolidation design) or 0.5 h before the 24-h retention trial (retrieval design). The subjects were assigned to either the nonshock condition or the high FS (1.0 mA) condition. Eye plexus blood was collected 30 s after the learning trial or 30 s after the retention trial. The results indicated that (1) intracerebroventricular injection of anti-VP serum, given before either the learning or the retention trial, markedly reduced reentry latency compared with rats treated with control serum, indicating a marked impairment of both consolidation and retrieval; (2) there were no differences in irAVP levels, assessed after the training trial, between rats pretreated with anti-VP or control serum; and (3) irVP levels assessed after the retention trial were significantly lower in rats pretreated with anti-VP serum than in controls, whether injections were given before PA training or retention testing; moreover, the decrease in irAVP was more marked when anti-VP serum was given before the retention trial rather than the training trial. Summarizing the results of this study: (1) plasma AVP assessed after the 24-h retention trial was significantly higher in the FS than in the NS groups for WNO males (experiments 1 and 2) and for both HONO and WNO females (experiment 3), and this level was positively associated with reentry latency scores (experiments 1 and 2), which in turn reflected the FS intensity used in the learning trial (experiments 1 and 2); (2) plasma AVP assessed after the PA learning trial was not influenced by FS in WNO males (experiments 1 and 2), HEDI males (experiment 3), or HONO and WNO females (experiment 3); nor was there a relationship between this level of plasma

124

Barbara B. McEwen

AVP and reentry latency scores (experiments 1 and 2); (3) anti-VP serum, given 0.5 h before the learning and the retention trial, significantly impaired memory consolidation and retrieval, respectively (experiment 4); and (4) moreover, anti-VP serum injected 0.5 h before either the training or retention trial impaired the ability of conditioned stimuli, encountered in the retention test, to release VP into the circulation (experiment 4). The authors concluded that the results of this study are in accord with the interpretation that although hormonal AVP is released by memorymediated fear of the test environment, the painful FS itself does not significantly influence this release. More importantly, the results indicate that there is no basis for concluding that peripherally circulating VP during the learning trial contributes to memory storage in this task.

D. Correlational Studies 2: Avoidance Retention and AVP Levels in the CSF The presence of central VP-ergic and OT-ergic binding sites in various limbic structures suggests that central VP and OT systems may play important roles in memory storage. If so, one might expect that CSF levels of these peptides would correlate with retention behavior, whether CSF serves as a medium for transporting the peptide from sites of synthesis to sites of memory processing or whether it merely contains the spillover effects of VP and OT secreted at activated neural terminals. Several studies, described below, were carried out to determine whether CSF levels of VP and/or OT are related to PA retention. 1. Selected Studies a. Van Wimersma Greidanus et al. (1979a) Van Wimersma Greidanus et al. (1979a) examined irAVP in the CSF of rats tested in a single-trial, step-through passive avoidance task. The rats received either no FS, a lowlevel FS (0.25 mA, 3 s) or a high-level FS (1.0 mA, 3 s). CSF in a lateral ventricle was collected from freely moving rats via a previously implanted cannula, 5 min after the 24-h retention test but not after the FS training trial. The results indicated that whereas median reentry latency scores increased in accordance with increased FS intensity, the no-shock and two footshock groups did not significantly differ in CSF level of irAVP. b. Mens et al. (1982a) Mens et al. (1982a) assessed irVP and irOT levels in the CSF at various times during PA testing. CSF was withdrawn from the cisterna magnum of freely moving rats via previously implanted cannulas. Samples were collected during the adaptation trial, 20 min after the FS learning trial, and 3 min after the onset of the 24-h retention test. Subjects in the OT-tested groups received no shock (NS group) or an FS of 0.25- or 0.75-mA intensity for 3 s. Subjects in the VP-tested groups received no shock or a single 0.50-mA FS for 3 s.

The Roles of Vasopressin and Oxytocin in Memory Processing

125

The results for the irOT-tested subjects indicated (1) no significant difference in CSF levels of irOT between the NS group and either the lowor high-FS groups when tested after the learning trial, or after the retention test trial; (2) no significant correlation between reentry latencies and OT levels in CSF measured after the retention test; and (3) marked differences in reentry latencies among the different shock groups, with maximum PA behavior (median reentry latency, 180 s) occurring at the 0.75-mA FS level. The results for the irAVP-tested subjects indicated: (1) no significant effect of the FS experience on CSF levels of irAVP and (2) a bimodal distribution of reentry latency scores for the shocked rats, with good avoiders exhibiting a median reentry latency of maximal duration (180 s) and nonavoiders exhibiting a median reentry latency of 68 s. In addition, (3) CSF levels of irAVP in avoiders were not significantly different from those in nonavoiders, and individual CSF levels of AVP were not correlated with avoidance reentry latencies in the 24-h retention test. c. Laczi et al. (1984) Laczi et al. (1984) determined CSF levels of irVP and irOT in male Wistar rats tested in a single-trial passive avoidance task. The rats received either no FS, a low-level FS (0.25 mA), or a high-level FS (1.00 mA) for 3 s. Retention test trials were given at both 24 and 120 h (5 days) after the learning trial. CSF was collected from the cisterna magnum in cannulated, anesthetized rats 30 s after the learning trial and the 24-h and 120-h retention test trials. Compared with the nonshocked rats, shocked rats exhibited longer reentry latencies in both retention tests, although the latencies were slightly lower in the 5-day test than in the 24-h test. Reentry latencies were positively correlated with FS intensity. When measured after the learning trial, CSF AVP levels were increased in shocked rats relative to the nonshocked controls, but this increase was statistically significant only for rats that received the 1.0-mA FS. When measured after the 24-h retention test, CSF irAVP was significantly increased for the low-level FS animals but was below the detection limit for the high-level FS-treated animals. After completion of the 5-day retention test, CSF irAVP was significantly increased in both FS groups and the amount of increase was related to reentry latencies, which in turn reflected intensity of the FS in the PA learning trial. The finding that irAVP in the CSF was below the detection limits in the 24-h retention test in rats that received a high level of FS required explanation. The authors suggested that the accessible pool of VP in the terminals of stimulated VP-ergic neurons may have been exhausted by the intense FS experience, and this prevented overspill in the CSF 24 h later. When tested 5 days later, the releasable pool of AVP was fully recovered, so that a significant increase in CSF irAVP was in evidence. This explanation rests on the assumption that AVP in the CSF represents spillover from central VPergic networks that deliver the peptide to memory storage sites in the brain.

126

Barbara B. McEwen

Although all three studies described above found a positive relation between PA behavior (reentry latencies) and FS intensity, they were not consistent with respect to a relationship between CSF levels and PA behavior. Of the two studies that observed CSF levels of VP and/or OT after the learning trial, Mens et al. (1982a) found no effect of an FS intensity of 0.25 or 0.75 mA on irOT, or of 0.50 mA on irVP level in CSF when it was sampled 20 min after the PA learning trial. On the other hand, Laczi et al. (1984), who assessed irVP level in the CSF immediately (30 s) after the learning trial, observed a significant increase in this level in the FS groups, the magnitude of which was positively related to the FS intensity. This discrepancy may have been due, in part, to differences in behavioral test procedure: Laczi et al. (1984) took precautions to habituate the subjects to handling and to the test conditions before experimental testing, because these factors could potentially mask the effects of the FS experience on irAVP levels in activated limbic sites and hence in the CSF (Laczi et al., 1983b). The absence of a relation between PA behavior and CSF level of VP in the studies by Van Wimersma Greidanus et al. (1979a) and Mens et al. (1982a), when tested in the 24-h retention test, were both interpreted as evidence of the argument that CSF is not involved as a medium of transport for VP and OT from sites of synthesis to sites involved in memory processing. The results obtained by Laczi et al. (1984), although differing in their specifics from those of the former two studies, nevertheless suggest a similar interpretation: that is, CSF appears not to function as a medium for transporting AVP to sites engaged in memory processing; rather, the changes in CSF AVP observed at different times during PA testing reflect spillover effects from VP-ergic circuitry activated during the testing process.

E. Correlational Studies 3: Avoidance Retention and AVP Levels in Selected Brain Structures The presence of central VP-ergic and OT-ergic fibers terminating in brain sites implicated in memory processing suggests the possibility that brain vasopressin may function as a neurotransmitter or neuromodulator to influence memory processing; in this case, a positive relationship between CSF levels of vasopressin and avoidance retention would signify diffusion of vasopressin from activated VP-ergic terminals into the CSF. To investigate the role of brain VP-ergic terminals in memory processing, studies have examined the relationship between avoidance retention and vasopressin levels in dissected brain structures. 1. Selected Studies a. Laczi et al. (1983a) Laczi et al. (1983a) measured the irAVP content in various midbrain–limbic system sites after a 24-h PA retention trial in male Wistar rats. The subjects received either no shock or a 3-s FS of 1.0-mA

The Roles of Vasopressin and Oxytocin in Memory Processing

127

intensity. Reentry latencies were observed to a maximum of 300 s in the 24-h retention test. Immediately thereafter each animal was killed and the brain was removed and dissected. irAVP content was measured in several extrahypothalamic brain sites (medial and dorsal raphe nucleus, locus coeruleus, lateral septum, and central amygdala nucleus) believed to mediate the effects of AVP on memory processing (Bohus et al., 1982; Kovacs et al., 1979a; Van Wimersma Greidanus et al., 1979b; see Chapter 4). The results demonstrated that (1) reentry latencies were significantly higher in shocked rats than in nonshocked controls; (2) no significant differences in irAVP levels between shocked rats and nonshocked controls occurred in the medial and dorsal raphe nuclei; and (3) relative to controls, irAVP levels in the shocked rats were significantly decreased in the lateral septum, and increased in the central amygdala and locus coeruleus of the limbic–midbrain areas. Suggested interpretations for these results were as follows: (1) the postretention decrease in septal irAVP, coupled with the similar decrease in hippocampal irAVP reported by Laczi et al. (1983b; see below), could indicate unrecovered low levels in these brain sites caused by AVP activation during memory consolidation. These researchers further noted that several lesion and microinjection studies (Kovacs et al., 1979a; Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4) also support a role for septal and hippocampal sites in mediating the influence of VP on memory consolidation; and (2) the increase in irAVP that occurred in the central amygdala nucleus and the locus coeruleus could have been related to the activation of neurons in these areas during the PA retention trial itself. That is, activation of VP circuitry in these areas may relate to a role in retrieval (amygdala; Van Wimersma Greidanus et al., 1979b, described in Chapter 4) and in arousal (stress)-related activation (locus coeruleus; Mason and Iversen, 1978). b. Laczi et al. (1983b) Laczi et al. (1983b) measured irAVP in the hippocampus and amygdala (i.e., amygdala plus overlying pyriform cortex) of male Wistar rats in association with various types of stress stimulation (handling, novelty, and anesthesia) as well as during acquisition and retention of a PA response. Four experiments were conducted. Experiment 1 tested the effects of each of the following stressors on irVP content in the hippocampus: handling the subjects, ether anesthesia (45 s duration), novelty (exposure to the PA apparatus without previous habituation and in the absence of FS), and pain (3-s FS at an intensity of 1.0 mA after entry into the dark chamber of a PA apparatus). The rats were killed immediately after these experiences. Hippocampal irAVP content was not significantly affected by handling alone or by handling followed by anesthetic treatment. Initial exposure to the PA test box alone significantly decreased the irVP level in the hippocampus, but the addition of a painful FS

128

Barbara B. McEwen

to this novel exposure produced no further change in hippocampal irVP content. Experiment 2 tested the effects of a 1.0-mA FS experience on hippocampal and amygdalar irAVP levels in rats previously adapted to the PA test apparatus. When killed immediately after the PA learning trial, hippocampal but not amygdalar irAVP was significantly decreased in shocked, relative to nonshocked, rats. In experiment 3, hippocampal irAVP was measured in rats killed immediately after the 24-h PA retention session. Approximately half of these animals were given ether anesthesia for 45 s before sacrifice. For statistical comparisons, the animals that received an FS (1.0 mA) in the PA learning trial were classified as either poor avoiders (reentry latencies less than 100 s) or good avoiders (reentry latencies greater than 100 s). Compared with the nonshocked controls, there was significantly less hippocampal irVP content in the good, but not the poor, avoiders. In experiment 4, hippocampal and amygdalar irAVP contents were measured in rats killed either immediately before or after the 120-h (5-day) retention test of a PA task. The rats received either no FS or a 3-s FS of 1.0-mA intensity during the learning trial. All shocked animals were classified as good and poor avoiders, as in experiment 3, and compared with nonshocked controls for irAVP content. When measured after the 5-day retention test, hippocampal irAVP was significantly decreased in good but not in the poor avoiders, whereas irAVP level in the amygdala was not significantly affected in either the good or poor avoiders. When killed immediately before the 5-day retention test, there were no significant differences between the shocked and nonshocked rats for either hippocampal or amygdalar irAVP. The overall results of this study were interpreted as indicating that (1) hippocampal irAVP is influenced by some forms of stress (footshock and novelty) but not by others (ether anesthesia); (2) the FS stress-induced change in hippocampal irAVP appears to be transient because it was not present 5 days later in rats killed before the retention test; (3) hippocampal irAVP content was related to avoidance performance because good avoiders exhibited a reduced level of hippocampal irAVP at both the 24- and 120-h retention trials; and (4) immunoreactive VP content in the amygdala was not associated with PA performance. The failure of this latter study (Laczi et al., 1983b) to observe a change in amygdalar irVP content during retention testing differed from the results of their former study (Laczi et al., 1983a), which indicated an increase in amygdalar AVP level during PA retention testing. It is possible that the more extensive fragment of the amygdala (i.e., including the overlying pyriform cortex), tested in the later study (1983b), masked the increased irAVP in the more restricted central amygdaloid nucleus observed in the former study (1983a).

The Roles of Vasopressin and Oxytocin in Memory Processing

129

The opposing directional changes in irAVP content in the two sets of limbic structures indicated by the studies of Laczi et al. (1983a, 1983b) may be related to the observation that AVP in the septal–hippocampal area has been observed to play a role in memory consolidation whereas that in the amygdala appears to be implicated in memory retrieval (Bohus et al., 1982).

F. Section Summary Taken together, the studies reported in this section support the view that central VP-ergic and OT-ergic circuitry, not peripherally circulating hormonal VP and OT, is physiologically involved in memory storage and retrieval. Specific lines of supportive evidence are as follows: first, even though intensity of the aversive unconditioned stimulus is significantly correlated with passive avoidance behavior, there is no significant relationship between intensity of the aversive stimulus and amount of VP and/or OT released into the blood, or between reentry latencies and plasma levels of VP and/or OT (Laczi et al., 1984; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a); second, the studies relating CSF levels of VP and/or OT to PA behavior (Laczi et al., 1984; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a) are perhaps best interpreted as indicating that fluctuation in CSF levels of these peptides observed during PA acquisition and retention reflect spillover effects from activated central VP-ergic and OT-ergic circuitry; and third, studies that have assessed levels of VP content in limbic areas implicated in memory processing have demonstrated altered levels of irAVP content in these structures in shocked but not unshocked subjects during PA retention (Laczi et al., 1983a,b).

IV. Vasopressin-Induced Increase in Behavioral Arousal Is Not Essential for Its Effect on Memory Processing

__________________________________________________________________________________________________

A. Introductory Comments Peripherally injected VP, in quantities used in behavioral experiments, produces pressor effects. It has been theorized by Koob and associates (Chapter 6) that the pressor-induced arousal effects of peripherally injected AVP and related peptides account for the retention effects observed in various conditioning tasks. De Wied and colleagues argue that, whether peripherally or centrally injected, AVP influences memory processing by a direct influence on central memory sites. They have used two procedural strategies to demonstrate that the pressor and behavioral arousal effects accompanying peripherally administered vasopressin may contribute, but are not essential to, the ability of the peptide to facilitate memory processing.

130

Barbara B. McEwen

In one experimental strategy, De Wied and colleagues use metabolic derivatives that are virtually devoid of the antidiuretic, pressor, and corticotrophic effects of the parent peptide but nevertheless produce the behavioral effect of the parent peptide. These AVP derivatives include desglycinamide vasopressin (DG-AVP and DG-LVP) and a number of C-terminal metabolic fragments such as [pGlu4,Cys6]AVP(4–8), [Cyt6]AVP(5–8), and [Cyt6]AVP(5–9). The second strategy has been to show that on those occasions when a vasopressin analog does increase behavioral arousal, this arousal effect may influence the short-term memory processes involved in learning but is not essential for the effect of the peptide on long-term memory storage. 1. Strategy 1: Dissociation of the Behavioral and Endocrine Effects of Peripherally Administered Vasopressin—Use of DG-AVP and Other C-Terminal VP Metabolites a. Selected Study: Gaffori and De Wied (1985) Gaffori and De Wied (1985) tested the effects of AVP and DG-AVP on a measure of behavioral arousal (open field behavior) and on acquisition and extinction in a pole-jump active avoidance task performed by male inbred Wistar rats. On day 1 of testing, the rats were placed in the open field for 3 min, and 5 min later were given 10 acquisition trials in the pole-jump shock avoidance task. Acquisition training was continued on days 2 and 3 (10 acquisition trials/day), and followed by 3 days of extinction testing (days 4, 5, and 8; 10 trials/day). Depending on the group, the subjects received a single subcutaneous injection of AVP (3 g/rat), DG-AVP (3 g/rat), or an equal volume of physiological saline (saline) either 15 or 60 min before the open field test on day 1 of training (i.e., 20 or 65 min before the first trial of avoidance training). When injected 15 min before testing, AVP but not DG-AVP significantly influenced open field behavior whereas neither peptide affected this behavior when injected 60 min before testing. The AVP-induced modifications in open field behavior patterns indicated that except for locomotor/rearing activity in the center of the field, which was significantly increased by AVP, all other field behaviors (grooming, fecal boluses, and locomotor/rearing activity in the arena periphery) were significantly decreased by the peptide. Subjects treated with AVP 20 min before the first acquisition trial made significantly more avoidance responses on day 2 of training compared with saline-treated controls. This did not occur if AVP was given 60 min before training, nor did it occur after DG-AVP treatment. Regardless of the time of injection, AVP and DG-AVP each exerted a long-term effect on maintenance of the conditioned avoidance response (i.e., significantly increased resistance to extinction on days 5 and 8). In discussing these results the authors noted that (1) although a peripheral injection of 3 g of AVP altered open field activity 15 min after

The Roles of Vasopressin and Oxytocin in Memory Processing

131

treatment, it is not clear that this can be interpreted as an increase in behavioral arousal. Thus, an increase in locomotor activity in the center of the arena combined with an overall decrease in grooming behavior and defecation is generally interpreted as indicative of decreased emotionality (Gispen and Isaacson, 1981; Hall, 1934; Whimbey and Denenberg, 1967); (2) the decreased overall level of general activity could have been related to an increase in blood pressure (BP) and bradycardia, which have been observed 10 and 30 min after AVP injection (e.g., De Wied et al., 1984a), but these physiological measures were not taken in this study; (3) it is also possible that the AVP-induced changes in physiology that contributed to the open field behavior were also responsible for the improved learning behavior of the subjects treated with AVP 20 min before day 1 of acquisition behavior; (4) however, the observation that resistance to extinction was equally effective in subjects treated with AVP 65 min before training (when the effects of AVP on open field behavior had disappeared), or with DG-AVP (which lacks the endocrinological effects of AVP), indicates that any arousal influence of this peptide is independent of its long-term effect on behavior. The authors, concluded that ‘‘   although peripheral effects of AVP may contribute to its memory-modulating effects, such peripheral effects are not essential’’ (Gaffori and De Wied, 1985, p. 443). DG-LVP-induced facilitation of memory consolidation has also been observed in an active avoidance task by De Wied et al. (1972) and in a sexually rewarded appetitive task by Bohus (1977; see Chapter 2). In addition, several studies have provided data on the comparative memory-facilitating effectiveness of AVP(1–9) and other C-terminal metabolites in active and passive conditioned avoidance paradigms. Examples of these findings include the following: (1) De Wied et al. (1987) administered C-terminal fragments that were more potent than AVP(1–9) in facilitating passive avoidance memory consolidation and retrieval, memory consolidation (posttraining peptide effect on response extinction) in a conditioned polejump avoidance task, and memory retrieval (i.e., ability to inhibit experimentally induced retrograde amnesia); (2) Gaffori and De Wied (1986) observed that peripherally injected C-terminal fragments were more potent than AVP(1–9) in their ability to facilitate PA memory consolidation and retrieval; (3) Kovacs et al. (1986; see Chapter 5) observed that C-terminal fragments are more potent than AVP(1–9) in facilitating PA memory consolidation, whether injected peripherally, intracerebroventricularly, or intracerebrally into selective limbic system brain sites; (4) Burbach et al. (1983a; see Chapter 5) demonstrated that when intracerebroventricularly administered, C-terminal fragments [pGlu4,Cyt6]AVP(4–9) and [pGlu4,Cyt6]AVP(4–8) were far more potent than the parent peptide in facilitating PA memory consolidation and retrieval. Further, unlike AVP(1–9), these fragments showed no pressor activity when peripherally administered (intravenously)

132

Barbara B. McEwen

over the entire dose range tested; and (5) De Wied et al. (1984a) reported a similar high degree of behavioral potency when the C-terminal fragment [pGlu4,Cyt6]AVP(4–8) was peripherally administered in a test of PA memory consolidation. 2. Strategy 2: Evidence That the Arousal Effect of Peripherally Injected VP Is Not Essential for Its Influence on Memory Storage a. Selected Study: Skopkova et al. (1991) Skopkova et al. (1991) investigated whether the long-term behavioral effects of DG-AVP are the result of an initial increase in behavioral arousal during the learning phase. The subjects, male Wistar rats of a genetically nonselected strain, were rated as high or low in nonspecific excitability on the basis of exploratory activity displayed in an open field test. The subjects received 3 days of acquisition training in a shuttlebox avoidance task (10 trials/day), followed by 2 days of extinction testing (10 trials/day). A single injection of placebo or DGAVP was administered subcutaneously 40 min before the first acquisition trial at a dose of either 0.1, 0.3, or 1.0 g/rat. The low and high doses of DGAVP were given to manipulate the arousal level of the subject during learning. The inference that DG-AVP influences arousal level was based on a previous observation that it stimulated exploratory behavior in an open field test (Skopkova et al., 1987). Results indicated that the major peptide effects on performance occurred on the first day of acquisition and on both days of extinction, and involved both the lowest (0.1 g) and highest (1.0 g) dose levels of the peptide. Of interest to this discussion were the interactional effects between these dose levels and performance of the high- and low-activity subjects. Acquisition of the shuttlebox avoidance response was facilitated in low-activity rats given the lowest dose of DG-AVP and impaired in high-activity rats given the highest dose of the peptide. During extinction, however, the peptide produced a dose-dependent facilitation of retention in both high- and low-activity rats. Moreover, the 0.1-g dose of DG-AVP, which had facilitated acquisition in the low-activity subjects, resulted in faster extinction in these subjects than in the highactivity group, indicating that the short-acting arousal effect of the peptide on acquisition behavior did not exert a long-term influence on this behavior. Interpretation of these results was based on proposals that arousal level is an important aspect of learning, and is related to performance efficiency by an inverted U-shaped function. Accordingly, it was theorized that in low-activity subjects the low dose of DG-AVP increased arousal to a level that greatly facilitated learning performance, whereas in high-activity subjects the high dose of the peptide increased arousal to a level that impeded it. The observations on extinction (retention) behavior were interpreted as indicating that the direct influence of the peptide on long-term memory was independent of its short-term arousal effect.

The Roles of Vasopressin and Oxytocin in Memory Processing

133

V. Peripherally Administered Neurohypophysial Peptides and Central Memory Processing

________________________________________________

A. Does Peripherally Injected VP or OT Reach Central Memory-Processing Sites? An important question in behavior pharmacology is whether peripherally injected peptides such as VP and OT can produce centrally mediated behavioral effects, given a blood–brain barrier (BBB) that presumably prevents these peptides from entering the brain. The BBB issue is discussed in detail in Chapter 14. At this point we can note two opposing theoretical interpretations of the means by which peripherally administered vasopressin influences memory storage and retrieval. De Wied and colleagues argue that some tiny amount of the pharmacological dose of the parent peptide, or one of its behaviorally active metabolic fragments, enters the brain and directly activates central vasopressin receptors that mediate the influence of the peptide on memory storage and retrieval. In contrast, Koob and colleagues argue against the likelihood that vasopressin can penetrate the BBB and suggest instead that the pressor response induced by the increased level of the peripherally circulating peptide raises the subject’s behavioral arousal, which is then responsible for the observed effect on retention behavior (this view is discussed in Chapter 6). De Wied and colleagues cite neuroanatomical observations as well as behavioral findings from several of their studies in support of their theoretical position, as described below. De Wied and colleagues have presented several lines of evidence consistent with the view that peripherally administered neurohypophysial peptides modulate memory storage and retrieval by a direct influence on central memory-processing sites. First, for a given effect on retention much smaller doses of the neurohypophysial peptides are required when injected intracerebrally than peripherally (De Wied, 1976; Kovacs et al., 1979a,b, 1986). Microgram dose levels are required for peripheral injection (e.g., Ader and De Wied, 1972; Bohus et al., 1972; De Wied et al., 1984a), nanogram amounts are needed for intracerebroventricular injections (e.g., Bohus et al., 1978b; Kovacs et al., 1978), and picogram amounts are required when directly microinjected into specific brain sites (e.g., Kovacs et al., 1979a). Second, De Wied and colleagues have consistently demonstrated that the endocrine-related (pressor/aversive) functions of peripherally injected vasopressin are not required for its effects on retention, because metabolic derivatives lacking these functions nevertheless facilitate memory storage and/or retrieval in appetitive (Bohus, 1977; Vawter et al., 1997) as well as in active (De Wied et al., 1972; Gaffori and De Wied, 1985; Skopkova et al., 1991) and passive (e.g., De Wied et al., 1991) avoidance paradigms. A third line of experimental support derives from studies that have utilized vasopressin agonists and antagonists to test the proposition that

134

Barbara B. McEwen

peripheral VP directly influences central VP receptors that mediate its effect on memory processing. Two of these studies are described below. The first (De Wied et al., 1984a) used agonist–antagonist interactions to investigate whether peripheral VP receptors mediate the pressor but not the behavioral actions of the peptide. The second (De Wied et al., 1991) used agonist– antagonist interactions to further characterize the central VP receptor and to distinguish it from peripheral V1,V2, and OT receptors. 1. Selected Study: De Wied et al. (1984a) De Wied et al. (1984a) conducted a study to differentiate between pressor and behavioral effects induced by peripherally administered VP. The study tested the interaction between the potent VP antagonist d(CH2)5[Tyr (Me)]AVP and the parent peptide AVP(1–9) and between the antagonist and the metabolic fragment [pGlu4,Cyt6]AVP(4–8) on both blood pressure and passive avoidance retention. Results indicated that peripherally administered AVP(1–9) produced a pressor effect and facilitated PA retention whereas the VP fragment produced only the PA retention effect. When centrally (intracerebroventricularly) administered, both the parent peptide and the VP fragment facilitated PA retention whereas neither increased blood pressure. When peripherally administered, the antagonist blocked both the pressor and the retention effect of peripherally administered AVP(1–9) as well as the retention effect of peripherally administered VP fragment. When centrally administered, the VP antagonist blocked the retention effect of the centrally administered peptides and, in addition, the retention but not the pressor effect of peripherally administered AVP(1–9). The results were interpreted as follows. First, the relatively large dose of VP antagonist used for peripheral administration (3 g/rat, subcutaneous) penetrated the BBB sufficiently to influence the central VP receptor, which is structurally similar to the peripheral VP receptor. Second, the very small dose of VP antagonist used for central injection (3 ng/rat, intracerebroventricular), although large enough to diffuse from the ventricle to central VP memory receptor sites, was not sufficient to reach the peripheral VP pressor receptors. Thus the centrally injected antagonist was able to block the behavioral but not the pressor effects exerted by peripherally administered AVP. It was concluded that the receptors mediating the behavioral effects of peripherally administered VP are in the brain, and those involved in the pressor response are in the periphery.

VI. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Continued

____________________________________________________________________________

This section continues the discussion of the theoretical propositions codifying the views of De Wied and colleagues on the roles of vasopressin and oxytocin in memory processing. The studies described in this chapter

The Roles of Vasopressin and Oxytocin in Memory Processing

135

provided findings relevant to propositions 1 and 3, delineated in Chapter 2, and to propositions 4, 5, and 6 which are added here.

A. Proposition 1: VP Facilitates Memory Consolidation and Retrieval The study of subjects deficient in endogenous vasopressin (e.g., Brattleboro rats and normal rats injected with anti-VP serum) is especially pertinent to the physiological role of vasopressin in memory processing. Preliminary studies by De Wied and colleagues with Brattleboro rats led them to conclude that central levels of endogenous vasopressin directly contribute to memory processing: (1) Brattleboro HODI rats were severely disturbed in long-term retention in a single-trial passive avoidance task (Bohus et al., 1975; De Wied et al., 1975) and in multitrial shuttlebox and pole-jump shock avoidance tasks (Bohus et al., 1975); (2) this disturbance was interpreted as due to a deficiency of VP at central memory-processing sites, and not a secondary result of the diabetes insipidus, because DG-LVP treatment normalized PA retention without correcting the diabetes insipidus (De Wied et al., 1975); (3) the retention impairment was reflected in the absence of a pituitary–adrenal axis stress response evoked by the conditioned stimuli present at the 24-h PA retention test (Bohus et al., 1975); (4) experimental testing for threshold responsiveness to a range of FS intensities ruled out pain sensitivity deficit as a cause for the retention impairment (Bohus et al., 1975; De Wied et al., 1975); (5) moreover, there was no deficiency in the normal pituitary–adrenal axis stress response evoked by the painful FS stimuli (Bohus et al., 1975). Later work with Brattleboro rats by De Wied and associates (De Wied et al., 1988) replicated the earlier-found deficits in retention in the active and passive avoidance tasks and, in addition, found significant impairment in retention of an aversive reversal discrimination problem in a T-maze, appetitive black–white discrimination in a Y-maze, and impairment in both working and reference memory in the hole board food search task. The Brattleboro rat model does not provide a straightforward means by which to learn about the nature of the contribution of VP to memory processing. A number of hormonal and metabolic secondary effects of diabetes insipidus (Bailey and Weiss, 1981) and alterations in temperament, emotion, and arousal level have been observed in these rats (Brito, 1983; Williams et al., 1983a,b). These factors themselves may influence behavioral performance in a variety of learning situations (Bailey and Weiss, 1981). For example, an altered baseline arousal level in HODI rats, inferred from study of their open field behavior (Bohus et al., 1975; De Wied et al., 1988; Williams et al., 1983a,b), has been suggested by some researchers as the cause of the impaired retention observed in VP-deficient rats (e.g., Herman et al., 1986b). Although De Wied and colleagues accept that an altered

136

Barbara B. McEwen

arousal level may disrupt attention and the short-term memory processing involved in a variety of cognitive tasks, they maintain that the vasopressin deficiency at central memory-processing sites is responsible for the memory storage deficits that they have consistently observed in HODI rats (De Wied et al., 1988).

B. Proposition 3: VP and OT Have No Major Role in the Learning Phase of Memory Processing De Wied and colleagues have reported that contrary to the memory deficit, Brattleboro rats do not exhibit persistent impairment in the attentional and short-term memory processes required for successful learning. Their early research showed that Brattleboro HODI rats successfully learned the PA response, because testing immediately after the learning trial indicated reentry latencies at near maximal value. When tested 3 h later, the reentry latencies, although shorter, were still significantly longer than those of nonshocked controls (Bohus et al., 1975; De Wied et al., 1975). Moreover, corticosterone activity, interpreted as a stress response to the conditioned fear stimuli, was positively correlated with the reentry latency data (Bohus et al., 1975). HODI rats also attained the designated learning criterion during the acquisition period in multitrial shuttlebox and polejump shock avoidance tasks, although at a slower rate than normal Wistar rats (Bohus et al., 1975). They also exhibited normal learning in the original and reversed discrimination of an aversive T-maze task (De Wied et al., 1988). However, De Wied and colleagues have observed significant impairment in HODI and HEDI rats in acquisition of an appetitive black–white discrimination, the pole-jump task, and in working memory in a hole board food search task (De Wied et al., 1988). Because arousal effects associated with exogenous AVP may influence learning behavior (e.g., Gaffori and De Wied, 1985; Skopkova et al., 1991), the alteration in arousal level inferred from open field test behavior in Brattleboro DI rats (De Wied et al., 1988; Herman et al., 1986b) may be the basis for the learning deficits that are inconsistently observed in these VP-deficient rats.

C. Proposition 4: Central VP-ergic and OT-ergic Circuitry and Not Peripherally Circulating Hormones Are the Primary Means by Which Neurohypophysial Peptides Influence Memory Processing According to proposition 4, feedback from peripherally circulating neurohypophysial peptides does not play an important role in memory storage; rather, it is the central VP-ergic and OT-ergic circuitry that mediates this function.

The Roles of Vasopressin and Oxytocin in Memory Processing

137

Three lines of experimental evidence support this proposition, one of which derives from studies that have neutralized peripheral and central stores of vasopressin. For example, Van Wimersma Greidanus et al. (1975a) observed that an intracerebroventricular injection of anti-VP serum that neutralized central levels of vasopressin significantly impaired passive avoidance memory consolidation, whereas a peripheral injection that neutralized plasma VP levels had no effect. A second line of support derives from the correlation of avoidance retention behavior with endogenous VP or OT localized either peripherally (in the plasma) or centrally (collected from cerebral spinal fluid or selected dissected brain sites). A number of studies have found that plasma AVP levels, assessed after daily learning sessions in a multitrial active avoidance task or after a single passive avoidance learning trial, are related neither to rate of extinction in the active avoidance task (Van Wimersma Greidanus et al., 1979a) nor to reentry latency scores in the passive avoidance task (Laczi et al., 1983c; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a). Moreover, there were no significant differences in postlearning plasma AVP levels between shocked and nonshocked groups in a passive avoidance learning trial (Laczi et al., 1983c). Similar results have been obtained in studies that assessed plasma OT level during passive avoidance learning (Mens et al., 1982a). Because hormonal levels of these neurohypophysial peptides were not differentially increased from control values during the learning trials it is unlikely that hormonal feedback at this time could contribute to memory consolidation. Thus, these findings support the proposition that peripheral levels of these peptides do not contribute to memory storage in these tasks. Studies that assessed CSF levels of AVP or OT after a PA learning trial, and again after retention test trials given 24 and/or 120 h later, suggest that CSF does not transport these peptides from sites of synthesis to sites of activity in the brain. Instead, the peptide levels in CSF represent peptides that have been released in the brain by activated VP-ergic and OT-ergic synapses (Laczi et al., 1984; Mens et al., 1982a). Laczi et al. (1983a,b) interpreted significant changes that occurred in irAVP levels in midbrain–limbic sites in shocked animals in a 24-h PA retention trial as support for putative roles of this VP-ergic circuitry in memory consolidation (septal/hippocampal area) and retrieval (central amygdala). A third line of support for this proposition derives from observations suggesting that peripheral and central vasopressin receptors have separate functions, with the former mediating only the endocrinological effects of the peptide, and the latter only the retention effects. This support includes observations that (1) VP-binding sites are present in memory-processing limbic system structures (Chapter 1); (2) centrally (intracerebroventricularly) injected AVP(1–9), at a dose level that facilitates avoidance retention, lacks the pressor/bradycardial effects characterizing peripherally (subcutaneously) administered AVP(1–9), suggesting that the central receptors that

138

Barbara B. McEwen

mediate the behavioral effects of the peptide are not involved in producing its peripheral pressor effects (De Wied et al., 1984a); (3) subcutaneously or intracerebroventricularly injected C-terminal VP fragments produce potent behavioral but no endocrinological effects, suggesting that these fragments activate central/behavior-mediating, but not peripheral/endocrinologically mediating VP-ergic mechanisms (De Wied et al., 1984a); and (4) a neurohypophysial peptide–receptor complex in the brain differs in structural/ functional character from V1, V2, and OT receptors in the body periphery (De Wied et al., 1991; see Chapter 5). Although the preceding discussion indicates substantial support for proposition 4, there is the puzzling finding that surgical removal of the posterior/intermediate lobes of the pituitary gland (neurohypophysectomy) impairs retention in avoidance paradigms (De Wied, 1965; see Chapter 2). If hormonal vasopressin has no important role in memory processing, why are neurohypophysectomized animals impaired in conditioned avoidance response retention and why is the impairment normalized by vasopressin replacement therapy? One possible answer may come from the observation that this surgery results in substantial structural damage to the hypothalamic nuclei (supraoptic and paraventricular) that produce the neurohypophysial hormones (Moll and De Wied, 1962). Because cells in these nuclei send neural output to extensive brain sites, especially in the brainstem, it is possible that some of these neural outputs serve a direct or indirect role in memory processing. As a result, damage to these hypothalamic nuclei may result in reduced VP transmitter output in this circuitry, which would then be increased by VP treatment.

D. Proposition 5: VP and OT Modulate Memory Processing Directly and Not by an Indirect Influence on Behavioral Arousal The idea that a vasopressin effect on retention is secondary to an influence on the behavioral arousal level of the subject is prominent in the theoretical views of both Koob and colleagues (Chapter 6) and Sahgal and colleagues (Chapter 7). However, early in their behavioral research with neurohypophysial peptides, De Wied and colleagues [De Wied, 1971 (Chapter 2); Van Wimersma Greidanus et al., 1975a] observed that although both vasopressin and ACTH [including ACTH(4–10)] prolonged extinction of a conditioned response, ACTH had only a short-term influence (while the peptide was present in the system) whereas AVP exerted a long-term effect, lasting for days and weeks after AVP treatment was discontinued. These observations led the authors to the tentative conclusion that whereas ACTH and related peptides may influence memory processing via an effect on temporary supportive functions such as behavioral arousal, neurohypophysial peptides were more likely to act on consolidation processes and

The Roles of Vasopressin and Oxytocin in Memory Processing

139

therefore instigate a more permanent change in the central nervous system (also see Van Wimersma Greidanus et al., 1975b; Chapter 4). Subsequent research was designed to respond directly to the challenges offered by arousal-based theories of vasopressin and memory processing. One such line of investigation included studies using vasopressin analogs lacking the pressor effects that, according to Koob and associates, produce the arousal changes responsible for the memory effects of the peripherally administered peptide. Numerous studies have reported that peripheral administration of the peptide analog DG-LVP (DG-AVP), which lacks the endocrine (hence pressor) effects of the parent peptide, can effectively facilitate memory consolidation in an active avoidance task (De Wied et al., 1972; Gaffori and De Wied, 1985; this chapter) and a sexually rewarded appetitive task (Bohus, 1977; see Chapter 2). Moreover, peripherally administered C-terminal fragments, demonstrated to lack pressor effects over a wide range of dose levels (Burbach et al., 1983a; see Chapter 5), have been shown to be far more potent than the parent peptide in facilitating memory consolidation in a pole-jump avoidance task (De Wied et al., 1987; see Chapter 5) and memory consolidation and retrieval in a passive avoidance task [De Wied et al., 1984a, 1987 (Chapter 5); Gaffori and De Wied, 1986 (Chapter 5)], and in protecting against experimentally induced retrograde amnesia (De Wied et al., 1987; see Chapter 5). More recently, Skopkova et al. (1991) showed that although DG-AVP apparently does have arousing properties that can exert an immediate effect on acquisition, its influence on extinction is due to an effect on memory consolidation. Further, because of the virtual lack of pressor effects of this VP analog, the behavioral effect cannot be ascribed to peripheral influences as theorized by Koob and associates.

E. Proposition 6: The Effect of Exogenously Administered VP and OT on Memory Processing Is Due to Action Exerted at Central and Not Peripheral Receptor Sites Proposition 6 represents a second key difference between the theoretical view of De Wied and colleagues and that of Koob and colleagues. According to the De Wied group, when vasopressin (or oxytocin) is peripherally injected (typically with a supraphysiological dose), a sufficient fraction of the injected peptide, or one of its behaviorally active metabolites, is able to cross the blood–brain barrier (BBB), enter the brain, and directly access central receptors involved in memory processing. This proposition is rejected by Koob and associates (Chapter 6), who propose instead that the injected vasopressin acts at peripheral vascular receptors that mediate the memory effect via pressor-induced changes in behavioral arousal. The issue of the ability of the peptide to cross the BBB remains to be settled (see

140

Barbara B. McEwen

Chapter 14), but De Wied and colleagues have offered indirect evidence in support of this proposition. Their argument that only a tiny fraction of the injected peptide needs to penetrate the BBB and reach appropriate brain receptors is supported by data demonstrating that increasingly smaller quantities of the parent peptide [AVP(LVP)(1–9) or OT(1–9)] are required for a retention effect as the treatment route proceeds from peripheral to intracerebroventricular to intracerebral (local brain site) injection. Thus, for a comparable effect on avoidance behavior, a microgram quantity is needed for peripheral injection [e.g., Ader and De Wied, 1972; Bohus et al., 1972 (Chapter 2); De Wied et al., 1984a], a nanogram (0.001 g) quantity is required for intracerebroventricular injection (e.g., Bohus et al., 1978a,b; see Chapter 2), and a picogram (0.001 ng) quantity is needed for intracerebral injection (Bohus et al., 1982; Kovacs et al., 1979a,b; see Chapter 4). Although this proposition admits the possibility that sufficient amounts of behaviorally active metabolites of the parent peptide may cross the BBB into the brain, there has been no direct evidence that the endogenous metabolite fragments observed from in vitro and in vivo studies of the rat brain are also produced in peripheral tissues. However, in a more recent publication, these authors did report evidence of the presence of endogenous DG-AVP in the plasma of Wistar rats (Laczi et al., 1991). Also consistent with this proposition is evidence of dissociation between the peripheral receptors that mediate the endocrine effects of vasopressin and the central receptors that are involved in its memory-processing effects. Thus, as reported in a previous section of this chapter, peripheral injections of DG-AVP and other metabolites lacking the endocrine effects of the parent peptide effectively enhanced memory consolidation for both avoidance (Gaffori and De Wied, 1985) and appetitive (Bohus, 1977; see Chapter 2) learned behaviors, and memory retrieval of a conditioned passive avoidance response (e.g., De Wied et al., 1991). Moreover, findings of studies that employed combinations of peripherally and centrally injected vasopressin agonists and antagonists to produce interactional effects support the theory that a VP receptor localized in the body periphery mediates its effects on blood pressure while a receptor of similar structure, but localized within the brain, mediates its effects on memory storage (De Wied et al., 1984a,b). De Wied and colleagues (1991) have also used agonist–antagonist interactions to further characterize and distinguish central from peripheral neurohypophysial peptide receptors as described in Chapter 5.