Expansion of Vasopressin⧸Oxytocin Memory Research II: Brain Structures and Transmitter Systems Involved in the Influence of Vasopressin and Oxytocin on Memory Processing

Barbara B. McEwen

Expansion of Vasopressin/ Oxytocin Memory Research II: Brain Structures and Transmitter Systems Involved in the Influence of Vasopressin and Oxytocin on Memory Processing

I. Chapter Overview

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This chapter continues the discussion of the eclectic approach to vasopressin/oxytocin (‘‘VP/OT’’) memory-processing research. In contrast with research presented in Chapter 9, the studies described herein directly manipulated central levels of these neuropeptides. Taken together, these investigators used a variety of aversive and appetitive paradigms in studies designed primarily to identify the brain structures in which VP and/or OT acts (Alescio-Lautier et al., 1989; Engelmann et al., 1992a,b; Everts and Koolhaas, 1999; Herman et al., 1991; Ibragimov, 1990; Metzger et al., 1989, 1993), and the neurotransmitters with which it interacts (Baratti et al., 1989; Boccia and Baratti, 2000; Faiman et al., 1987, 1988, 1991; Hamburger-Bar et al., 1984) in its contribution to memory processing. Several of these investigators (Engelmann et al., 1992a; Ermisch et al.,

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1986; Ferguson et al., 2000) also examined the putative role of endogenous VP and/or OT in memory processing.

II. Central Neural Structures Involved in VP and/or OT Effects on Memory Processing

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A. The Hippocampus De Wied and colleagues provided several lines of evidence indicating that a number of limbic system structures are involved in mediating the influence of vasopressin and oxytocin on memory processing in aversive motivational tasks (see Chapter 4). Ibragimov (1990) obtained further information along this line in a study of the effects of chronic intrahippocampal injections of these peptides on memory processing in rats tested in an active avoidance paradigm. Alescio-Lautier, Metzger and colleagues (Alescio-Lautier et al., 1989; Metzger et al., 1989, 1993) have extended this research by studying the role of dorsal or ventral hippocampal VP in memory retrieval, using an appetitive go/no-go brightness discrimination task and an inbred strain of mouse. These studies are described below. 1. Effects of Lysine Vasopressin and OT and Metabolites on Memory Processing in an Aversive Paradigm ",5,0,4,0,105pt,105pt,0,0>a. Selected Study: Ibragimov (1990) Ibragimov (1990) compared the effects of chronic intrahippocampal application of lysine vasopressin (LVP) and OT, and of the vasopressin and oxytocin fragments desglycinamide-arginine vasopressin (DG-AVP) and prolyl-leucyl-glycinamide (PLG), on acquisition and extinction of a shuttlebox footshock active avoidance (AA) response. Sixty minutes before daily test trials, independent groups of adult male CFY rats were treated with physiological saline or a 0.5-, 2.0-, or 4.0-ng dose of LVP or OT, or a 4.0-ng dose of DGAVP or PLG, throughout the 6 days of acquisition and extinction testing in the AA task. With one exception (LVP was intraventricularly administered during extinction testing), all treatments were microinjected into the ventral hippocampus via a preimplanted cannula. The results indicated that relative to saline controls: (1) intrahippocampally applied LVP tended to facilitate acquisition, an effect that was statistically significant for the lowest dose level, and intracerebroventricularly administered LVP severely retarded response extinction, an effect that was significant at all dose levels; (2) intrahippocampally injected OT significantly retarded the formation of the conditioned avoidance response at all dose levels, and showed a nonsignificant acceleration of extinction at the highest (4.0 ng) dose level; and (3) intrahippocampally injected DG-AVP tended to facilitate acquisition and retard extinction of the AA response, whereas

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similarly applied PLG produced the opposite effects. These opposing influences did not attain statistical significance for response acquisition, but did so for response extinction. Taken together, these results suggest that chronic pretesting application of LVP and of the desglycinamide VP analog (DG-AVP) exert similar effects on AA behavior, producing a relatively weak facilitated learning effect but a stronger enhanced retention effect (i.e., greater resistance to extinction). These findings also suggest that both OT(1–9) and its C-terminal peptide PLG act in opposition to the VP family of peptides in their influence on AA behavior, retarding the rate of learning and attenuating retention in this paradigm. The finding that PLG produced an OT-like rather than the VP-like action on memory processing, observed by Gaffori and De Wied (1988), is another example of the puzzling inconsistent effects obtained regarding the role of OT in memory processing. These discrepant findings for OT and memory processing receive further commentary in Chapter 15. 2. Effect of VP on Memory Processing in an Appetitive Paradigm a. Selected Studies i. Alescio-Lautier et al. (1989) Alescio-Lautier et al. (1989) used the go/no-go appetitive visual discrimination task with two major objectives: first, to determine whether hippocampal VP circuitry is involved in mediating retention in this behavioral paradigm; and second, to learn whether this same circuitry contributes to retention effects induced by central administration of exogenous AVP. Mice of the inbred BALB/c strain served as subjects and three experimental tests were conducted, each of which used a retrieval design. The apparatus and behavioral training/testing procedure for this successive go (Sþ), no/go (S) black/white (B/W) discrimination task are described in Chapter 9. Retention performance was evaluated by a discrimination ratio (i.e., the sum of the running times for Sþ divided by the sum of the running times for both Sþ and S trials) calculated for each subject for the retention test session given 24 days after original learning. The smaller the discrimination ratio, the better the retention. The role of endogenous dorsal hippocampal VP in mediating retention of the learned discrimination was tested in four groups of mice. For the two experimental groups, endogenous VP was neutralized by a bilateral dorsal hippocampal injection of anti-VP serum, diluted with artificial cerebrospinal fluid (aCSF) at a ratio of either 1:50 or 1:10. The two control groups received a similarly placed injection of normal rabbit serum diluted with aCSF at the same ratio levels. Two experimental tests were conducted for the second objective, using this same task. First, two independent groups of mice were tested for retention after intracerebroventricular administration of either AVP (2 ng/kg) or

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physiological saline. Second, four independent groups of mice received an intracerebroventricular injection of AVP (2 ng/kg) in addition to either intrahippocampally injected anti-VP serum or normal rabbit serum diluted with aCSF at either a 1:50 or 1:10 ratio. Treatments were administered either 10 min (AVP or saline, intracerebroventricular) or 20 min (anti-VP or normal rabbit serum) before the retention test. The results of this study indicated (1) no impairment in memory retrieval after immunoneutralization of dorsal hippocampal VP whether diluted with a CSF at the 1:50 or 1:10 level; (2) enhanced memory retrieval induced by intracerebroventricularly administered AVP (i.e., mean discrimination ratio in the retention session was significantly smaller for the AVP recipients than for the saline-treated controls); and (3) an impairment of the AVPinduced retrieval effect after treatment with the less diluted (1:10) but not the more diluted (1:50) anti-VP serum solution. The authors noted that the failure of dorsal hippocampal immunoneutralization of vasopressin to impair retention in this task was in sharp contrast to data obtained by Kovacs et al. (1982a; see Chapter 4) and by Veldhuis et al. (1987; see Chapter 4). These latter researchers observed that intrahippocampally injected VP antiserum, at even a weak dilution (1:50), impaired memory consolidation (Kovacs et al., 1982a) and retrieval (Veldhuis et al., 1987) in a step-through passive avoidance task in rats. However, the fact that the earlier studies used footshock stress, absent in this appetitive visual discrimination task, may have contributed to the discrepant findings, because release of hippocampal VP may not have occurred in this study in a degree sufficient to mediate memory retrieval of the learned discrimination. Further, histochemical observation in the present study indicated that the anti-VP serum remained localized in the dorsal hippocampus, whereas Kovacs et al. (1982a) found that the serum had spread from the dorsal hippocampus into the lateral septum and ventral hippocampus. Thus it is possible that the retention impairment after vasopressin immunoneutralization observed by Kovacs et al. (1982a) involved the ventral, as opposed to, or in addition to, the dorsal hippocampus. The finding that memory retrieval was improved by AVP, injected intracerebroventricularly, was consistent with the observation that subcutaneously injected AVP also improved memory retrieval in this task (Alescio-Lautier and Soumireu-Mourat, 1990; see Chapter 9). The fact that intracerebroventricular administration of a 2-ng dose of AVP had the same behavioral effect as a subcutaneous injection at the 1-
g dose level indicates that the peptide was 500 times more effective when administered centrally (intracerebroventricularly) than peripherally (subcutaneously). The increased effectiveness of the peptide when centrally administered has also been reported for rats tested in an aversive motivational task (e.g., De Wied, 1976; and see Chapters 2–5), and has been cited by De Wied and colleagues as support for the ‘‘VP/OT Central Memory Theory.’’

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The observation that intrahippocampal injection of less diluted anti-VP serum (1:10 ratio) blocked the enhanced retrieval produced by intracerebroventricularly injected AVP indicated involvement of dorsal hippocampal VP receptor sites in the retention effect induced by exogenous AVP. This finding is consistent with the demonstration that dorsal hippocampal lesions impair the retention improvement induced by intracerebroventricularly administered AVP in mice (Alescio-Lautier and Soumireu-Mourat, 1986) and rats (Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4). In conclusion, whereas endogenous hippocampal AVP was not necessary for memory retrieval in this task, AVP binding to dorsal hippocampal sites seems to be required for the behavioral effect of intracerebroventricular AVP treatment. That is, the dorsal hippocampus is involved in mediating the effects of exogenous AVP on retention in this appetitive task. ii. Metzger et al. (1989) Metzger et al. (1989) assessed the effect of AVP microinjected into the dorsal or ventral hippocampus on memory retrieval for the successive, go/no-go B/W discrimination task in BALB/c mice. This study used the same task, training and testing procedure, and measure of retention (discrimination ratio) as employed by Alescio-Lautier et al. (1989). Saline or AVP (25 pg/mouse) was bilaterally microinjected into either the dorsal or ventral hippocampus 10 min before the retention test. A putative AVP effect on general activity level was evaluated after completion of retention testing by injecting saline or AVP (25 pg/mouse) into the dorsal or ventral hippocampus 10 min before placing the subject in a circular runway, where locomotor activity was recorded every 10 min for 3 h. Partial forgetting in the retention test occurred in both the VP- and saline-treated subjects, as has been previously observed in this task (e.g., Alescio-Lautier and Soumireu-Mourat, 1986). AVP injections into either the dorsal or ventral hippocampus improved memory retrieval relative to the saline controls. However, peptide placement in the ventral hippocampus led to significantly greater improvement compared with placement in the dorsal hippocampus. In addition, locomotor activity was significantly depressed, relative to saline controls, by AVP microinjected into the ventral, but not the dorsal, hippocampus. These authors noted that the greater effectiveness of AVP when delivered to the ventral rather than the dorsal hippocampus of rats has also been reported by Kovacs et al. (1986) in a passive avoidance paradigm. It was suggested that this may be because AVP-binding sites are more abundant in the ventral hippocampus than in the dorsal hippocampus (Van Leeuwen and Wolters, 1983). This difference in number of VP-binding sites may account for the greater sensitivity (depression) of locomotor behavior to the effects of VP microinjected into the ventral hippocampus as compared with the dorsal hippocampus. The VP-induced alteration in locomotor activity can

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be dissociated from the effect of the peptide on memory retrieval because the improved memory retrieval was exhibited simultaneously as a decrease in S (stimulus not followed by reinforcement) and an increase in Sþ (stimulus signaling availability of reinforcement) running speed. iii. Metzger et al. (1993) Metzger et al. (1993) designed three experiments to study the putative role of ventral hippocampal AVP in the retrieval and relearning of successive (go/no-go) B/W discrimination in BALB/c mice. In experiment 1, either AVP or anti-VP serum was microinjected into the ventral hippocampus (VH) to increase or neutralize, respectively, endogenous VP levels within this brain structure. The effects of these treatments on retrieval and relearning of the visual discrimination were tested shortly thereafter. The remaining two experiments were concerned with the degree to which involvement of the VH vasopressin system in these memoryprocessing activities depends on the integrity of the medial amygdaloid nucleus (AME). In experiment 2 immunohistochemical (IHC) techniques were used to localize the cell bodies in the AME that give rise to the VP-ergic fibers that terminate in the VH. The rationale for this experiment is the evidence that AVP innervation of the VH comes from the AME in rats (Sofroniew, 1985a). In experiment 3, this pathway was lesioned before a microinjection of AVP into the VH, and the effect of the lesion on the ability of the microinjected AVP to influence retrieval and relearning of the B/W discrimination was evaluated. In experiment 1, the procedure used during initial training in this discrimination task was identical to that described by Alescio Lautier et al. (1989). Performance was expressed as the average running time for 6 go (Sþ) and 6 no-go (S) trials for each group in a given session (12 trials). Learning consisted of a decrease in running times for Sþ trials, and an increase for S trials. Retention testing occurred 24 days after the 3 daily sessions (12 trials/session) of acquisition training and consisted of an additional session of 12 trials under the original learning conditions. Surgical implantation of the cannula assembly into the ventral hippocampus (VH) for bilateral injection of the treatment solution was carried out after the completion of original learning or preliminary locomotor activity practice. Ten minutes before the retention test, each of four independent groups of mice received a bilateral intrahippocampal injection of VP (25 pg/rat), saline (Sal), anti-VP serum (1:10 dilution), or normal rabbit serum (NRS). Performance in trial 1 of the retention test was identified as a retrieval effect, and that on subsequent test trials as indicative of the rate of relearning. The learning data (running times for the go and no-go trials) were analyzed by a repeated measures multivariate analysis of variance (MANOVA) that computed the main effect of time (daily session) and its interaction effect with group and reinforcement. Then 4 (groups)  2 (reinforcement or not) ANOVAs for each daily session were computed. These analyses

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assessed the similarity of initial learning across the treatment groups. The retention test data were analyzed by a repeated measures MANOVA to determine the main effect of time (chronological series of trials) and its interaction effect with group and reinforcement. The 4 (groups)  2 (reinforcement or not) ANOVAS for each trial were computed. To test treatment effects on activity level, separate groups of animals were food deprived, placed in the same apparatus (each runway was arbitrarily divided into four squares), and assessed for locomotor activity (running times, number of squares crossed) as well as for grooming, rearing, and defecations. Activity level testing was carried out for three daily 5-min sessions, and 24 days later for an additional 5-min session. Before the 24-day test session, the groups received the same treatments used earlier, thus testing activity level under conditions comparable to those used for visual discrimination training and retention testing. Locomotor activity data (square crossings and rearings) were analyzed by a repeated measures MANOVA, which evaluated across-group changes over the 5-min period. ANOVAs were then computed for defecations, groomings, and running times. Postmortem examinations after the completion of retention testing verified cannula placement in the VH, and showed that the area of diffusion of the injected antiserum in the five test animals remained within the confines of the VH. Results of the various analyses of the learning and retention test data can be summarized as follows: (1) the absence of significant differences in original learning among the subsequently defined treatment groups indicated that learning performance was not an influential factor in the group differences observed in the retention test; (2) both control groups (Sal and NRS treatments) exhibited a substantial amount of spontaneous forgetting (poor retrieval) and moderate relearning; their relearning performance was comparable to that obtained in session 2 of original learning; (3) AVP microinjected into the VH reduced the spontaneous forgetting that normally occurs in this task, that is, the levels of both retrieval and relearning for this treatment group were comparable to those for session 3 of original learning; (4) neutralization of VH vasopressin impaired retrieval and prevented relearning. Thus, mice receiving anti-VP serum microinjections into the VH performed no better in retention performance than they had at the onset (session 1) of original learning, suggesting that they had forgotten the previously learned discrimination; and (5) in contrast, the NRS-treated mice, although exhibiting poor performance in the retrieval trial, did relearn the discrimination over subsequent go/no-go trials, demonstrating some savings. Results of the analysis of the locomotor activity scores in experiment 1 indicated that neither AVP nor anti-VP serum treatment affected locomotor activity (i.e., squares crossed per unit time, rearings, or autonomic

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activity), with the exception that both these treatment groups exhibited increased running times relative to their corresponding controls. The authors attributed this locomotor decrement to an interaction between treatment and the food-deprived/nonrewarded status of the animal, rather than to treatment-induced hypoactivity. This interpretation was consistent with the following: (1) although the subjects were food deprived during performance of both the discrimination task and the locomotor activity test, they were rewarded in accordance with the reinforcement contingency during the former task, but were never rewarded during the latter test; and (2) there were no significant differences between the treatment group and the corresponding controls on the number of squares traversed in the locomotor test. In experiment 2 IHC staining (incubation with VP antiserum) was used postmortem to localize the VP-ergic neuronal cell bodies and fibers in the amygdala–hippocampal pathway that mediated the behavioral performance observed in this study. The brains of naive BALB/c mice from three groups were studied postmortem: (1) group 1 [six normal mice whose brains were studied by IHC staining to locate VP-immunoreactive (VPir) fibers in the VH]; (2) group 2 [2 days before examination of VP neurons in AME, eight mice received an intracerebroventricular injection of colchicine (12, 24, or 48
g) and the remaining four mice received no colchicine pretreatment]; and (3) group 3 (five mice received a unilateral AME lesion 22 days before postmortem staining for VP-ergic fiber endings in the VH). The results of experiment 2 were as follows: (1) in group 1, VPir fibers were detected in the CA4–dentate gyrus region, but were less dense than in the CA1–ventral hippocampal field. Thus, VP fibers from the AME appeared to reach the VH via the amygdalohippocampal area and entered CA1 and CA2 pyramidal cell layers; (2) in group 2, IHC neuronal staining enhancement was best after the 24-
g dose of colchicine, whereas the VP neurons in the brains of non-colchicine-pretreated mice could not be stained; and (3) in group 3, the AME lesions were similar in size to those given to mice in experiment 3. IHC staining for VP indicated an almost complete disappearance of VPir fibers in the CA1 and CA2 fields of the VH ipsilateral to the lesion; however, VPir fibers were intact contralaterally, and also bilaterally, in the CA4–dentate gyrus region. Taken together, these results indicated an ipsilateral VP-ergic projection from the AME that originated in the dorsal portion of the structure, passed through the amygdalo–hippocampal area, and ended in CA1 and CA2 fields and in the ventral subiculum of the VH. However, this VP-ergic input into the VH may not be exclusively from the AME, because the lesion may have destroyed some VP-ergic fibers of passage originating in other brain structures. In summary, the results of experiment 2 indicate that the AME is the source of VP innervation to the CA1–CA2 VH fields and to the ventral subiculum regions of the VH, but not to the CA4–dentate gyrus region of this structure.

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Experiment 3 used the go/no-go visual discrimination task to test the effects of AVP injected into the VH on retrieval and relearning processes of mice previously lesioned in the medial amygdala. The apparatus and procedure for both the visual discrimination task and the locomotor activity test were the same as for experiment 1. AME-lesioned and sham operates were tested in the visual discrimination task. Three treatment subgroups were formed for the AME-lesioned mice, depending on intra-VH injection: noninjected, saline injected, or AVP injected (25 pg bilaterally). First, comparison between the performance of the AME-lesioned group and sham operates indicated significant impairment of retrieval, but not rate of relearning, in the lesioned subjects. Second, comparison between VP- and saline-injected AME-lesioned mice indicated that VP injection into the VH enhanced both retrieval and relearning. Taken together, these two results suggest that VP-ergic projections from the AME to the VH affect retrieval by means of influencing postsynaptic receptors that remain intact after the AME lesion. The fact that the AME lesion did not affect relearning suggests that VP inputs from sites other than the AME, and ending in the CA4– dentate gyrus region (not affected by the AME lesion), may mediate the influence of VP on relearning. The results of the locomotor activity test indicated that the sham operates exhibited greater running times than did the lesioned group, whether treated or nontreated; thus AVP treatment after an AME lesion did not increase running times as it did for the nonlesioned subjects in experiment 1. In summary, the results of this study indicated that (1) increasing VP levels in the VH, by microinjection of the peptide, enhanced both retrieval and relearning of the successive B/W discrimination (experiment 1); (2) decreasing the level of endogenous VP in this structure, by immunoneutralization, markedly impaired both retrieval and relearning in this task (experiment 1); and (3) VP-ergic cell bodies in the medial amygdala project to the CA1–CA2 fields in the ventral hippocampus and contribute to retrieval by means of postsynaptic influence (experiments 2 and 3).

B. The Septal Area 1. Effects of AVP and AVP Receptor Antagonists on Memory Processing in an Active Avoidance Paradigm a. Selected Study: Engelmann et al. (1992a) Engelmann et al. (1992a) investigated the effects of enhancing or reducing intraseptal AVP activity on the acquisition of a pole-jump avoidance response in male Wistar rats. The subjects were trained in a pole-jump avoidance task for 3 successive days (one 30-min session per day, 10 trials/session). In this task, the animals were scored for the number of trials per session in which they successfully performed an avoidance response (completed a pole jump before the end of

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the 8-s auditory conditioned stimulus). Group differences in average number of successful avoidance responses per session were statistically analyzed by a one-way ANOVA followed by the Newman–Keuls test for individual group comparisons. Treatment solutions were infused into the mediolateral septum (MLS) of rats via a microdialysis probe that had been implanted after training session 1 [see Engelmann et al., 1994 (Chapter 13), for a discussion of the microdialysis technique]. A nontreatment group served as a control for the effect of the microdialysis probe on task performance. The rats with probe implants were infused via the MLS with one of the following treatment solutions during training sessions 2 and 3: (1) artificial cerebrospinal fluid on its own (aCSF treatment group), (2) aCSF containing synthetic AVP (approximately 0.2 ng; AVP treatment group), (3) aCSF containing the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (approximately 5.0 ng; V1ant treatment group), or (4) aCSF containing the V1/V2 receptor antagonist [1-(-mercapto-,-pentamethylenepropionic acid)-2-(O-ethyl)-d-tyrosine, 4-valine]arginine vasopressin [d(CH2)5[d-Tyr(Et)]VAVP, approximately 5.0 ng; subsequently referred to as the V1/V2ant treatment group]. The results were as follows: (1) histological study of implant localization carried out at the end of each experiment indicated that the probes had been correctly placed in the MLS; (2) implantation of the microdialysis probe per se did not influence task performance [i.e., there were no significant differences between the aCSF-treated rats and the untreated rats in number of conditioned responses (CRs) during any of the training sessions]; (3) the infusion of AVP into the MLS did not influence the rate of acquisition in this pole-jump test (i.e., the number of CRs per session performed by the AVP-treated rats did not significantly differ from those shown by the aCSFtreated rats); (4) intraseptal infusion of both the V1 and V1/V2 antagonists significantly impaired acquisition of the pole-jump response (i.e., both the V1ant and the V1/V2ant treatment groups exhibited significantly fewer successful CRs per session than did the untreated and aCSF-treated groups during sessions 2 and 3); and (5) the V1/V2 antagonist was equal in potency to the V1 antagonist in its impairment of avoidance learning (there were no significant differences between the two antagonist-treated groups in the number of CRs per session during sessions 2 and 3). In discussing these findings the authors made the following comments: first, increasing intraseptal AVP, by microdialysis of synthetic AVP, beyond that produced by endogenous release in this stressful situation did not significantly influence pole-jump avoidance learning. Bohus et al. (1978b) also found that centrally (intracerebroventricularly) injected AVP failed to influence pole-jump avoidance learning but observed that it did enhance retention in this task. Their findings were interpreted as consistent with the proposal of De Wied and colleagues that AVP does not play an important role in the acquisition phase, but it does in the consolidation phase of

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memory processing (see Chapter 2). However, the findings in the present study suggest that V1-mediated VP-ergic neurotransmission in the MLS does contribute to learning, at least in the pole-jump avoidance paradigm, because blocking this transmission impaired this learning. As an alternative explanation for the failure of centrally administered AVP to enhance avoidance learning, Engelmann et al. (1992a) suggested that the presence of the footshock during acquisition induced the release of central AVP that was sufficient for learning. An increase in this level by treatments such as an intraseptal infusion of AVP may raise it above the optimal value and therefore fail to improve acquisition. This would not pertain during extinction when centrally applied AVP might be expected to improve retention as observed by Bohus et al. (1978b). Second, the present findings could not rule out the possibility that, in addition to a V1 receptor, a V2 receptor is also involved in mediating the septal–AVP influence on active avoidance learning. This latter possibility is consistent with evidence obtained from in vitro studies that, in addition to the V1subtype of septal AVP receptor (Raggenbass et al., 1987; Shewey and Dorsa, 1988), a V2 receptor subtype is present, and may produce a positive feedback action of AVP on its own release (Landgraf et al., 1991a). The authors concluded that their findings support the following hypothesis: ‘‘following its release in the septum, endogenous AVP may be involved in the facilitation of the acquisition and storage of information in stressful situations represented by the acquisition period of pole-jumping behavior in rats’’ (Engelmann et al., 1992a, p. 56). 2. Effects of AVP and/or AVP Receptor Antagonists on Memory Processing in a Spatial Learning Task a. Selected Studies i. Engelmann et al. (1992b) Engelmann et al. (1992b) studied the effects of increasing and decreasing septohippocampal AVP neurotransmission on spatial learning in Long-Evans hooded male rats tested with the Morris water maze (MWM). Treatment solution was delivered into the mediolateral septum (MLS) via previously implanted microdialysis probes. Behavioral testing began 2 days after probe implantation and consisted of 3 days of acquisition training (1 session of 12 trials/day) in the MWM. In addition to an untreated group, three treatment groups received either artificial cerebrospinal fluid alone (aCSF group), aCSF containing AVP (AVP group), or aCSF containing the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (referred to as the AAVP group). The treatment perfusion began 7 min before the first trial and continued at a rate of 3
l/ min throughout the session, resulting in the delivery of about 0.2 ng of AVP or 5.0 ng of AAVP into the MLS over the 30-min perfusion interval. In a given learning trial, the rat was placed in the pool at one of four fixed starting positions and scored for escape latency (time required to reach

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the underwater target platform hidden from view by the opacity of the water). The average escape latency scores of each 12-trial session for each subject were used for statistical evaluation by a two-way ANOVA (treatment  days) with repeated measures on the last factor followed by post hoc paired t tests. At the end of behavioral testing, microdialysis probe location was histologically verified. The ANOVA indicated significant main effects of treatment and days but no significant interaction. Follow-up t tests indicated more specific findings. These statistical comparisons indicated that (1) all groups learned the task as indicated by the significant decrease in escape latency between the first and second, and the second and third, sessions; (2) microdialysis probe infusion per se did not interfere with acquisition of the MWM because the aCSF-infused group learned the maze at the same rate as did the untreated subjects; (3) decreasing septal–VP transmission had no effect on maze learning (i.e., no significant difference between AAVP- and aCSF-treated rats on escape latency within and across training sessions); (4) increasing the level of intraseptal AVP impaired maze performance as indicated by (a) a significantly lengthened escape latency in AVP-infused rats, relative to the other groups tested, during each of the three training sessions; and (b) a significantly slower rate of maze learning by the AVP treatment group relative to the other groups on all 3 days. The present results were discussed in relation to other relevant findings in the research literature. First, failure of the microdialysis technique itself to interfere with normal performance in this spatial learning task has also been observed with an active avoidance task (Engelmann et al., 1992a; this chapter). Second, the inability of the VP receptor antagonist to influence maze acquisition suggests that V1 receptor-mediated VP-ergic transmission in the septal area is not critically involved in spatial memory processing, at least as measured in this task. In contrast, this VP-ergic neurotransmission appears to be important for mediating learning/memory in an active avoidance paradigm (Engelmann et al., 1992a; this chapter) and in a test of social recognition memory (Dantzer et al., 1987; see Chapter 13). Third, the disturbance to spatial learning/memory in the MWM by intraseptally infused AVP has not been observed for other types of memory processing. Intraseptal administration of exogenous AVP did not impair retention of an active avoidance response (Engelmann et al., 1992a), and improved it when tested with a passive avoidance task (Kovacs et al., 1979a; see Chapter 4) and the social recognition memory test (e.g., Dantzer et al., 1988; see Chapter 13). The authors concluded that endogenous AVP (at least that influencing the V1 receptor in the MLS) is not essential for spatial learning in the MWM, whereas the excessive presence of synthetic AVP interferes with it. These findings, together with others noted in their discussion, were interpreted as consistent with other evidence reviewed by O’Keefe and Nadel (1978) in support of the thesis that different brain structures and

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hence neurotransmitter/neuromodulatory systems contribute to spatial and nonspatial learning. ii. Everts and Koolhaas (1999) Everts and Koolhaas (1999) infused a V1/V2 receptor antagonist into the lateral septum (LS) of male Wistar rats to examine the role of septal VP-ergic neurotransmission in spatial memory processing tested in the MWM. The V1/V2 receptor antagonist used in this study, d(CH2)5[d-Tyr(Et)]VAVP, was shown to be as potent as the most commonly used V1 antagonist, d(CH2)5[Tyr(Me)]AVP, in a pole-jump avoidance task by Engelmann et al. (1992a). The former blocks both V1 and V2 types of vasopressin receptor and was selected for study because of the demonstration of the V2 receptor in the hippocampus and other brain sites (Hirasawa et al., 1994; Kato et al., 1995), and the suggestion that it may also be present in the septum (Engelmann et al., 1992a; Landgraf et al., 1991a; Ramirez et al., 1990). Saline or the V2/V1 antagonist (2 ng/
l, enough to ensure a total blockade of both receptor types) was bilaterally infused into the LS via a preimplanted cannula/osmotic minipump assembly throughout behavioral testing. The MWM (a circular polyester pool containing opaque water and divided into four ‘‘imaginary’’ quadrants) was placed in a large observation room provided with a number of extramaze cues. Twelve training trials (3 trials/day; 1-h intertrial interval) were given over 4 days. The animals were allowed 2 min to find the hidden escape platform located in quadrant A. If the platform was not found within this time limit, the rat was placed on the platform for 30 s. During trial 10 the platform was switched to a new location for the rest of the test. The first 9 trials assessed rate of learning the location of the platform relative to extramaze cues, and trials 10–12 assessed memory for its former location and relearning its new one. In each trial the animals were assessed on the following dependent measures: (1) the time taken and distance swum until 2 min had passed; (2) time required to reach the platform (escape latency), and (3) time spent in each of the four quadrants. The results were as follows: (1) both treatment groups reached asymptotic performance on the dependent measures by trial 9, and in trial 10 showed retention of the platform’s original location by their disturbed behavior (increase in travel distance and swimming speed) when it was moved to a new location, which they quickly learned; and (2) statistical testing indicated that the VP antagonist did not impair learning/retention in this task [no significant differences between the two treatment groups on any of the measures of performance (traveled distance, average swimming speed, latency to reach the escape platform, or time spent in any of the four quadrants)]. In discussing their findings the authors noted that whereas research has supported a role for the lateral septum in spatial memory processing

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(M’Harzi and Jarrard, 1992), only more recently has attention been directed to a possible septal VP contribution. These findings are in conformity with those of Engelmann et al. (1992b), who found that microdialysis application of a V1 receptor antagonist into the mediolateral septum during spatial learning in the MWM was without effect. Taken together, this observation and the present results suggest that septal AVP is not causally involved in spatial learning, at least that mediated by either V1 or V2 receptors.

C. The Parvocellular Hypothalamic VP-ergic System 1. Introductory Comments There are two sets of parvicellular (small-sized) VP-ergic neurons in the paraventricular nucleus (PVN) of the hypothalamus, which influence neuroendocrine and autonomic output during stress (these neurons and their projection pathways are described in Chapter 1). One set sends fibers to the median eminence and influences adrenocorticotropic hormone (ACTH) release from the pituitary gland. A second set projects to autonomic nervous system (ANS) centers in the brainstem and spinal cord and influences sympathetic and parasympathetic outflow, especially in connection with cardiovascular regulation. These systems are activated by certain stressors and undoubtedly contribute to neuroendocrine and physiological activities conducive to adaptive responding to these stressors. Feedback from these peripheral activities during stress could influence arousal level and thereby contribute to the learning and memory effects of AVP hormonal treatment, in accordance with theoretical views of Koob and associates (see Chapter 6) and Sahgal and associates (see Chapter 7). However, it is the influence of the extrahypothalamic VP-ergic system, present in the midbrain–limbic structures of the central nervous system, which may be most relevant to the thesis of De Wied et al. (see Chapters 2–5) that AVP exerts a direct influence on memory storage and retrieval, independent of its role in central arousal. Because the parvicellular VP-ergic systems in the PVN and the extrahypothalamic VP-ergic systems are separately localized in the brain, they can be independently manipulated. This strategy was used in the research described below. 2. Selected Study: Herman et al. (1991) Herman et al. (1991) designed a study to determine whether parvicellular VP-ergic neurons in the hypothalamic PVN contribute to memory processing by means of arousal-related behavioral processes. Adult female rats of the selectively bred Roman high-avoidance (RHA) strain were used as subjects to permit comparison with a previous study (Herman et al., 1986b). The parvicellular VP-ergic neurons were selectively destroyed by ibotenic acid (IBO) lesions whereas the adjacent magnicellular VP-ergic

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neurons, involved in fluid and electrolyte balance, were spared. Twelve of the 18 subjects received the IBO lesion; the remaining 6 were given sham operations. After recovery from surgery the subjects were tested for open field behavior, acquisition of a multimodal sensory discrimination, spatial short-term memory, and approach-avoidance behavior. Open field behavior was tested over four consecutive days (1 trial/day) during which the subjects were assessed for number of premarked central and perimeter squares entered, number of rearings, amount of grooming (mild stress), and autonomic signs of stress (number of fecal boli). After open field testing, the subjects were placed on a food-restricted diet; 1 week later they were adapted for 8 days to enter and eat in the accessible goal box of a T-maze apparatus; and then they were successively trained and tested in this apparatus using paradigms designed to test sensory discrimination learning, short-term spatial memory in a delayed nonmatching-to-sample paradigm, and approach-avoidance behavior. The discriminant stimulus for the sensory discrimination task was a plastic grid floor insert that provided tactile, visual, and perhaps olfactory cues for the subject; depending on the trial, it was placed over the pressboard floor of one or the other goal box. The number of correct choices on each of 4 training days (12 trials/day) was tabulated for each subject. Training in the delayed nonmatching-to-sample (DNMS) paradigm began on the day after completion of sensory discrimination testing. For this task, six trials (each trial consisted of an information run followed 20 s later by the choice run) were given each day for 10 days. During the information run (the sample) one door was open to permit entry into a goal box where food was available. During the choice run, both doors were open but food was available only in the goal box not containing it during the information run. For analysis the data were grouped in blocks of 12 trials. The number of correct choices per 12-trial block (session), and the number of sessions to reach the accuracy criterion (10 correct responses out of any 12 consecutive trials), were tabulated for each subject. Training in the approach-avoidance task began on the day after completion of the DNMS task. For this task, the pressboard inserts were removed to expose the metal grid floor, enabling delivery of electric shock, and the left goal box was closed off throughout testing. During the approach phase (six trials/day for 3 days), the subjects were trained to run rapidly and consistently to the right goal box and eat the food reward within 15 s, and were doing so by the third day. After the eighteenth trial, an intense shock (1.5 mA) was delivered across the grid floor and the metal food cup for each contact with the food cup during the 60-s confinement in the goal box. During postshock testing, which began 24 h later and lasted for six consecutive days (three trials/day), food was available in the goal box but no shock was given. The subjects were scored for latency to eat in the goal box (up to a default time of 60 s) in the last preshock and first postshock trials, and for

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running speed [(1/latency)  100] in the last preshock trial and during each day of the postshock (avoidance response extinction) period. The results were as follows: (1) histological examination indicated that 8 of the 12 ibotenic acid-lesioned rats met the criterion for a successful lesion (elimination of the vast majority of identifiable parvicellular PVN neurons while sparing significant numbers of magnicellular neurons). These subjects were included in the behavioral analyses; (2) lesioning affected neither drinking behavior nor body weight maintenance, verifying that hypothalamic magnicellular VP-ergic secretory neurons were spared; (3) total ambulation, ambulation in the centrally located squares, and number of rearings in the open field test were greater in the lesioned mice than in the sham operates; (4) both groups of subjects met the sensory discrimination learning criterion, but the lesioned group exhibited a nonsignificant tendency toward poorer performance (reduced percentage of correct discriminations) relative to the sham operates on each of the daily test sessions; (5) both groups achieved criterion performance in the DNMS task [performed the task at above-chance levels by session 5 (fifth block of 12 trials)], but the lesioned subjects performed more poorly (fewer correct choices per session) than the sham operates in each daily session, a difference that reached statistical significance in sessions 2 and 3; and (6) both groups were similar in the avoidance response exhibited in the first postshock trial (i.e., significant increase in goal approach response latency relative to preshock levels) and in their rates of extinguishing the learned avoidance response. The authors inferred that the lesion reduced fear and cautiousness in the novel open field environment because the greater levels of total and central ambulation and incidence of rearing behavior observed in the lesioned subjects, has been found to be negatively correlated with indices of ‘‘fear’’ in rats (e.g., Archer, 1973). This finding suggests that the parvicellular VP system in the PVN is normally involved in mediating fear, hence an increased arousal level. On the other hand, this system does not appear to play any essential role in memory processing because the lesion did not prevent criterion performance in the sensory discrimination or the DNMS tasks, retention of the shock experience (postshock avoidance responding), or subsequent extinction of the avoidance response. The lesion did slightly retard acquisition of the discrimination and impair short-term memory in the DNMS task, which was interpreted as reduced behavioral efficiency due to a lesion-induced reduction in behavioral arousal. Earlier research by Herman et al. (1986b; see Chapter 3) tested a group of RHA rats genetically deficient in endogenous AVP (i.e., homozygous for diabetes insipidus; RHA-DI) in several of the behavioral tasks used in this study. In comparison with normal RHA rats, RHA-DI rats exhibited behaviors similar to those observed for the lesioned mice in this study (i.e., increased levels of central and peripheral ambulation and incidence of rearing in an open field; good memory for the shock experience in the

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approach-avoidance paradigm and impaired performance in the DNMS task). Because both the RHA-DI rats and the lesioned rats were deficient in parvicellular VP in the PVN, their behavioral commonalities suggest a role for this VP-ergic system in both emotional behavior (open field test) and performance efficiency in cognitive tests (multimodal discrimination learning, spatial short-term memory). In summary, the results of this study are consistent with the hypothesis that the VP-ergic parvicellular system in the hypothalamic PVN influences cognitive performance via an influence on behavioral arousal, an effect that occurs in conjunction with the system’s joint influence on endocrine and autonomic responsiveness to a variety of environmental stressors.

III. VP and/or OT Interaction with Central Neurotransmitter Systems and Memory Processing

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De Wied and colleagues have obtained supportive evidence that the influence of vasopressin on long-term memory is mediated by interaction of the peptide with catecholaminergic (CA-ergic) mechanisms (see Chapter 4). Most of this research has focused on the locus coeruleus–noradrenergic (LC–NA-ergic) projection system, although a few studies included midbrain dopamine (DA) projections. Investigators outside the immediate De Wied et al. sphere of influence extended the research on the DA system, and also initiated study on acetylcholine (ACh) mechanisms in the effect of vasopressin on memory processing, as indicated in the following sections.

A. The Nigrostriatal DA System The possibility that VP released in the caudate nucleus may interact with the striatal DA system in its influence on memory processing was suggested by research observations reported in Chapter 4 (e.g., Kovacs et al., 1977; Van Heuven-Nolsen and Versteeg, 1985). 1. Selected Study: Hamburger-Bar et al. (1984) Hamburger-Bar et al. (1984) pursued this line of inquiry in an investigation of the effect of vasopressin on shuttlebox avoidance learning in DAlesioned male rats. At 5 days of age, rats in the DA lesion group received an intracisternal injection of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), after an intraperitoneal injection of desipramine that spared noradrenergic neurons. This treatment had previously been shown to result in a persistent reduction of brain DA content (70% reduction in the whole brain, 95% reduction in the striatum) (Smith et al., 1973). Rats in the nonlesioned control group were injected with vehicle.

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At 2 months of age, both groups of rats received subcutaneous injections of physiological saline, LVP (1
g/rat), or the VP derivative 1-desamino-8d-arginine vasopressin (DDAVP, 20
g/rat), 1 h before the 15-trial learning session on each of 4 consecutive days of acquisition training. The doses selected for use were based on the 20:1 ratio for behavioral activity of the peptide and its derivative reported by Walter et al. (1978). Extinction testing was carried out on day 7, 3 days after the rats reached a learning criterion of 50% avoidance responding and the last treatment. The authors had previously shown that this VP treatment did not increase open field locomotor activity (Hamburger-Bar et al., 1983). An independent group of DA-lesioned and nonlesioned rats that had been treated with DDAVP or saline and tested as described above were killed 1 month after acquisition training (3 months of age). Brain tissue samples were extracted and tested for AVP levels in the pituitary, hypothalamus, hippocampus, and caudate nucleus. The behavioral results for the nonlesioned rats and the lesioned rats were as follows: (1) the saline-treated nonlesioned group demonstrated better learning performance than did the saline-treated lesioned group [significantly greater number of conditioned avoidance responses (CARs) in the latter group on test days 1 and 2]. However, the lesioned group had ‘‘caught up’’ by the third day of testing and the two groups showed no further significant differences in learning or in subsequent extinction testing; (2) comparisons between the treatments given to the nonlesioned rats indicated that, relative to saline treatment, LVP, but not DDAVP, significantly enhanced learning on test day 4 and retarded extinction (significantly greater number of CARs on day 7); and (3) comparisons between the treatments given to the DA-lesioned rats indicated that relative to saline treatment, LVP and DDAVP significantly enhanced learning on each day of training and retarded extinction. The histological testing indicated that (1) the DA lesions changed VP levels only in the pituitary, where VP content was significantly reduced. DDAVP treatment helped to restore a normal level of pituitary VP, and it was suggested that this effect was probably due to compensatory changes in VP turnover induced by the interference of DDAVP with water metabolism (Boer and Swab, 1983); and (2) the amount of VP in the caudate nucleus was significantly correlated with learning averaged over the 4 days of training in the nonlesioned controls (r ¼ 0.69) as well as in the saline-treated (r ¼ 0.64) and DDAVP-treated (r ¼ 0.60) lesioned rats (correlations between caudate levels of VP and rate of extinction were not tested). The authors interpreted their findings as follows: (1) comparison of the learning/retention performance of the lesioned and nonlesioned rats suggested normal levels of brain DA are important for early acquisition but not for the elaboration and maintenance of memory processing in this task; (2) the effects of LVP on learning/extinction were in accord with the De Wied et al. proposal that whereas VP consistently facilitates memory consolidation and retrieval, it has no important influence on learning except in

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circumstances in which learning is impaired (see Chapters 2 and 3); (3) the failure of DDAVP to influence memory processing in the normal controls may have been due to the weak behavioral efficacy of the drug relative to LVP (i.e., the dose may have been sufficient for enhancing learning in VP-sensitive DA-damaged rats, but not in normal controls); and (4) the correlational data combined with the ability of both VP analogs to enhance memory processing in the DA-lesioned rats suggest that, although VP release in the caudate nucleus influences one or more phases of memory processing in this task, DA is not an essential mediator in this influence. The following commentary on this study seems warranted. First, although these findings indicated that DA had no important role in regulating the release of VP in the caudate nucleus as it did for VP in the pituitary gland, they did not rule out the converse. In fact, evidence that VP is able to modulate the release of DA in the caudate nucleus was reviewed in Chapter 4 (Van Heuven-Nolsen and Versteeg, 1985; Versteeg et al., 1979). Second, the finding that the DA-lesioned rats given LVP treatment showed enhanced retention relative to those injected with saline could signify that VP increased DA release from whatever DA fibers remained intact in the nigrostriatal projection system after the lesion. However, it could also signify that the LVP treatment enhanced retention by means other than a VP–DA interaction, for example, by a VP–NA interaction (Kovacs et al., 1979b; see Chapter 4) or by a VP–ACh interaction (Baratti et al., 1989; Faiman et al., 1987, 1988, discussed below). Third, the absence of a significant difference in retention between the saline-injected lesioned versus saline-injected nonlesioned rats suggests that DA was not importantly involved in memory consolidation as it was during early acquisition in this paradigm.

B. The Cholinergic System 1. VP–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm a. Introductory Comments Faiman and colleagues (1987) noted that cholinergic mechanisms have been implicated in the modulation of memory processing (Flood et al., 1981), that cholinergic input influences the release of hormonal vasopressin (Hatton and Mason, 1985), and that before 1987 there were no studies examining a potential interaction between VP and ACh mechanisms in memory processing. Accordingly, beginning in 1987, these researchers (Baratti et al., 1989; Faiman et al., 1987, 1988, 1991) carried out a number of studies designed to investigate this possibility. In these studies, the experimental protocols examined VP–cholinergic interactional effects using peripherally administered vasopressin, and cholinergic agonists and/or antagonists for the two major types of cholinergic receptors: muscarinic and nicotinic receptors. The subjects, adult male Swiss mice,

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were trained in a single-trial passive (inhibitory) avoidance task with footshock as the negative reinforcer and tested for reentry latency 48 h after the posttraining treatment. The VP–ACh aspects of these studies are described below; other aspects of these studies were reviewed in Chapter 9. b. Selected Studies i. Faiman et al. (1987) Faiman et al. (1987) formed 10 independent treatment groups that received posttraining subcutaneous injections of either physiological saline, LVP (0.03
g/kg), 1 of 4 cholinergic antagonists [methylatropine (0.5 mg/kg, peripheral muscarinic antagonist), atropine (0.5 mg/kg, central muscarinic antagonist), hexamethonium (5 mg/kg, peripheral nicotinic antagonist), mecamylamine (5 mg/kg, central nicotinic antagonist)], or each of these cholinergic antagonists combined with LVP. The subjects were tested for reentry latency 48 h after the inhibitory learning trial and the posttraining treatment. The results indicated that LVP, on its own, facilitated memory consolidation (i.e., significantly prolonged reentry latency relative to saline controls), whereas none of the cholinergic antagonists given alone did so. When coinjected with LVP, only the central nicotinic receptor antagonist mecamylamine prevented the LVP-induced facilitation of memory consolidation. That mecamylamine (the central nicotinic receptor antagonist) but not hexamethonium (the peripheral nicotinic receptor antagonist) prevented the LVP-induced facilitation of retention indicates that the interactional effect occurred at a central and not a peripheral level. However, the specific nature of this vasopressin–cholinergic interaction was not clear from the results. The authors noted that: either (1) the nicotinic receptor antagonist interacted with some central effect produced by the pressor or aversive properties of peripherally acting LVP, which itself was unable to cross the blood–brain barrier (BBB) and directly interact with central VP-ergic systems (Ermisch et al., 1985a; and see Chapter 14); or (2) LVP did reach central VP receptors, as surmised by De Wied and colleagues (1984a, 1991; see Chapter 5), and the antagonist interacted with these centrally activated VP-ergic systems. ii. Faiman et al. (1988) Faiman et al. (1988) examined a potential vasopressin–cholinergic interaction in memory retrieval in BALB/c mice tested in the passive (inhibitory) avoidance paradigm (see Chapter 9). This study employed the same four cholinergic blockers and dose levels used by Faiman et al. (1987). Independent groups of mice were subcutaneously injected with either saline, LVP (0.03
g/kg), one of the four cholinergic blockers, or LVP combined with each cholinergic antagonist. The injection was administered 20 min before the 48-h retention test. LVP facilitated memory retrieval (prolonged reentry latency) in this task. At the dose levels used, none of the cholinergic antagonists, injected alone, was effective.

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When coinjected with LVP only the central nicotinic antagonist mecamylamine prevented the LVP-induced facilitation of memory retrieval. These findings complement those obtained in the earlier study (Faiman et al., 1987), demonstrating that cholinergic–vasopressinergic interactions operate at the central rather than the peripheral level in mediation by the peptide of retrieval as well as consolidation of memory for the single-trial experience in this inhibitory avoidance paradigm. iii. Baratti et al. (1989) Baratti et al. (1989) designed a study to investigate the ability of an osmotic stimulus to influence retention in an inhibitory avoidance task and, if so, to determine whether cholinergic blocking agents can antagonize this effect. The osmotic stimulus, intraperitoneal injection of hypertonic saline, had previously been shown to release endogenous hormonal and central vasopressin in rats [Koob et al., 1985a (see Chapter 6); and see Lebrun et al., 1987]. An initial experiment tested the effects of various doses of hypertonic saline on retention in shocked and unshocked mice tested in this paradigm. Four treatment groups received a subcutaneous injection of physiological saline immediately after the training trial and 10 min later, depending on the group, an intraperitoneal injection of physiological saline (controls) or hypertonic saline (0.25, 0.50, or 1.00 M NaCl). Reentry latency was tested in the apparatus, under nonshock conditions, 48 h later. The results indicated that for the shocked mice, hypertonic saline, at a dose of 1.00 M, enhanced retention (prolonged reentry latency) relative to the saline controls. A subsequent experiment tested the effects of each of four cholinergic antagonists on the enhanced retention induced by 1.00 M NaCl. The cholinergic receptor antagonists and dose levels used in this study were the same as those previously used by these researchers (Faiman et al., 1987, 1988). Independent groups of mice received an intraperitoneal injection of hypertonic saline (1.00 M NaCl solution) 10 min after a subcutaneous injection of physiological saline, or 10 min after a subcutaneous injection of each of the four cholinergic blocking agents. Given alone, the osmotic stimulus released sufficient endogenous vasopressin to facilitate memory consolidation, as it had done in the initial experiment. This memory enhancement effect remained intact when the osmotic stimulus was combined with three of the four cholinergic antagonists; it was reversed only by mecamylamine, the centrally acting cholinergic nicotinic receptor antagonist. The authors interpreted these results as suggesting that (1) endogenous vasopressin released by the osmotic stimulus enhanced retention of the learning trial and (2) this vasopressin was localized at central receptors that interacted with cholinergic mechanisms contributing to memory formation in this task. These results, together with the observation that vasopressin may act as a modulator for catecholaminergic transmission in this task (Versteeg and Van Heuven-Nolsen, 1984; and see Chapter 4,

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Section III), are consistent with the proposal that central vasopressin might interact with both cholinergic and catecholaminergic mechanisms during memory processing and require further study. iv. Faiman et al. (1991) Faiman et al. (1991) designed several experiments to further clarify the nature of the interaction between vasopressin and cholinergic mechanisms in memory consolidation of inhibitory (passive) avoidance responding in male Swiss mice. The rationale for these experiments was based on the proposition that if peripherally administered vasopressin influences memory consolidation by activating nicotinic cholinergic mechanisms, then the following experimental outcomes should be expected: (1) a peripherally administered nicotinic receptor agonist (nicotine) should exert the same effect as LVP on inhibitory avoidance retention (experiments 1 and 2); (2) a nicotinic-induced influence on retention should be blocked by a nicotinic receptor antagonist (experiment 3); (3) posttraining treatment with LVP and a nicotinic agonist should produce similar time gradient effects on retention (experiment 4); (4) subthreshold doses of LVP and a nicotinic agonist should have additive effects on retention (experiment 5); and (5) LVP-induced retention effects should be blocked by pretreatment with either a V1 receptor antagonist or a nicotinic receptor blocker, whereas a nicotinic-induced retention effect should be blocked by a nicotinic receptor blocker but not by a V1 receptor antagonist (experiment 6). Experiments 1 and 2 tested retention effects induced by immediate posttraining subcutaneous injections of physiological saline or various doses of LVP (0.003–1.00
g/kg, experiment 1) or of nicotine (1.00–30.00
g/kg, experiment 2). These treatments were given to mice that received a footshock, as well as to nonshocked mice, to test for possible nonspecific effects of the drugs. LVP and nicotine, when given to the shocked mice, produced similar inverted U-shaped dose–response curves for retention of the passive avoidance (PA) task. Thus, a posttraining injection of a midrange dose of LVP or of a central nicotinic receptor agonist (nicotine) significantly facilitated PA retention (i.e., prolonged reentry latencies) whereas higher and lower levels had no significant effect on these scores. These treatments did not affect posttraining reentry latency for the nonshocked subjects, indicating an absence of nonspecific pharmacological effects on this behavior. Experiment 3 demonstrated that nicotinic cholinergic mechanisms were involved in memory processing in this avoidance task paradigm. Different groups of mice received an immediate posttraining subcutaneous injection of physiological saline, nicotine (10.0
g/kg), a cholinergic antagonist [i.e., atropine (0.5 mg/kg), methylatropine (0.5 mg/kg), hexamethonium (5.0 mg/kg), or mecamylamine (5.0 mg/kg)], or each of the cholinergic antagonists plus nicotine (10.0
g/kg) as a single injection. Nicotine enhanced memory consolidation, but none of the anticholinergic agents

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influenced retention when injected alone. When combined with nicotine, the central nicotinic receptor antagonist mecamylamine prevented the nicotinic retention effect. However, neither the nicotinic receptor antagonist hexamethonium, which crosses the blood–brain barrier poorly, nor the muscarinic receptor antagonists atropine and methylatropine, prevented the influence of nicotine on retention. Time-dependent effects were tested in experiment 4. Separate groups of mice received posttraining physiological saline, LVP (0.03,
g/kg, subcutaneous), or nicotine (10.0
g/kg, subcutaneous) either 0, 30, or 180 min after the single training trial. Posttraining injections of either LVP or nicotine prolonged reentry latencies relative to saline controls if given within 30 min, but not when delayed 180 min after training. Thus, similar time gradient effects on retention were found for LVP and a nicotinic agonist. In experiment 5, separate groups of mice were injected subcutaneously with physiological saline, LVP (0.003
g/kg), physostigmine (35.0
g/kg), nicotine (1.5
g/kg), or LVP combined with physostigmine or nicotine as a single injection, immediately after training. Physostigmine, an anticholinesterase, is a cholinergic agonist because it inactivates the enzyme (acetylcholinesterase) that destroys acetycholine. When given alone none of these drugs, at these dose levels, significantly affected reentry latency. However, when given with the ineffective dose of LVP each of the cholinergic agonists, physostigmine and nicotine, significantly increased reentry latency. Thus, combining subthreshold doses of LVP and either cholinergic agonist did produce additive effects on retention. In experiment 6, six independent groups of mice received, immediately posttraining, a subcutaneous injection of physiological saline, LVP (0.03
g/kg), a V1 antagonist (0.01
g/kg), mecamylamine (5.0 mg/kg), or LVP combined with either the V1 antagonist or mecamylamine as a single injection. Six independent groups of mice received the same treatments cited above except that nicotine (10.0
g/kg) instead of LVP was tested. When injected alone, neither the V1 antagonist nor mecamylamine influenced reentry latency. The retention effect of posttraining LVP was prevented when it was combined with either the V1 antagonist or the cholinergic blocker mecamylamine. On the other hand, the prolonged reentry latency produced by nicotine when given alone was prevented by mecamylamine but not by the V1 antagonist. The fact that mecamylamine by itself, at the dose level used, did not affect retention but blocked the LVP-induced facilitation of retention, indicated that the antagonism between LVP and the nicotinic receptor blocker was not due to a nicotinic blockade of the release of endogenous vasopressin. The finding that mecamylamine at a higher dose level (10 mg/kg, subcutaneous) impaired retention (Faiman, 1990, as cited in Faiman et al., 1991) is consistent with the participation of nicotinic cholinergic receptors in memory modulation.

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The authors concluded that a cholinergic system is involved in inhibitory avoidance retention and suggested that the enhanced consolidation induced by vasopressin treatment was due, at least in part, to action of the peptide on central nicotinic cholinergic mechanisms important to memory formation. 2. OT–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm a. Introductory Comments Two separate lines of evidence stimulated the study presented below. First, numerous findings have been consistent with the proposition that, whereas VP and VP fragments enhance memory consolidation and retrieval in appetitive and avoidance learning paradigms, OT tends to attenuate this memory processing (see Chapters 2–5). Two studies by Boccia, Baratti, and colleagues, using mice, confirmed the proposed OT amnestic role in memory processing. Boccia and Baratti (1999) showed that OT impaired retention of a ‘‘nose poke’’ habitation response. Boccia et al. (1998) observed that when immediately administered after the footshock learning trial, OT impaired, whereas a selective OT receptor antagonist on its own enhanced, retention in this single-trial inhibitory avoidance paradigm. These effects were dose dependent, producing significant effects at midrange dose levels and produced reciprocal U-shaped dose– response curves. Pretreatment with a dose of the OT antagonist, which did not affect retention by itself, prevented the effects of OT on retention. This latter finding, combined with the observation that pretreatment with a V1a vasopressin receptor antagonist failed to influence the effect of OT on retention, led the authors to propose that the OT amnestic effect in this paradigm was probably due to an interaction of OT with a specific OT type of receptor (but see De Wied et al., 1991; Chapter 5). Second, there have been a number of studies indicating that OT, like VP, modulates synaptic transmission in catecholaminergic transmitter pathways implicated in memory processing in limbic subcortical and cortical structures (see Chapter 4). Given the evidence obtained by this research team suggesting that the facilitative role of VP in retention of learned inhibitory behavior may be mediated, at least in part, by its positive modulatory influence on central cholinergic mechanisms involved in memory processing (see above), it was deemed feasible to investigate the possibility that OT might also interact with these cholinergic mechanisms, and if so to characterize the nature of this interaction. b. Selected Study: Boccia and Baratti (2000) Boccia and Baratti (2000) investigated potential interactive effects on memory storage between oxytocin (OT) and an OT receptor antagonist and drugs known to affect the cholinergic system centrally and/or peripherally. Elands et al. (1988b) considers the arginine vasotocin (AVT) analog (CH2)5[Tyr(Me)2,Thr4,

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Tyr(NH2)9]OVT (OVT, ornithine vasotocin) to be a highly potent OT receptor antagonist. It was used as the putative OT receptor antagonist (AOT) in this study. Three major experiments were conducted using independent treatment groups of adult male Swiss mice. In all the experiments with OT or the vasotocin analog AOT, the drug was subcutaneously injected after the training trial and tested for its effects on PA retention 48 h later. The first set of experimental tests were attempts to replicate earlier findings by these investigators concerning the effect of OT, AOT, and their interaction on PA retention (Boccia et al., 1998; see Chapter 9). In this experiment, independent treatment groups of mice were used in each of three experimental tests. The first test examined the effects of OT, AOT, or their interaction on PA retention when treatments were given within 10 min of the training trial. Specifically, each of five pairs of test solutions was subcutaneously injected: the first, immediately after the training trial, the second, 10 min later. Depending on the group, the two sets of injected test solutions were as follows: (1) saline–saline (saline controls), (2) saline–OT (0.10
g/kg); (3) AOT (0.03
g/kg)–saline; (4) AOT (0.30
g/kg)–saline, or (5) AOT (0.03
g/kg)–OT (0.10
g/kg). The second test examined the effects of posttraining subcutaneously injected saline, OT (0.10
g/kg), or AOT (0.30
g/kg) on PA retention when the training-treatment delay was extended to 3 h. The third test examined the possibility that nonspecific proactive effects of these peptides lasted more than 48 h. For this test, three treatment groups were trained under nonshock conditions and subcutaneously injected with saline, OT (0.10
g/kg), or AOT (0.30
g/kg) immediately after the nonshock training trial. The results were as follows: (1) when administered within 10 min of the footshock training trial, OT significantly retarded, and the higher dose of AOT significantly enhanced, PA retention (reduced and increased reentry latencies, respectively) relative to saline controls. Moreover, pretreatment with AOT at a dose (0.03
g/kg) that had no PA retention effect on its own blocked the amnestic effect of OT in this paradigm (reentry latency of OT under this condition did not differ from that of saline controls). This finding confirms the ability of this AVT agonist to serve as a highly effective and perhaps selective OT receptor antagonist (Boccia et al., 1998; Manning and Sawyer, 1993); (2) when administered 3 h after the footshock training trial, neither OT nor its receptor antagonist influenced PA retention (reentry latencies did not significantly differ among OT-, AOT-, and saline-treated groups). Taken together, the findings of the first and second experimental tests are consistent with the idea that these peptides affect a process underlying storage of recently acquired information (McGaugh, 1989); and (3) neither peptide influenced reentry latency when the subjects were trained under nonshock conditions, indicating that nonspecific proactive effects of OT and AOT on retention were not responsible for the results obtained in the first experimental test.

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The second experiment examined the possibility that the amnestic action of OT, observed in the first experiment, might involve an interaction between OT and a peripheral and/or central cholinergic transmitter system. To this end, central (physostigmine) and peripheral (neostigmine) anticholinesterase drugs were tested for their effects on PA behavior and for their ability to reverse the amnestic action of OT on this behavior. Anticholinesterase drugs promote cholinergic activity at postsynaptic sites because they prevent the hydrolysis of acetycholine produced by action of the enzyme acetylcholinesterase (Taylor, 1996). Ten treatment groups were formed. Each of five groups of mice received a posttraining subcutaneous injection of saline followed 10 min later by intraperitoneally injected saline, physostigmine (35, 70, or 150
g/kg), or neostigmine (150
g/kg). Each of five additional treatment groups received a subcutaneous injection of OT (10
g/kg) immediately after the PA training, followed 10 min later by an intraperitoneal injection of saline, physostigmine, or neostigmine at the dose levels designated above. For this second experiment, statistical comparisons indicated that (1) OT (saline–OT group) produced its expected amnestic effect (significantly decreased reentry latencies) in the 48-h PA retention test; (2) the low dose of physostigmine (saline–physostigmine low-dose group) had no effect on retention but produced a partial (not significant) attenuation of the amnestic effect of OT (OT–physostigmine low-dose group); (3) the two higher doses of physostigmine (saline–physostigmine high-dose group) significantly enhanced PA retention, and fully reversed the PA amnestic action of OT when injected 10 min after OT (OT–physostigmine high-dose group); (4) neostigmine had no effect on PA behavior when given alone, nor did it modify the amnestic action of OT when accompanying OT treatment. Taken together, these findings suggest an OT–cholinergic interaction at central but not peripheral cholinergic receptor sites. The third experiment tested the ability of centrally and/or peripherally acting cholinergic receptor antagonists to impair the memory-facilitative effect of AOT on PA behavior observed in the first set of experiments. Depending on the treatment group, the subject received an intraperitoneal injection of saline (controls), atropine (0.5 mg/kg), methylatropine (0.5 mg/kg), mecamylamine (5.0 mg/kg), or hexamethonium (5.0 mg/kg) immediately after the training trial, followed 10 min later by a subcutaneous injection of saline or AOT (0.30
g/kg). These researchers cited evidence indicating that atropine and mecamylamine, but not methylatropine and hexamethonium, can readily cross the blood–brain barrier. Accordingly, the former act at central, and the latter at peripheral, cholinergic receptor sites. Comparisons with the saline control condition indicated that (1) AOT significantly enhanced retention (increased reentry latencies) in the 48-h PA retention test; (2) neither anticholinergic drug on its own, at the dose levels used here, influenced PA retention; (3) pretreatment with the centrally, but

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not the peripherally, acting cholinergic receptor antagonists prevented enhancement by AOT of PA retention. Taken together, the findings that only (1) centrally acting anticholinesterases were able to reverse the OT-induced PA amnestic action (experiment 2) and (2) centrally acting cholinergic antagonists prevented the AOTinduced facilitated memory effect on PA behavior (experiment 3) strongly suggest that an OT–cholinergic interaction contributes to the OT influence in memory processing. It can be concluded that the pharmacological evidence obtained in the second and third experiments is clearly consistent with the authors’ speculation that ‘‘oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response’’ (Boccia and Baratti, 2000, p. 217).

IV. Endogenous AVP and/or OT and Memory Processing

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A. Endogenous AVP and Avoidance Learning 1. Selected Study: Engelmann et al. (1992a) Engelmann et al. (1992a) performed two experiments in their investigation of the effects of increasing and decreasing endogenous levels of peripheral and central AVP on pole-jump footshock avoidance learning. Experiment 1, reported in Section II.B.1, involved pharmacological manipulation of intraseptal AVP neurotransmission during acquisition of the avoidance response. That experiment showed that increasing septal AVP concentration beyond its normal level had no influence on avoidance learning, but that blocking normal VP receptor transmission in this brain site impaired it. In experiment 2, herein reported, central and peripheral levels of endogenous VP were increased by osmotic stimulation (peripherally administered hypertonic saline), and central as well as peripheral VP receptor transmission was blocked by a peripherally administered lipophilic V1 receptor antagonist. The procedure for training and performance evaluation in experiment 2 was the same as described for experiment 1 (Section II.B.1). After completion of the first training session, the subjects tested in this experiment were randomly assigned to one of four treatment groups. The animals in each group received three intraperitoneal injections containing two types of test solutions [the solution mentioned first was injected twice (immediately before the second and third training sessions), the one mentioned second was injected once (immediately after the second training session)]: (1) Iso-Iso

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group [isotonic saline (0.14 M NaCl)–isotonic saline]; (2) V1ant-Hyper group [V1 antagonist (10
g)–hypertonic saline (2.0 M NaCl)]; (3) V1antIso group (V1 antagonist–isotonic saline); and (4) Iso-Hyper group (isotonic saline–hypertonic saline). It was found that all four treatment groups improved their performance in this avoidance learning across the successive training sessions. Statistical analysis indicated no significant differences between the four treatment groups in number of CRs per session performed in any of the training sessions. The authors related these experimental findings to relevant studies conducted by other researchers as well as to those obtained in experiment 1 of this study. It was noted that the failure of peripherally administered hypertonic saline to influence pole-jump acquisition behavior appears to be at variance with findings by Koob et al. (1985a), in which similar treatment facilitated retention (delayed extinction) in the same behavioral task. Because the osmotic stimulus is known to release endogenous AVP in both central and peripheral compartments (Landgraf et al., 1988), the retention effect observed by Koob et al. (1985a) could theoretically have been mediated by activation of AVP in either or both compartments (see Chapter 6). However, they attributed the enhanced retention effect to the pressor-associated arousal action of peripheral AVP, released by the osmotic stimulus. In support of this interpretation was their further observation that pretreatment with a subcutaneously injected V1 antagonist that blocks the peripheral pressor effects of AVP also prevented the memory-enhancing effect of osmotic stimulation (Koob et al., 1985a). However, one major difference between the two studies is that the findings of the present study are relevant to acquisition of the avoidance response, whereas those of Koob et al. (1985a) concerned retention of this learned behavior. The findings of experiment 2 are consistent with a number of experimental findings by De Wied and colleagues, and with their viewpoint that suggests that, although endogenous VP has a definitive role in mediating memory storage and retrieval, it is not important for learning except under special circumstances (see Chapter 2). The findings of experiment 2 are also partly in accord with those obtained in experiment 1 of this study. Thus, increasing the concentration of AVP centrally (experiments 1 and 2) as well as peripherally (experiment 2) did not affect acquisition behavior. Engelmann et al. (1992a) proposed that the failure of intraseptal infusion of AVP to influence avoidance learning (experiment 1) might have been due to an increase in the concentration of septal VP beyond that conducive to learning. This explanation would seem equally applicable to endogenous VP released by the osmotic stimulus. As to the failure of the lipophilic V1 antagonist to influence this behavior, the view held by De Wied and colleagues, that VP is not normally implicated in learning as it is in memory storage and retrieval, could equally

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explain this result. However, the results of experiment 1 suggested the converse, that is, that endogenous VP, at least that present in certain brain sites, does have a role in avoidance learning because interfering with the normal neurotransmission of the peptide impaired this learning (experiment 1). If so, then the failure of the peripherally administered V1 antagonist to impair avoidance learning (experiment 2) might have been due to the fact that the amount of the V1 antagonist that reached central receptors was insufficient to interfere with the VP contribution to this behavior.

B. Endogenous AVP and OT and Memory Processing in an Aversive Paradigm 1. Selected Study: Ermisch et al. (1986) Ermisch et al. (1986) examined endogenous levels of peripheral and central AVP and OT in male Wistar rats selected for high and low learning/ memory performance in a Y-maze aversive brightness discrimination task. These subjects were tested in this footshock avoidance discrimination task during 4 sessions (22 trials/session). The first session was termed the ‘‘training’’ (T) session and the following three were termed ‘‘relearning’’ (R1, R2, and R3) sessions. R1, R2, and R3 occurred 1 day, 5 days, and 6 weeks after T, respectively. The number of errors in the training session (Te) and in the relearning sessions (Re1, Re2, and Re3) were used to calculate the relearning index [i.e., RI ¼ (Te – Re/Te)  100]. The results indicated (1) progressive improvement (reduced errors) over the successive relearning sessions for all tested rats, including those selected from the highest and lowest end of the learning/relearning performance gradient (the high- and low-performance groups); and (2) these two groups significantly differed from each other in terms of RI in each relearning session. Two months after completion of behavioral testing, the high- and lowperformance groups were killed and examined postmortem for endogenous AVP and OT levels in plasma, and in the posterior pituitary and four areas of the brain (the motor cortex, hippocampus, septum/striatum [contained the septal nuclei, bed nucleus of the stria terminalis, caudate nucleus, putamen, and pallidum], and the hypothalamus). The results of this postmortem analysis were as follows: (1) the AVP levels in the septum/striatum and the posterior pituitary of the high-performance group exceeded those of the lowperformance group; (2) relative to the low-performance group, the OT level in the high-performance group was significantly higher in the septum/striatum and lower in the hippocampus; and (3) there were no significant differences between the two groups in OT or VP peptide level in the motor cortex, the hypothalamus, or the plasma. The authors commented on the results of this study as follows: (1) the peptide levels observed at postmortem probably reflected genetically

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determined differences between the two performance groups rather than a differential response to task-related stressful events, because 2 months intervened between the end of behavioral testing and the postmortem analysis; (2) the pattern of high endogenous AVP and OT levels in septum/striatum neurons and low OT levels in hippocampal neurons might be prerequisites for high performance in the brightness discrimination task; and (3) although endogenous AVP and OT are likely involved in memory processing in a brightness discrimination learning task, they are not necessarily similarly involved in other learning tasks. For example, in a preliminary study involving a pole-jump active avoidance paradigm, these authors observed no evidence of the extremely high and low performance scores exhibited in this brightness discrimination test. In addition, the authors noted that a number of other research groups have also attempted to relate high and low performance in learning/memory tasks to differences in VP and/or OT levels in various brain areas. Lecesse (1983) reported that in mice, drugs that enhanced avoidance performance also resulted in increased AVPir levels in the lateral septum and dorsal raphe nucleus. Hamburger-Bar et al. (1984; this chapter) found that in rats, improved learning ability in an active avoidance task was correlated with enhanced levels of AVP in the caudate nucleus. In their studies with rats, discussed in Chapter 3, Laczi and colleagues observed that immediately after the retention trial in a passive avoidance (PA) task, rats that showed good PA behavior also exhibited decreased levels of AVP in the lateral septal nucleus (Laczi et al., 1983a) and dorsal hippocampus (Laczi et al., 1983b). The decreased levels were thought to reflect task-associated increases in AVP release at peptidergic terminals (Laczi et al., 1983a,b, 1984). However, unlike the findings of Lecesse (1983), Hamburger-Bar et al. (1984), and Laczi et al. (1983a,b, 1984), which probably reflected AVP responses to a fearful situation, the findings of this study were thought to reflect differences in ‘‘genetically determined potency of neurons to produce, transport and release AVP and OT’’ (Ermisch et al., 1986, p. 27).

C. Endogenous OT and Spatial Memory 1. Selected Study: Ferguson et al. (2000) Ferguson et al. (2000) compared male mice mutant for the OT gene (OT /) with wild-type mice, which are normal for the OT gene (OT þ/þ) on social recognition memory (SRM). As discussed in Chapter 13, the OT/ group was impaired in SRM. The two groups were also tested on spatial memory to determine whether it is dependent on a normal OT genotype, as had been found for SRM. Separate subgroups were tested in the Morris water maze (MWM), and in a two-trial Y-shaped maze under red light illumination (see Fig. 1).

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FIGURE 1 Performance in spatial memory test. Morris water maze data depict the decline in the mean (1 SEM) distance [(a) effect of repeated testing: F(4, 290) ¼ 26.4, p < 0.05] and latency [(b) effect of repeated testing: F(4, 290) ¼ 20.5, p < 0.05] required for mice mutant for the oxytocin gene (Oxt/; open symbols) and wild-type mice (Oxtþ/þ; solid symbols) to locate a submerged platform averaged over four repeated trials within five successive daily sessions. We detected genotype-dependent differences for latency [F(1, 290) ¼ 4.28, p < 0.05] and swim speed [F(1, 290) ¼ 9.04, p < 0.05], but not for distance traveled. Analysis did not detect interaction effects on any measures of water maze performance. Also portrayed is the amount of time spent in the platform quadrant during a 2-min probe trial on day 6 (c). We did not detect genotype-dependent differences. (d) Performance in the two-trial Y-maze test. We did not detect significant genotype-dependent differences in the behavior of mice during the familiarization trial. During the test trial, subjects showed a preference for exploring the new arm expressed as amount of time (mean  1 SEM) and activity allocated to the new versus two familiar arms during the recall trial [effect of arm preference—duration, F(2, 28) ¼ 6.62, p < 0.05; effect of arm preference-distance traversed, F(2, 28) ¼ 5.87, p < 0.05]. We did not detect significant genotype-dependent differences. *Significant difference between the new arm and arm 1; þsignificant difference between the new arm and arm 2. Source: Ferguson et al., 2000 (Fig. 3, p. 286). Copyright ß 2000 by the Nature Publishing Group. Reprinted with permission.

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Spatial navigation in the MWM was observed over five successive days [4 trials/day with a 10-min intertrial interval (ITI)]. Initial placements in the opaque water varied systematically across the four trials. Time and distance required to locate the submerged platform were recorded as the dependent measures of learning. A 2-min probe trial with no platform present was given on day 6. Although OT / mice swam faster and exhibited shorter latencies than did OT þ/þ mice, the two genotypes were comparable in learning the maze (significant decline in time and distance required to reach the platform over successive trials), and in remembering the location of the platform (both genotypes spent equal amounts of time in the platform quadrant in trial 5). The OT / and OT þ/þ mice tested in the Y-shaped maze (made of clear Plexiglas with guillotine doors isolating each of the three arms) were given two trials separated by a 30-min ITI. During the first trial (familiarization phase), one arm of the Y-shaped maze was closed off, and the mice were placed in one of the two remaining arms and allowed to explore the maze for 5 min. During the second trial (retrieval phase), the door was removed and the mice were allowed free access to all three arms. A significant preference for the novel arm was interpreted as evidence of spatial recognition (Contarino et al., 1999). There was no significant difference between the two genotypes in Y-maze performance, and OT / as well as OT þ/þ mice exhibited spatial recognition (each genotype showed a significant preference for the novel arm relative to each of the two remaining arms). Taken together, the results of this study indicated that mice genetically deficient in OT were not impaired in spatial memory. The authors concluded that their data ‘‘indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory’’ (Ferguson et al., 2000, p. 284). One should not conclude from this study that endogenous OT has a physiological role in modulating only memory underlying SRM. There is evidence that OT is involved in modulating avoidance retention (relevant evidence cited in various chapters of this text, especially in Chapters 2–5). Moreover, the failure of mice lacking endogenous OT to show a specific memory deficit does not preclude the possibility that endogenous OT has a physiological role in modulating that memory system under normal circumstances. A host of other classic neurotransmitters and peptides interact in complex ways to influence spatial memory and may compensate for the absence of OT. Finally, for a number of other reasons caution is warranted in interpreting findings from research using genetic knockout models (see Section II of Chapter 3, and Bohus and De Wied, 1998). Chapter 11 summarizes the research studies presented herein and relates their findings to the theoretical views described in earlier chapters of this text.