Expansion of Olfactory-Based Social Recognition Memory Research: The Roles of Vasopressin and Oxytocin in Social Recognition Memory

Barbara B. McEwen

Expansion of Olfactory-Based Social Recognition Memory Research: The Roles of Vasopressin and Oxytocin in Social Recognition Memory

I. Introductory Remarks

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This chapter updates research on the roles of vasopressin (VP) and oxytocin (OT) in rodent olfactory-based social recognition memory (SRM). As noted in Chapter 12, Dantzer, Bluthe, and colleagues launched this burgeoning field of inquiry in the late 1980s. After a brief description of the test paradigms used to assess SRM, evidence is discussed that confirms and expands the research findings presented in Chapter 12.

II. Test Paradigms for Assessing SRM

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A. Social Recognition Test The conventional paradigm used to test SRM in rats and mice is the social recognition test (SRT) described in Chapter 12. A significant reduction in the duration of social investigatory behavior of a stranger juvenile when it Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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is reintroduced in a second 5-min exposure period after a designated interexposure interval (IEI) is operationally defined as SRM. Control test sessions, in which a novel juvenile is presented after the IEI, are generally included to ensure that any reduced investigative behavior of the same juvenile by the subject is not merely the result of satiation, fatigue, or nonspecific drug effects. Preliminary testing in this paradigm is typically included for the purpose of subject selection and can be illustrated by reference to a study by Sekiguchi et al. (1991a): the adult rat was presented with the juvenile twice daily for 5 days. The IEI on days 1 and 2 was 5 min, on days 3 and 4 it was 30 min, and on day 5 it was 120 min. The same juvenile was presented in the second presentation trial, except for day 4, when a different juvenile was used. Only the animals that reliably investigated the juveniles and did not display aggressive or sexual behavior toward them were used. As a further precaution against the danger of aggressive behavior in the resident subjects, the juveniles may have been periodically replaced (e.g., every 10 days or so) (Arletti et al., 1995). Under nontreatment conditions, intact adult male mice and rats recognized the reencountered juvenile after an IEI of 30 but not 120 min; whereas female rats appeared to recognize the juvenile after IEIs of 120 but not 180 min (Bluthe and Dantzer, 1990).

B. The Social Discrimination Test Engelmann et al. (1995) developed the social discrimination test (SDT) as an alternative paradigm for testing this type of memory. In the SDT the familiar juvenile (presented in the first investigative encounter) and a novel (different) juvenile are simultaneously presented during the second investigative encounter. SRM is indicated when the time spent investigating the familiar juvenile is significantly less than for the novel juvenile. It is argued that the advantage of this procedure is that the use of the novel juvenile as a comparative test stimulus provides a built-in control that reduces the number of sessions needed for a given experimental series. Like the SRT, this procedure has verified that a single investigative exposure of the juvenile results in a short-term memory that is present after an IEI of 30 but not 120 min the male rat.

C. The Multitrial Social Recognition Test A more recently developed paradigm, the multitrial recognition test, has been used to test SRM in both male and female mice (Ferguson et al., 2000). The mouse in its home cage is presented with the same conspecific social test stimulus in each of four 1-min social investigative trials (10-min intertrial interval, ITI). SRM is operationally defined as a significant decline in the duration of social investigative activity over the successive 1-min encounters

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with the same social stimulus. A different conspecific social stimulus is presented in a fifth trial as a control for nonspecific effects. Prolonged social investigation of the stranger is interpreted as renewal of interest that had waned with increasing familiarity of the previous conspecific. Another control for nonspecific effects used by Ferguson et al. (2000) with this paradigm was the presentation of a different conspecific test stimulus in each of the four 1-min presentation trials. The conspecific test stimuli used by Ferguson and colleagues included adult male mice, as well as ovariectomized and intact female mice.

III. Effects of Peripherally and/or Centrally Administered VP, OT, or Their Metabolic Fragments on SRM in Laboratory Rats and Mice

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A. Vasopressin and Related Peptides 1. General Comments The pioneer studies by Dantzer, Bluthe, and colleagues (see Chapter 12), which showed that peripherally administered AVP enhances SRM in male rodents, have been confirmed and extended by the research described below. Popik et al. (1991) have drawn parallels between the effects of arginine vasopressin (AVP) in the social recognition paradigm and in other learning paradigms in their studies using AVP derivatives. Sekiguchi et al. (1991a) further clarified the contribution of exogenous AVP to this form of memory by their analysis of time–effect, structure–activity, and dose–response relationships. In structure–activity testing, the investigator attempts to learn which part of the peptide molecule is specifically responsible for the physiological or behavioral effect under study. Popik and Van Ree (1992) also carried out a structure–activity analysis to further characterize the memory-enhancing effect of exogenous AVP in the social recognition test. Their findings led them to suggest that social recognition depends on two types of memory processes, short-term and long-term, and they appear to be differentially sensitive to the facilitating effects of various AVP-related peptides. 2. Peripheral Administration a. Selected Studies i. Popik et al. (1991) Popik et al. (1991) investigated the effects of peripherally administered desglycinamide-arginine vasopressin (DG-AVP) and AVP(4–8) on SRM in Wistar male rats. It was of interest to determine whether these AVP derivatives, which lack the endocrine effects of the parent peptide, would facilitate this SRM as they were shown to with avoidance retention [De Wied et al., 1972, 1987; Gaffori and De Wied, 1986; Kovacs et al., 1986 (see Chapter 5)].

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The SRT, with varying IEIs, was used to assess SRM in three experiments. Two measures of social investigative behavior were used: (1) the total duration of social investigation, which included proximate orientation to the juvenile and direct contact activity (e.g., sniffing, social grooming, inspection of body surface); and (2) duration of each of the following social behaviors: close following, explorative sniffing other than that of the anogenital area, anogenital sniffing, and social grooming. Drugs or placebo were subcutaneously injected 1 min after the first investigative encounter. Differences in investigative time between the first and second 5-min encounters were computed separately for the rats presented with the same and different juveniles during the second exposure period. The first experiment was designed to examine the effects of IEI duration (15, 30, 60, or 120 min) on SRM. The results were as follows: (1) social investigation of the same juvenile during the second encounter was significantly reduced compared with that of the first encounter for an IEI of 15 or 30 min, but not of 60 or 120 min; and (2) no significant difference in investigative time between the two encounters was found when a different juvenile was presented during the second encounter. In the second experiment the investigators used the SRT with IEIs of 60 and 120 min to examine the effect of two AVP derivatives on SRM. A peripheral injection of placebo, DG-AVP (6.0
g/kg, subcutaneous), or AVP(4–8) (1.0
g/kg subcutaneous) was given 1 min after the first presentation trial. A pilot experiment had found that AVP (3
g/kg, subcutaneous) enhanced SRM [significantly reduced social investigative time (SIT) in the second investigative trial, but not for the placebo-treated rats]. The results of experiment 2 were as follows: (1) placebo-treated rats exhibited no difference in SIT between the first and second encounters when either the same or a different juvenile was tested; (2) DG-AVP produced a small but significant decrease in SIT during the second encounter, when the same juveniles were presented after an IEI of 120 min, but not 60 min; (3) the AVP(4–8)-treated rats exhibited significantly decreased SITs in the second encounter with the same juvenile after both IEIs; and (4) no effect of either peptide was found when a different juvenile was presented in the second investigative trial. In the third experiment the investigators examined the degree to which each of the four types of social investigative behaviors monitored in this study (anogenital exploration, close following, sniffing, and grooming) was associated with the influence of DG-AVP on SRM. The subjects were presented with the same or a different juvenile during the second presentation trial after an IEI of 30, 60, or 120 min. The results indicated that (1) half of the first presentation trial was spent in social investigation of the juvenile, and most of this involved anogenital sniffing (75%), with considerably less time involved with the remaining social behaviors [close following (2%), sniffing (13%), and grooming (10%)]; (2) placebo-treated rats significantly reduced their social investigative behavior of the familiar juvenile after a

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30-min, but not a 60- or 120-min, IEI, and this was entirely attributable to a reduction in anogenital sniffing. The SIT was not reduced when a different juvenile was presented during the second investigative trial; and (3) DGAVP-treated rats spent significantly less time investigating the same juvenile after a 30- or 120-min IEI, but not after a 60-min IEI. This reduction in investigative behavior was entirely due to a decrease in anogenital exploration, because time spent on other social behaviors was not decreased. DGAVP did not produce changes in the duration of investigative behavior when a different juvenile was tested during the second presentation trial. Taken together, these results have confirmed the short-term duration of SRM observed in laboratory rats (e.g., Thor and Holloway, 1982) (experiment 1), and the importance of olfactory investigation of the anogenital area for this recognition (Carr et al., 1976; Sawyer et al., 1984), because this was the major social behavior observed during the first presentation trial and was the one reduced during the second presentation of the familiar juvenile (experiment 3). Moreover, they have shown that other measures of social investigative activity, such as grooming, sniffing per se, and following the juvenile, appeared not to be important for SRM because their duration did not change from the first to the second presentation trial (experiment 3). The study further showed that, in addition to peripherally administered AVP (pilot test), its derivatives DG-AVP (experiments 2 and 3) and AVP(4–8) (experiment 2) enhanced SRM and AVP(4–8) was a far more potent enhancer than DG-AVP (induced a stronger effect at a six-times lower dose level) (experiment 2). The study also showed that the AVP-induced enhancement of SRM was not dependent on its peripheral endocrine effects because DGAVP and AVP(4–8), which lack these effects, also enhanced SRM. Moreover, this finding is in accord with similar findings for memory tested in other retention paradigms [e.g., Bohus, 1977; Vawter and Van Ree, 1995; Vawter et al., 1997 (see Chapter 2); and De Wied et al., 1987; Gaffori and De Wied, 1986; Kovacs et al., 1986 (see Chapter 5)]. The failure of DG-AVP-treated rats, in contrast to AVP- and AVP(4–8)treated rats, to enhance SRM after a 60-min IEI could not be explained. This finding was even more puzzling given the ability of DG-AVP to enhance SRM after a longer (120 min) IEI. The authors noted that Gaffori and De Wied (1986) reported differences between various VP analogs with respect to their time-dependent effects on avoidance retention. However, the time-dependent effects pertained to training-treatment intervals over which the peptides demonstrated memory facilitation, and may have resulted from differences among the peptide analogs in ‘‘metabolism, rates of distribution to the various body compartments or brain structures involved in the behavioral effects of the peptides’’ (Popik et al., 1991, p. 1033). This explanation does not seem applicable to the present study, because the training-treatment interval was the same for all the tested peptides, and it does not explain the ability of DG-AVP to enhance SRM tested with a 120-min, but not a 60-min, IEI.

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ii. Sekiguchi et al. (1991a) Sekiguchi et al. (1991a), noting the reports of the ability of exogenous AVP to facilitate SRM in the male rat (Dantzer et al., 1987, 1988; Le Moal et al., 1987; see Chapter 12), designed several experiments to further analyze this influence by studying time–effect, structure–activity, and dose–response relationships. Adult male Wistar rats were tested in the SRT with male juvenile rats (4–5 weeks of age) as social test stimuli. The duration of investigative behavior in each presentation trial was determined by adding the times engaged in each of the following specific activities directed toward the juvenile: close following, social sniffing of the body surface other than the anogenital area, anogenital investigation, and social grooming of the juvenile’s body other than the anogenital area. Experiment 1 tested time–effect relationships. Placebo (physiological saline) or DG-AVP (6
g/kg, subcutaneous) was injected immediately after the first investigative trial and the same juvenile was re-presented in the second trial after an IEI that differed for independent groups of rats (1, 2, 4, 6, 8, 24, or 48 h). The results indicated that DG-AVP treatment resulted in social recognition for all IEIs except for those of 1 or 48 h (investigative time during the second trial was significantly reduced after IEIs of 2, 4, 6, 8, or 24 h). In contrast, the placebo controls showed no significant reduction in the duration of second trial investigative behavior for any of these IEIs. Experiment 2 tested structure–activity relationships. After the first investigative trial the subjects were subcutaneously injected with placebo or a 6-
g/kg dose of one of the following AVP analogs: AVP(4–8), AVP(4–9), AVP (5-8), or AVP(5–9). The same juvenile was re-presented in the second trial after a 120-min IEI. Three of the tested analogs, AVP(4–9), AVP(5–8), and AVP(5–9), enhanced SRM (investigative behavior of the same juvenile in the second trial was significantly reduced from that in the first trial). The effect of AVP(4–8) on SRM was in the expected direction but did not reach statistical significance. Placebo treatment did not facilitate SRM. Experiment 3 tested dose–response relationships. DG-AVP (0.0, 0.2, 0.6, 2.0, 6.0, or 20.0
g/kg) or AVP(4–8) (0.0, 0.2, 0.6, 2.0, or 6.0
g/kg, subcutaneous) was injected 1 min after the first presentation trial with a 120-min IEI. DG-AVP at the two highest dose levels (6.0 and 20.0
g/kg), and AVP(4–8) at a dose level of 2.0
g/kg enhanced social recognition (SIT was significantly reduced from the first to the second trial). Placebo-treated controls did not recognize the reencountered juvenile. The following points were made during discussion of these results: 1. DG-AVP (6 mg/kg, subcutaneous) induced a long-term enhancement of SRM, extending it from its normal duration of 30 min (Popik et al., 1991; Thor and Holloway, 1982) to 24 h (experiment 1). Other attempts to prolong recognition time, such as increasing the duration of the first presentation, or the number of encounters on one day or on several subsequent days, have not been successful (Sekiguchi et al., 1991b). These observations

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together with the present findings suggest that long-term action of DG-AVP is more related to the memory processes involved than to the amount of information received. 2. It is noteworthy that this long-term effect on SRM required that the juveniles and adults remain in the same experimental room during the 24-h IEI. The effect was absent when the animals were returned to the animal house, and this may have been due to interference from other environmental stimuli encountered during the IEI, that is, an example of the retroactive inhibitory effect demonstrated by Dantzer et al. (1987). Although not stated by the authors, the continued presence of background stimuli (visual, auditory, airborne odors) in attendance at the initial social encounter might also have interacted with the peptide to keep SRM processing active during the IEI. 3. The social recognition enhancement induced by DG-AVP was also observed for the AVP analogs AVP(4–9), AVP(5–8), and AVP(5–9) (experiment 2). These observations suggest that portion 5–8 of the vasopressin molecule is the active site for this effect, and is in accord with findings on structure–activity relationships reported in studies with active and passive avoidance paradigms [De Wied et al., 1987; Kovacs et al., 1986 (see Chapter 5)]. 4. Peripherally administered AVP(4–8) was somewhat more potent than DG-VP in facilitating SRM, enhancing it at a lower dose level (2
g/kg) than did DG-AVP (6.0 and 20.0
g/kg) (experiment 3). This, too, is consistent with findings on active and passive avoidance tasks, although the difference in potency between these two peptides is more pronounced in these two latter paradigms. iii. Popik and Van Ree (1992) Popik and Van Ree (1992) used the SRT with a 24-h IEI to determine whether peripherally administered AVPrelated peptides can extend the duration of SRM over a 24-h period, and if so, to characterize the active part of the molecule responsible for this ability. The design of this study was stimulated, in part, by observations that SRM in wild rats (Thor, 1979), after a single encounter with a conspecific juvenile, may endure significantly longer then the 30-min interval suggested by the study of nontreated laboratory-bred rats (Thor and Holloway, 1982). It is possible that in the laboratory SRM lasts longer than 30 min, but is too weak to be demonstrated by the paradigm used. The design of the study was also influenced by the memory-modulating effects of AVP and OT in the SRM paradigm (e.g., Dantzer et al., 1987) and in particular by the demonstration by Sekiguchi et al. (1991a) that peripherally administered DG-AVP extended SRM in male rats from 30 min to 24 h, thus producing the VPinduced memory persistence observed in other task paradigms [Bohus, 1977 (see Chapter 2); Kovacs et al., 1986 (see Chapter 5)].

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The subjects were sexually experienced adult male Wistar rats, and the social test stimuli were male juvenile (3- to 4-week old) conspecifics. The juvenile was removed after the first presentation trial, and SRM was evaluated after a 24-h IEI in the second presentation trial with the same or a different juvenile as the social test stimulus. Peptide or placebo was given immediately after the first presentation. The peptides tested were as follows: AVP(1–9), numerous AVP fragments [AVP(1–8), AVP(1–7), AVP(1–6), AVP(1–5), AVP(4–9), AVP(4–8), and AVP(7–9)], OT(1–9), and the OT fragment OT(1–6). With the exception of OT(1–9) and AVP(7–9) each of these peptides was injected at two dose levels (0.3 and 3.0
g/rat, subcutaneous). OT(1–9) was tested at the 3.0-
g/rat dose level and also at a dose level of 0.75 ng/rat (subcutaneous), on the basis of the observation that low doses of OT facilitated SRM after an IEI of 2 h (Popik et al., 1992). The subjects in each peptide dose group served as their own placebo controls (i.e., ‘‘each peptide treatment was placebo controlled’’; p. 568). More specifically, this was accomplished by a cross-over design whereby half the subjects in a peptide dose group received the peptide, and the remainder received the placebo (physiological saline) on the first test day; the same subjects received the reversed treatment on the second test day. A placebo-controlled recognition index was computed for each peptide treatment. The social investigative time (SIT) during the first and second encounters was determined for each resident rat. These data were entered into a formula that comprised the recognition index (RI) for each rat: RI ¼ ([SITsecond encounter (peptide)/SITfirst encounter (peptide)] – [SITsecond encounter (placebo)/ SITsecond encounter (placebo)])  100. A significant negative value indicated SRM after peptide treatment as compared with placebo treatment. These values were averaged for each experiment (peptide treatment/placebo control) and statistically analyzed in a two-way analysis of variance (ANOVA). The results of the analysis of the placebo-controlled RIs of the subjects treated with different peptide fragments indicated that (1) SRM (a significant decrease in SIT during the second encounter) was present after treatment with the 3.0-
g dose of the following peptides: AVP(1–8), AVP(1–6), and AVP(1–7), all of which contain the covalent ring structure of the peptide molecule; (2) of these peptides, only AVP(1–6), the covalent ring structure of the AVP molecule, induced social recognition at the low dose level (0.3
g/ rat); (3) those AVP peptide molecules that lacked an intact covalent ring [AVP(1–5), [pGlu4,Cyt6]AVP(4–8), [pGlu4,Cyt6] AVP(4–9), and AVP(7–9)] did not influence social recognition (i.e., did not significantly reduce SIT during the second encounter); and (4) treatment with OT(1–6), which does contain an intact ring structure, did not result in social recognition, thereby indicating the specificity of the influence of VP on social recognition; moreover, neither the 0.75-ng nor 3.0-
g dose of OT(1–9) affected the placebocontrolled RI values after an IEI of 24 h.

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In discussing these findings the authors noted the following points: 1. Several of the VP peptides exerted long-term effects on SRM, as has been demonstrated for retention in avoidance paradigms (De Wied, 1971; and see Chapters 2–5), ‘‘supporting the assumption that memory processes are involved in social recognition’’ (Popik and Van Ree, 1992, p. 570). 2. The observed importance of the covalent ring structure for the longterm effects of VP on SRM supports the proposal that it contains the primary site for the memory-enhancing activity of the molecule, although a second active site for this process may be present in the linear part of the molecule (Van Ree et al., 1978). More specifically, Van Ree et al. (1978) have reported that AVP(1–6) enhances memory consolidation in avoidance paradigms, but in contrast to the C-terminal linear component, does not facilitate memory retrieval (i.e., prevent experimentally induced amnesia). 3. Earlier findings have indicated the ability of AVP(4–8) and AVP(4–9) to enhance memory consolidation in avoidance learning tasks at considerably lower doses than that of the parent peptide (De Wied et al., 1987; see Chapter 5), and to promote social recognition after an IEI of 2 h [Popik et al., 1991; Sekiguchi et al., 1991a (see above)]. These observations along with the present evidence of their inability to enable SRM to extend over a 24h IEI led the authors to postulate that SRM involves two different memory processes, one short-term and another long-term in nature, which are differentially sensitive to the facilitating effects of various VP-related peptides. This interpretation receives support from the authors’ unpublished observation that the covalent ring structure of AVP was not active when an IEI of 2 h was used.

B. Oxytocin and Related Peptides 1. Section Overview The amnestic property of peripherally and/or centrally administered OT in active and passive avoidance conditioning tasks has been well documented (see Chapter 2), and has also been reported for the nonstressful social recognition test (Dantzer et al., 1987). Moreover, a memory-impairing action for endogenous OT has been demonstrated in avoidance paradigms after peripheral and central administration of OT antiserum [Bohus et al., 1978b (see Chapter 2); Kovacs et al., 1979a (see Chapter 4)]. A study by Popik and Vetulani (1991) found a similar effect on SRM after peripheral administration of high doses of two OT receptor antagonists. Whereas the foregoing evidence supports postulated amnestic properties of OT, there have been reports that OT does not always impair memory because the neuropeptide prevented puromycin-induced amnesia (Walter

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et al., 1975; see Chapter 2) and exerted a bimodal effect on avoidance behavior (Gaffori and De Wied, 1988; see Chapter 2). Results of most of the studies described below suggest a dose-dependent modulation of SRM, whereby low doses of exogenous OT facilitate SRM, and the higher doses, more frequently used in behavior study, impair it (Popik et al., 1992). Structure–activity analyses, used to clarify this dose–response effect of exogenous OT on SRM, have led to proposals of physiological mechanisms that may be responsible for it (Popik et al., 1996). The study by Arletti et al. (1995) confirmed this dose-dependent SRM enhancement effect of OT, and demonstrated its effectiveness in aged as well as young rats. Using centrally injected OT, Benelli et al. (1995) found the same low-dose enhancement effect of OT that had been observed after peripheral administration of the peptide. 2. Peripheral Administration a. Selected Studies i. Popik and Vetulani (1991) Popik and Vetulani (1991) used the SRT to learn whether peripheral administration of two OT antagonists, [10 -(10 -thio-40 -methylcyclohexane)-acetic acid1]-oxytocin (MeCAOT) and [10 -(10 -methyl-40 -thiopiperidine)-acetic acid1]-oxytocin (MePAOT) act by themselves as memory-enhancing factors, and interfere with the amnestic action of OT when combined with this peptide. MeCAOT appears to be a peripheral OT antagonist, because it and not MePAOT blocked the action of OT in an isolated rat uterus preparation (Rekowski et al., 1987). This study defined a peptide-induced memory-impairing effect as loss of social recognition after a 20-min IEI, and a memory enhancement effect as the presence of juvenile recognition after a 60-min IEI. The putative OT amnestic effect was tested with a 20-min IEI; the putative memory-enhancing effect of the antagonistic treatment on its own was tested with a 60-min IEI. Both antagonists were peripherally administered at two dose levels (12 and 24
g/ kg, subcutaneous). A given subject received one or the other dose of the peptide. When tested on its own the OT antagonist was injected immediately after the first encounter. For antagonist–OT interactional effects on SRM, the lowest memory-disrupting dose of OT (determined by pretest results), was injected 2 min after injection of the OT antagonist (administered 1 min after the first presentation trial). The four main results were as follows: (1) injected on their own, the high dose level of both OT antagonists enhanced SRM (i.e., SIT was significantly reduced during the second investigative trial when the same juvenile was presented after a 60-min IEI); (2) there was no change in SIT between the first and second investigative trials if a different juvenile was presented after the 60-min IEI, even if the subjects received the high dose of the OT antagonists; (3) injected on its own, OT, over a dose range of 6 to 750 mU/kg, impaired normal SRM (no significant reduction in SIT between

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the first and second investigative trials, a reduction that occurred for placebo controls when the same juvenile was presented after the 20-min IEI); and (4) MePAOT at both dose levels, and MeCAOT at the higher dose level, antagonized the amnesia induced by the least effective amnestic dose of OT (6 mU/kg). The following points were made during the discussion of these results: (1) the results verified the OT-antagonistic property of MePAOT and MeCAOT. This was indicated by their memory-enhancing effects when injected alone, and their ability to interfere with the amnestic effects induced by appropriate doses of peripherally administered OT; (2) the results support a physiological role for OT in the modulation of SRM as appears to occur for memory tested in other learning paradigms. That is, the enhancement of SRM induced by peripheral injections of the OT antagonists was presumably due to interference with OT-ergic neurotransmission, just as the enhancement of memory in avoidance paradigms induced by intracerebroventricularly injected OT antiserum was presumably due to the resulting reduction of endogenous OT (Bohus et al., 1978b; see Chapter 2); and (3) the greater effectiveness of MePAOT in antagonizing the centrally mediated amnestic action of OT, together with its inability to influence a peripheral action of the hormone (Rekowski et al., 1987), indicates a distinction between OT central and peripheral actions, and suggests that separate and possibly independent mechanisms are involved. ii. Popik et al. (1992) Popik et al. (1992) used the SRT to examine the effects of a wide dose range of peripherally administered OT on SRM in male rats. This study was stimulated by the observation that the doses of peripherally administered OT that have been observed to attenuate SRM (i.e., greater or equal to 24 ng/kg; Popik and Vetulani, 1991) probably result in rather high plasma levels of OT, compared with physiological levels of the hormone (Mens et al., 1983). Accordingly, this study used doses of OT lower than those effective in attenuating SRM. Also, OT and AVP have been shown to have opponent effects on memory tested in avoidance learning paradigms (e.g., Bohus et al., 1978b), as well as in the SRT (Dantzer et al., 1987). Therefore, AVP and arginine vasotocin (AVT), the ancestral neurohypophysial peptide, were included in this study for comparative purposes. AVP and AVT were tested at two dose levels (1.5 and 6.0 ng/kg, subcutaneous), and OT was tested over a dose range of 0.09 to 24.0 ng/kg (i.e., 0.09, 0.36, 1.5, 6.0, or 24.0 ng/kg) in experiments in which the same juveniles were presented in the second encounter, and at two dose levels (1.5 and 6.0 ng/kg) in those experiments using novel juveniles. The same 16 subjects were tested under all treatment conditions. Injections were given immediately after the first encounter, and 48 h intervened between successive treatments. Attenuated SRM was defined as the inability to recognize the preencountered juvenile after an IEI of 30 min, and facilitated SRM as the ability to

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recognize the juvenile after an IEI of 120 min. The criterion for SRM in a group of residents was a statistically significant (paired t test) shortening of the SIT on the second encounter with the familiar juvenile. Paired t tests indicated no change between SIT for first and second encounters with the same juvenile under placebo conditions, or at an OT dose level of 24 ng/kg, whereas OT treatment at doses ranging from 1.5 to 6.0 ng/kg facilitated SRM (induced a significant reduction in SIT during the second encounter). A one-way ANOVA of the
SIT scores (
SIT ¼ difference in SIT between the first and second encounter) indicated a significant treatment effect, and individual comparisons revealed a significant reduction in SIT during the second encounter relative to the placebo treatment condition, after OT doses of 1.5 and 6.0 ng/kg. The results of the control experiments with the novel juvenile indicated that an OT dose of neither 1.5 nor 6.0 ng/kg decreased SIT during the second encounter. In experiments with an IEI of 30 min, placebo treatment resulted in normal SRM (i.e., SIT was significantly reduced in the second, relative to the first, encounter with the juvenile). The rats also showed normal SRM after treatment with the two lower doses (1.5 and 6.0 ng/kg, subcutaneous) of OT but not with the higher dose (24 ng/kg, subcutaneous). In addition, the ANOVA indicated a significant difference in SIT scores between placebo treatment and OT treatment with the 24-ng/kg dose, but not with the 1.5- and 6.0-ng/kg doses for this experiment. In the experiments with the same juvenile and the 120-min IEI, comparison between placebo treatment and each of the dose levels used for AVP and AVT treatment conditions indicated no significant differences for the SIT scores obtained in the second encounter. The most important result of this study was the dose–response curve indicating that peripherally administered OT facilitates SRM when given in low doses, and impairs it in high doses. Noting that the passage of these peptides across the blood–brain barrier and their uptake by brain tissue is still under debate (see Chapter 14), these authors nevertheless interpreted their data as evidence that the peptide interacted with central brain sites implicated in the processing of SRM. Evidence that a central action of OT mediates this form of memory has been demonstrated in studies using intracerebroventricularly and microinjected peptides into specific brain structures (discussed below). Two other points were made in discussing these results. The first was their relevance for a physiological action of the peptide in SRM. Although the plasma level of OT was not directly measured in this study, it was suggested that the low dose levels used in this study produced plasma levels within the range of physiological values observed in animals and/or humans during or after a variety of self- and species-preservative encounters. These include increases in OT plasma level after restraint (Gibbs, 1984), during sexual activity (McNeilly and Ducker, 1972; Murphy et al., 1987), parturition

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(Fitzpatrick, 1961; Higuchi et al., 1985), and nursing of young (Higuchi et al., 1985). The second point was the generalizability of this dose–response effect to retention in a variety of situations involving adaptive behavior (including learning). The authors proposed that a mild increase in plasma OT, such as might result during an encounter with a conspecific juvenile, facilitates retention of the experience. On the other hand, high levels of circulating OT, such as those observed during the stressful experience of parturition in animals and humans (Fitzpatrick, 1961; Higuchi et al., 1985), attenuate retention of the experience. High levels of OT during labor (Fitzpatrick, 1961) and memory impairment for the pain experienced at this time (Kennett et al., 1982) have both been reported in humans. iii. Popik et al. (1996) Popik et al. (1996) used the SRT to investigate dose-dependent memory-facilitating and -attenuating effects of subcutaneously injected OT(1–9) and several OT-derived peptides in male rats. The OT-derived peptides were as follows: desglycinamide-OT [OT(1–8)]; tocinamide [OT(1–6)]; [pGlu4,Cyt6]OT(4–9) [OT(4–9)]; [pGlu4,Cyt6]OT(4–8) [OT(4–8)]; [Pro-Leu-Gly-NH2]OT(7–9) [PLG]; [Leu-Gly-NH2]OT(8–9) [LG], and glycine. The doses used for assessing SRM-facilitating effects were 0.6 and 6.0 ng/kg (except glycine, which was used at doses of 1.0 and 10.0 ng/kg). The SRM-attenuating effects were assessed with doses of 0.6 and 6.0
g/kg. The selection of these doses was based on previous studies [Popik and Vetulani (1991) and Popik et al. (1992); see above]. The subjects were tested under all treatment conditions in a cross-over design that ensured placebo-controlled treatment conditions; 2 days intervened between successive test treatments. Placebo or peptide was subcutaneously injected at the end of the first presentation trial. The same juvenile was reintroduced after a 30- or 120-min IEI. The change in social interest, expressed as the recognition index (RI), was calculated as the social investigative time (SIT) in the second presentation trial divided by that of the first trial and multiplied by 100: (second SIT/first SIT)  100. The placebocontrolled RI was calculated for each subject and equaled the RI of placebo treatment minus the RI of peptide treatment. Thus, positive values for this RI indicated SRM facilitation, and negative values indicated attenuation. The results were as follows: (1) social investigation of the juveniles by the residents was vigorous and lasted between 80 and 120 s (i.e., about 30% of the presentation interval); (2) an OT amnestic action on normal SRM was found after treatment with both high doses (0.6 and 6.0
g/kg) of OT(1–9), OT(1–8), OT(7–9), and OT(4–9), and with only the 0.6-
g/kg dose of OT(1–6) and OT(4–8), whereas neither of the high doses of OT(8–9) influenced this form of memory; and (3) SRM of the preencountered juvenile occurred after an IEI of 120 min in rats treated with low doses (0.6 and

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6.0 ng/kg) of the following OT-related peptides: OT(1–9), OT(7–9), and OT(8–9), whereas the other peptides and glycine were ineffective in this respect. The discussion points were as follows: 1. These findings together with those obtained by Popik et al. (1992; see above) support the concept that SRM is facilitated by low but not by high doses of peripherally administered OT. The observation that, unlike a low dose of OT(7–9), a high dose failed to enhance SRM (120-min IEI) offered further support for this concept. 2. The structure–activity component of this study indicated that different parts of the OT molecule are responsible for its dose-related attenuating and facilitating of memory effects. The memory attenuation induced by high doses of OT was mimicked by OT-related peptides with and without the C-terminal glycinamide. The SRM attenuation induced by OT(1–9), OT(1–6), OT(7–9), OT(4–9), and OT(4–8), but not by OT(8–9), indicates that the amino acid residues in region 5–7 of the OT molecule are particularly important for this amnestic action. The SRM facilitation induced by low doses of OT was mimicked only by peptides with the C-terminal glycinamide, such as OT(1–9), OT(7–9), and OT(8–9), suggesting that region 8–9 of the OT molecule was important for this function. However, the failure of low doses of OT(4–9) to facilitate SRM is not consistent with this interpretation and requires clarification. 3. The SRM-attenuating and -facilitating effects of OT and its Cterminal metabolites have been observed in other learning paradigms. Memory-attenuating effects for OT(1–9) and its C-terminal metabolites OT(4–8) and OT(4–9) have been observed in tests of retention involving active and passive avoidance behavior [Bohus et al., 1978b (see Chapter 2); De Wied et al., 1987 (see Chapter 5)]. Small C-terminal peptides of OT, such as PLG and LG, had memory-facilitating effects: reduced puromycin-induced amnesia in mice, and PLG facilitated reversal learning in rats in a brightness discrimination task (Rigter and Popping, 1976). In other studies, PLG and related peptides exhibited attenuating effects on retention, such as the facilitation of extinction in a conditioned taste aversion (Rigter and Popping, 1976). All these studies, however, used much higher dose levels than those used in the present study. 4. The effectiveness of low doses of PLG [OT(7–9)] and LG [OT(8–9)] in facilitating SRM in this study led these researchers to postulate that these, or structurally related metabolites of OT, are physiologically involved in memory processes. They cited evidence that such peptides may be normally generated in the brain by enzymatic processes (Burbach et al., 1983b; see Chapter 5) as consistent with this postulate. However, two facts detract from the strength of this postulate: (a) neither this study nor that of Popik et al. (1992) obtained an independent measure of the plasma levels of OT and OT metabolites produced by their treatments; and (b) given the

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blood–brain barrier (see Chapter 14), it is not clear that these peripherally administered peptides entered the brain and exerted direct effects on brain structures involved in SRM processing. iv. Arletti et al. (1995) Arletti et al. (1995) designed a study to test both SRM-enhancing effects and antidepressant effects of OT in aged male Wistar rats. Only the testing pertinent to SRM is discussed here. They noted that memory function in humans is usually compromised during normal aging (Rapp and Amaral, 1992) and that, in rats, OT improves SRM (Benelli et al., 1995; Popik et al., 1992). The subjects (26-month-old Wistar male rats) were tested for memory in the SRT (IEI of 120 min) with 4- to 5-week-old male juvenile conspecifics as social test stimuli. Placebo or OT (1.5, 3.0, 6.0, or 15.0 ng/kg; intraperitoneal) was peripherally administered at the end of the first presentation trial. The difference in the time spent investigating the juvenile (proximally oriented toward, or in direct contact with the juvenile) between the first and second presentations was calculated as
SIT (second SIT – first SIT); a negative
SIT value means a reduction in SIT during the second trial. The criterion for enhanced SRM was a statistically significant shortening in mean SIT in the second relative to the first presentation for a given treatment group. Placebo treatment did not enhance SRM (no significant
SIT after a 120min IEI). OT treatment at the dose levels of 3 and 6 ng/kg, but not at the dose levels of 1.5 or 15 ng/kg, enhanced SRM memory (significant
SIT after the 120-min IEI). Thus, depending on the dose, OT enhanced SRM in these aged rats as it has been observed to do in young rodents when administered peripherally (Popik et al., 1992; see above) or centrally (Benelli et al., 1995; see below). 3. Central Administration a. Selected Study: Benelli et al. (1995) Benelli et al. (1995) studied the effect of a wide dose range of intracerebroventricularly injected OT (1 ng to 1000 ng/rat) on SRM, and the ability of a selective OT antagonist, d(CH2)5[Tyr(Me),Orn8]vasotocin (VT), to block this effect. A decrease in SIT during the second encounter with the same juvenile after a 120-min IEI indicated improved memory. Experimental rats received a specific dose level of OT immediately after removal of the juvenile at the end of the first encounter; control rats received physiological saline by the same route, at the same infusion rate, and at the same time as the experimental rats. Tests were separated by a minimum of 48 h. In tests with the OT antagonist, the two peptides were injected either alone [i.e., OT (1 ng or 500 ng/rat, intracerebroventricular) or the OT antagonist (1 ng or 500 ng/rat, intracerebroventricular)] or together (OT, 1 ng plus OT antagonist, 1 ng; or OT, 500 ng plus OT antagonist, 500 ng) after removal of the juvenile in the first encounter.

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When injected together, the injection of the antagonist preceded that of OT by 5 min. The results were as follows: (1) OT at the low end of the dose range used in this study (10 ng to 1 ng) significantly improved SRM but had no effect or slightly impaired it at the high end (above 10 ng). The failure of these low doses to reduce SIT time when a novel juvenile was presented after a 120min IEI supports a mnemonic effect rather than a spurious nonspecific treatment effect; and (2) injected on its own, the OT antagonist at either dose level had no effect on SRM, but pretreatment with the antagonist at the same dose as the agonist blocked the memory-improving effect of the low dose (1 ng) of OT, and reversed the slight memory impairment induced by the high dose (500 ng) of OT. In the discussion, the authors commented on (1) the implication of these results for a physiological role of OT in the modulation of SRM in the male rat, and (2) possible reasons for opponent effects of high and low doses of OT on this memory. The effectiveness of minimally active doses of OT (within the range of physiological values) in preserving STM suggested that centrally released OT plays a physiological role in this processing, although the failure of the OT antagonist to block SRM when given alone did not corroborate this. Nevertheless, the ability of the antagonist to block the facilitated SRM induced by OT treatment makes clear that central OT receptors mediated this mnemonic effect. The opposing effects of the low and high doses of OT on SRM could be attributed to a number of causal factors. Thus, the two dose ranges may each have recruited or activated different circuitries with opposite effects on memory processes, or activated different OT receptor subpopulations mediating opposing actions on social memory, or these effects may have resulted from dose-related opposing effects of OT on certain neurotransmitter systems involved in memory processes (see discussion by Kovacs, 1986 and Chapter 4).

IV. Sex Differences and the VP/OT Influence on SRM

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A. General Comments ‘‘Anatomical sexual dimorphy’’ characterizes the extrahypothalamic VP-ergic circuitry in the brains of rats. Anatomical studies with VP-staining techniques have shown that the amount of AVP mRNA in cell bodies of the medial amygdala and bed nucleus of the stria terminalis (BNST), and density of VP-ergic fiber projections from these nuclei, are greater in male than in female rats (De Vries and Al-Shamma, 1990; De Vries et al., 1985; Miller et al., 1989b; Van Leeuwen et al., 1985; see Chapter 1). Studies of De Vries, Wang, and colleagues (cited in Chapter 1) provided evidence that

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the steroidal hormonal environment during prenatal development exerts an organizational effect on this sexually dimorphic circuitry. Further support has been added by observations that the birth of AVP cells within various subdivisions of the BNST occurred in close proximity to the gestational days when testosterone level increased in the male fetus (Al-Shamma and De Vries, 1996; Baum et al., 1991), and that prenatal exposure to flutamide (androgen antagonist) reduced AVP immunoreactivity (AVPir) in the BNST of male rats (Axelson et al., 1993). Bluthe and Dantzer (1990; see Chapter 12) demonstrated a ‘‘functional sexual dimorphy’’ for SRM by showing that only male rats were dependent on extrahypothalamic VP-ergic circuitry for normal SRM (i.e., a peripherally applied AVP receptor antagonist disrupted this memory in male but not in female rats). In addition, they demonstrated that AVP-mediated SRM in the male was androgen dependent because social recognition in castrated male rats was as insensitive to the effects of AVP antagonists as that in intact females. The research discussed below confirms and extends these findings of Bluthe and Dantzer (1990): (1) Axelson et al. (1999) furthered our understanding of the influence of the prenatal hormonal environment on the ‘‘functional sexual dimorphy’’ operating in SRM; (2) Van Wimersma Greidanus and Maigret (1996) and Landgraf et al. (1995) indicated the importance of VP-ergic sexually dimorphic circuitry for SRM in the male; and (3) Engelmann et al. (1998) provided evidence that OT may be significant for SRM in the female. 1. Selected Studies a. Axelson et al. (1999) Axelson et al. (1999) carried out two experiments: in experiment 1 they determined the degree to which VP-mediated SRM in the male rat is influenced by previous sexual experience, and in experiment 2 they investigated the organizational actions of circulating androgens in the prenatal environment on SRM. In experiment 1, sexually experienced intact males (‘‘breeders,’’ which successfully copulated with sexually primed and receptive ovariectomized females during each of three test trials) and sexually naive intact males (‘‘virgins’’; denied copulation by nonreceptive ovariectomized females during such test trials) were tested in the SRT with a 30-min IEI. Each subject was tested for SRM under three testing/treatment conditions: familiarcontrol, unfamiliar-control, and familiar-antagonist. These three conditions differed as to whether a familiar (preencountered) or an unfamiliar (novel) juvenile was presented after the 30-min IEI, and whether the subject received a subcutaneous injection of physiological saline or a 30-
g/kg injection of the AVP receptor antagonist [deamino-Pen1,O-Me-Tyr2,Arg8]vasopressin (AVP-Ant treatment condition) immediately after the initial presentation. These test conditions were given to all subjects in a counterbalanced order, with each subject tested under a given condition every other day over 6 days.

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The SIT of the second presentation trial was scored as a percentage change from that of the first presentation trial. The results of experiment 1 showed that sexually experienced and virgin males behaved similarly in this SRT: (1) after vehicle treatment, both breeders and virgins recognized the familiar juvenile. Both groups significantly reduced SIT in the second relative to the first investigative trial with the familiar but not the unfamiliar juvenile (percent reduction for breeders and virgins was 69 and 64%, respectively); and (2) in the AVP-Ant treatment condition, both breeders and virgins failed to recognize the previously encountered juvenile. SIT during the second trial with the familiar juvenile did not differ significantly from that with the unfamiliar juvenile (SITs during the second encounter with the same juvenile were 94 and 87% of the level observed during the first encounter for the breeders and virgins, respectively). In summary, the results of experiment 1 indicated that prior sexual experience was not a requirement for either olfactory-based conspecific recognition, or for the role of AVP in this behavior. Both virgins and breeders recognized the familiar juvenile after a 30-min IEI and this depended on normal VP-ergic transmission, because AVP-Ant treatment blocked this SRM. In experiment 2, SRM was tested in four groups of subjects: (1) flutamide-TP males, male offspring of females that received an injection of the androgen antagonist (flutamide) on each day of the last 10 days of gestation; these offspring were treated with testosterone proprionate (TP; 50
g/rat, subcutaneous) within 8 h of birth; (2) flutamide-control males, male offspring of flutamide-treated females; these offspring received saline instead of TP after birth; (3) control males, nontreated offspring of females that were injected with saline instead of flutamide during gestation; and (4) normal females. At 25 days of age the subjects were weaned and at a later date serum samples were assayed for total testosterone and immediately thereafter each male was castrated and implanted with crystalline testosterone to provide physiological levels of the hormone (Smith et al., 1977). At 90 days of age the subjects were tested for SRM with the same treatment and test procedures used in experiment 1. The major findings of experiment 2 were as follows: (1) after vehicle treatment, both the control males and females recognized the familiar juvenile after the 30-min IEI (relative to the first encounter, the SIT with the familiar, but not the unfamiliar juvenile, significantly decreased by 45 and 57% in the control males and females, respectively); moreover, SRM did not differ between estrous and nonestrous females; (2) AVP-Ant treatment blocked normal SRM in the control males, but not in the females (AVPAnt-treated male controls increased SIT spent with the familiar juvenile during the second encounter relative to the first encounter, as also occurred with the novel juvenile after vehicle treatment. However, AVP-Ant-treated females recognized the familiar juvenile, as reflected by a 72% reduction in SIT); (3) whether or not they received day 1 treatment with vehicle or TP, the

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offspring of flutamide-treated females recognized the familiar juvenile during the second encounter, as did their male controls and female counterparts; and (4) however, like the females, both the flutamide-control males and flutamide-TP males were insensitive to the effects of AVP-Ant treatment, and recognized the familiar juvenile after the 30-min IEI (they decreased their SITs by 47 and 33%, respectively, during the second encounter). Taken together, the results of experiment 2 replicated the earlier findings of Bluthe and Dantzer (1990; see Chapter 12) that normal males show more interest than females in their initial investigation of juvenile conspecifics, and that unlike males, SRM in females is not dependent on sexually dimorphic extrahypothalamic VP-ergic circuitry. These results further showed that prenatal androgens were important for sex differences in VP-dependent SRM, but not for sex differences in interest shown during initial investigation of the juvenile. Flutamide-induced antagonism of normal androgen release in the prenatal environment prevented the VP-dependent SRM normally observed in intact males, and rendered their performance on the SRT equivalent to that of their female counterparts. However, this antagonism did not influence the sex differences in juvenile investigative behavior during the initial encounter. These findings and previous studies demonstrating that prenatal flutamide treatment reduced AVP immunoreactivity within the BNST (Axelson et al., 1993), and that septally released AVP is important for SRM in the male rodent [Bluthe et al., 1990; Dantzer et al., 1988 (see Chapter 12)] provide evidence that sex differences in AVP dependency are linked to sex differences in AVP content in cells of the BNST and their projections to the lateral septum. b. Engelmann et al. (1998) Engelmann et al. (1998) designed a study to determine whether endogenous OT is involved in the SRM of female rats. These animals were tested in their home cage during the activity phase of the light–dark cycle with the social discrimination paradigm (SDP) described earlier. Briefly, the resident was given an initial 4-min investigative trial with a juvenile conspecific (23–35 days old, both sexes) that was promptly removed from the resident’s cage at the end of the trial. After an IEI of 30, 60, or 180 min it was reintroduced to the resident female, along with a novel juvenile, for a second 4-min investigative trial. SRM was judged to be present if the duration of investigation of the familiar juvenile was significantly shorter than that of the novel one during the second trial. All subjects were tested under three types of testing conditions: (1) nontreatment sessions with IEIs of 30, 120, and 180 min; (2) treatment with intracerebroventricularly injected OT (1 ng/rat) or vehicle (Ringer’s solution), and IEIs of 120 or 180 min; and (3) treatment with intracerebroventricularly injected vehicle, the OT receptor antagonist desGly-NH2,d(CH2)5[Tyr(Me)2Thr4]OVT (100 ng), or the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (100 ng), and IEIs of 60 min. Treatments were administered via previously

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implanted cannulas immediately after removal of the juvenile during the first investigative trial. The 60-min IEI was chosen for testing the effects of the antagonists on SRM because this IEI was within the time interval during which the nontreated residents consistently recognized the preencountered juvenile, and it allowed a more sensitive detection of the effects of a blockade of OT-ergic and VP-ergic neurotransmission than the 30-min IEI. Investigative behavior was compared for the estrous versus the anestrous condition during nontreatment and treatment test sessions. The results were as follows: (1) the SIT during the first presentation was significantly shorter in the estrous than in the anestrous state; however, there was no significant difference in SRM between the two hormonal states; (2) the effects of the various treatments were also independent of the stage of the estrous cycle; (3) in the vehicle control condition, the SITs were significantly shorter in duration with the preencountered compared with the novel juvenile after IEIs of 30 and 120 min, but there were no SIT differences after an IEI of 180 min; (4) there were no significant differences between OT-treated females and vehicle controls in SRM tested with either the 120- or 180-min IEI; (5) in contrast to vehicle treatment, the OT antagonist blocked SRM after the 60-min IEI (i.e., OT treatment abolished the significant SITs for the familiar versus the unfamiliar juvenile observed for the vehicle control condition after the 60-min IEI); and (6) unlike OT, the VP antagonist failed to block SRM after this 60-min IEI. The investigators related these results to other findings and offered several explanations for their causes. First, the reduced duration of investigation during estrus was likely due to increased time spent in proceptive (ear wiggle, darting, and hopping) and receptive (lordosis) sexual behavior. Although the sex of the juvenile might have influenced this behavior, this was not thoroughly investigated in the study. Despite their reduced investigative curiosity, these estrous females showed normal recognition ability. In addition, the duration of the IEIs during which female rats demonstrated SRM for preencountered conspecifics (30–120 min), or not (180 min), replicate observations reported by Bluthe and Dantzer (1990; see Chapter 12) and further indicate that the duration of SRM is twice as long in female than in male rats tested under similar conditions (Engelmann et al., 1995; Landgraf et al., 1995). Second, the inability of OT treatment to influence SRM may have been due to a saturation of relevant binding sites by endogenous OT during the first encounter, so that reinforcement of this OT-ergic neural communication by supplemental exogenous OT was of no further consequence. If so, it is not clear why higher doses of OT with different treatment schedules have been shown to alter social behavior (Witt et al., 1992). In addition, it is not clear why, in contrast to OT, peripherally and centrally administered AVP should improve SRM in females (Bluthe and Dantzer, 1990; Engelmann and Wotjak, unpublished observations), as well as in males (Bluthe and Dantzer, 1990). According to Dantzer and colleagues (see Chapter 12) the

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improvement in SRM induced by exogenous AVP is due to activation of a non-androgen-dependent AVP that produces its effects on SRM via an interaction with the central arousal system. Third, the inability of the centrally administered V1 receptor antagonist to interfere with normal social recognition confirms the results obtained by Bluthe and Dantzer (1990) with this VP analog when peripherally administered. Further, the differential effects of the VP and OT receptor antagonists on formation of SRM implicate causal involvement of OT, but not VP, receptors in this processing. These findings, together with evidence that both VP and OT receptor antagonists show partial antagonism on each other’s receptors when present in the same brain structures (De Wied et al., 1991; see Chapter 5), raise a question concerning the brain site(s) at which endogenous OT acts to influence this processing in the female rat. It was proposed that whereas endogenous VP in the septum is important for mediating social recognition in male rats [Dantzer et al., 1988 (see Chapter 12); Landgraf et al., 1995], endogenous OT in the medial preoptic area might perform this role in the female rat. Evidence that this brain site is important in mediating social behavior in the female rodent (Popik and Van Ree, 1991; see below) is consistent with this suggestion. It was further proposed that local administration of the OT receptor antagonist via inverse microdialysis (retrodialysis) might clarify this issue. In concluding remarks, the authors explained the sex-differentiated dependency of SRM on VP and OT by proposing the following ‘‘working hypothesis’’: OT becomes progressively involved in SRM during ontogeny, and in females this situation persists and may be further developed and fine-tuned by female sex steroids, which contribute to the characteristic aspects of maternal behavior (Argiolas and Gessa, 1991; Pedersen and Prange, 1979; Pedersen et al., 1994). However, the increasing production by males of male sex steroids results in the greater degree of VP synthesis observed within their limbic brain, and this sexually dimorphic AVP is likely to be among the regulatory mechanisms involved in male social and territorial behavior (Bluthe et al., 1990; Compaan et al., 1993; Koolhaas et al., 1991). Consequently, ‘‘these mechanisms (including AVP) may dominate the original ones (including OT) in regulating social recognition abilities in male rats as well’’ (Engelmann et al., 1998, p. 93). The investigators cite a number of observations supportive of this working thesis (for further discussion see Engelmann et al., 1998).

V. Influence of Septal–Hippocampal VP and OT on SRM

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A. General Comments Evidence cited in earlier chapters has indicated that VP and OT, present in the septal–hippocampal system, influence memory processing in selfpreservative learning paradigms, and that the effects of these peptides on

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retention may involve an interaction with classic catecholamine (see Chapter 4) and cholinergic (see Chapter 10) mechanisms implicated in information processing, as well as via enhancement of glutamatergic neurotransmission (see Chapter 5). The studies presented below provide support that these peptide systems also play a role in memory processing underlying conspecific recognition, which is clearly important for reproductively related social interaction. Dantzer et al. (1988) were the first to demonstrate that exogenous AVP (0.1 ng) injected into the septal area enhanced (prolonged the duration of) normal SRM, and this finding was subsequently replicated by others (Popik et al., 1992) and led to the demonstration of similar but more potent effects of VP metabolites (Popik et al., 1992). Dantzer et al. (1988) also provided the first supportive evidence that endogenous septal AVP has an important role in SRM in the male rat (i.e., an intraseptal injection of a V1 receptor antagonist on its own prevented the normal expression of SRM). The research findings discussed below confirm and extend these research findings of Dantzer, Bluthe, and colleagues. 1. Selected Studies a. Van Wimersma Greidanus and Maigret (1996) Van Wimersma Greidanus and Maigret (1996) injected anti-AVP serum or anti-OT serum, intracerebroventricularly or locally, into several limbic brain sites to examine the putative involvement of endogenous AVP and OT in SRM processing in the male rat. This technique was previously used to investigate the role of endogenous VP and OT in avoidance learning paradigms [Bohus et al., 1978a; Van Wimersma Greidanus and De Wied, 1976a (see Chapter 2); Van Wimersma Greidanus et al., 1975b (see Chapter 4)]. Resident male rats were tested in the SRT. Separate groups received an intracerebroventricular or local injection of anti-AVP serum (AVP antiserum), anti-OTserum (OTantiserum), or normal rabbit serum (NRS; controls). Each intracerebroventricularly injected substance was delivered via an implanted cannula into the left lateral ventricle in a volume of 3
l (1:10 or 1:20 dilution) or 2
l (1:10 dilution) for AVP antiserum, and of 2
l (1:10 or 1:30 dilution) for OT antiserum. Local injections of AVP antiserum (2-
l volume, 1:20 dilution) or OT antiserum (2
l, 1:10 dilution) or NRS (1:20 or 1:10 dilution as controls for peptide antiserum treatments) were delivered via bilaterally implanted cannulas into the dorsal hippocampus (DH), ventral hippocampus (VH), dorsal septal region (DSR) or olfactory nucleus (ON). Treatment was administered immediately after removal of the juvenile in the first investigative trial. An IEI of 30 min was used with AVP antiserum treatment, and of 120 min with OT antiserum treatment, to test for impairment and preservation of normal SRM, respectively. A significant reduction in mean SIT in the second relative to the first encounter for a given treatment group (assessed by t tests for paired data)

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indicated SRM. The mean
SIT score for a given treatment group was calculated by subtracting the average SIT in the first encounter from that in the second trial (a negative value indicated an average reduction of SIT in the second trial). Comparisons between selected antiserum treatment groups and their NRS controls on mean
SITs were evaluated by the Newman–Keuls test (i.e., a significantly more negative mean
SIT value in a treatment versus a NRS control group indicates a treatment-induced improvement in SRM, whereas a significantly less negative value indicates the converse). Cannula placements were histologically verified at the end of the experiments. Results of the experiments with intracerebroventricularly injected NRS, AVP, and OT antisera were as follows: (1) all NRS-treated rats recognized the familiar juvenile after the 30-min IEI (significant reduction in SIT in the second presentation trial); (2) AVP antiserum at a dose of 3
l (1:10 or 1:20 dilution) but not at a dose of 2
l (1:10 dilution) impaired SRM after the 30-min IEI (i.e., compared with NRS treatment, the higher dose of AVP antiserum significantly increased SIT during the second trial and significantly decreased the negative
SIT scores); (3) treatment with 2
l of OT antiserum at a dilution of 1:10, but not 1:30, preserved SRM after a 120min IEI (i.e., decreased SIT during the second encounter, and induced a mean
SIT that was significantly more negative than that in NRS controls); and (4) the increased SIT observed with anti-OT serum at the 1:10 dilution during the second trial with the same juvenile did not occur in a control experiment that presented a different juvenile during that trial. Results with the locally injected substances indicated that (1) AVP antiserum (2
l in a 1:20 dilution), injected into the VH, impaired normal SRM (30-min IEI) (i.e., no significant difference in SIT between the first and second encounters with the same juvenile, and the
SIT value was significantly less negative than that for the NRS control group); (2) OT antiserum (2
l in a 1:10 dilution) preserved SRM (120-min IEI) when injected into the VH (i.e., compared with the NRS control condition, anti-OT serum significantly reduced SIT during the second relative to the first encounter, and produced a significantly greater negative mean SIT value); (3) AVP antiserum (2
l, 1:20 dilution), injected into the DH, impaired normal SRM (30min IEI) (i.e., compared with NRS treatment, AVP antiserum significantly lengthened SIT during the second relative to the first encounter and produced a significantly less negative
SIT); (4) OT antiserum (2
l, 1:10 dilution), injected into the DH, did not preserve SRM (120-min IEI) (no significant difference from controls with respect to either SIT during the second relative to the first encounter, or
SIT); (5) AVP antiserum (2
l, 1:20 dilution), injected into the DSR, impaired normal SRM (30-min IEI) (compared with NRS treatment, AVP antiserum produced a significantly smaller reduction in SIT during the second relative to the first encounter, and a significantly smaller
SIT value); (6) OT antiserum (2
l, 1:10 dilution), injected into the DSR, did not significantly influence SIT or
SIT relative to

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NRS controls; and (7) when injected into the ON, neither antiserum influenced SRM [no significant differences in SIT during the second relative to the first encounter, or in
SIT scores in AVP antiserum (2
l, 1:20 dilution)treated subjects relative to NRS controls (tested with a 30-min IEI) or in OT antiserum (2
l, 1:10 dilution)-treated subjects relative to NRS controls (tested with a 120-min IEI)]. Taken together, these results showed that (1) anti-AVP serum, injected intracerebroventricularly or microinjected into the DH, VH, and DSR, impaired SRM after a 30-min IEI, a time when NRS controls recognized the previously encountered juvenile; (2) anti-OT serum, injected intracerebroventricularly or microinjected into the VH, but not into the DH or DSR, preserved SRM for the 120-min test interval, a time when it was absent in NRS controls; and (3) microinjections of either antiserum into the ON did not influence SRM in the test paradigm. In their discussion of these results the authors made the following comments: (1) OT, released in the local limbic structures studied in this investigation, seems less involved than AVP in SRM; thus OT located within or released from the VH (this study) and the medial preoptic area (Popik and Van Ree, 1991), but not from the DH, DSR, or ON, was shown to have a role in SRM; (2) in those limbic areas where OT does exert an effect on SRM, its role is rather complex in nature. Thus, depending on the dose level, central administration of this peptide enhances, impairs, or has no effect on this form of memory (Benelli et al., 1995; Popik and Van Ree, 1991); (3) given the importance of olfactory cues in this paradigm (Carr et al., 1976; Popik et al., 1991; Sawyer et al., 1984), and the presumed importance of an intact vomeronasal system in VP-ergic modulation of SRM in rats (Bluthe and Dantzer, 1993), it was surprising to observe that apparently neither VP nor OT in the ON appears to be physiologically important in conspecific recognition memory; and (4) comparison between the present study and those investigating the physiological roles of VP and OT in avoidance paradigms has shown that, at least for VP, release of this peptide within the DH, VH, and septum is of physiological importance in mediating memory processing tested in the olfactory-based social recognition paradigm as well memory processing tested in aversive learning paradigms. b. Engelmann et al. (1994) Engelmann et al. (1994) noted that it had previously been shown that osmotic stimulation of the hypothalamic supraoptic nuclei (SON) caused it to release endogenous AVP (Landgraf and Ludwig, 1991). They designed two series of experiments to determine (1) whether this stimulation would also release endogenous AVP from a brain site (i.e., mediolateral septum, MLS) other than the SON, and (2) if so, to determine whether this increase in endogenous AVP was associated with enhanced SRM in the male rat. Osmotic stimulation was effected by administration of hypertonic artificial cerebrospinal fluid (aCSF containing

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1 M NaCl) via microdialysis into each SON. Endogenous AVP released in the dialysates collected from the SON during microdialysis, and in the perfusates collected from the MLS during push–pull perfusion, was measured by radioimmunoassay (RIA). Two surgical procedures were performed: (1) bilateral implantation of U-shaped microdialysis probes into each hypothalamic supraoptic nucleus (SON) for osmotic stimulation and measurement of AVP released from the SON and (2) implantation of the push–pull cannula into the MLS for measurement of AVP released from this brain site in response to the osmotic stimulation. Probe placements were confirmed by postmortem histology. An initial series of experiments tested the effects of osmotic stimulation of the SON on AVP release from the SON, and from the MLS in anesthetized rats. Endogenously released AVP (picograms per sample) was measured by RIA in dialysate and perfusate samples collected simultaneously over successive periods of 30 min. During osmotic stimulation, isotonic aCSF (0.15 M NaCl) was replaced by hypertonic aCSF (1.0 M NaCl). Figure 1 depicts the AVP content in the SON dialysates (Fig. 1A) and MLS perfusates (Fig. 1B) simultaneously collected during microdialysis of the SON with isotonic aCSF (samples 1 and 2), with hypertonic aCSF (sample 3), and during the poststimulation period (samples 4 and 5). Osmotic stimulation of

FIGURE 1 (A) AVP contents in 30-min dialysates sampled continuously (means þ/ SEM; data are pooled from left and right SON) in urethane-anesthetized male rats (n ¼ 12). Isotonic (0.15 M) was replaced with hypertonic aCSF (containing 1 M NaCl) during collection period of sample number 3. Note the typical ‘rebound’ increase in AVP release during the poststimulation period (**p < 0.01 vs all other dialysates, ANOVA; see also Fig. 2). (B) Simultaneously collected push-pull perfusates (means þ/ SEM) from the mediolateral septum (perfusion medium: isotonic aCSF). *p < 0.05 vs perfusions 1, 2 and 5, ANOVA. & microdialysis; & push-pull perfusion. Source: Engelmann et al., 1994 (Fig. 1, p. 392). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

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the SON with microdialysis-applied hypertonic aCSF produced a nonsignificant increase in the content of AVP released from the SON, and this was followed by a significant increase in this content, associated with the robust ‘‘rebound’’ effect that occurred in the poststimulation period (dialysis medium was changed from hypertonic to isotonic aCSF) (Fig. 1A). Concomitantly released AVP within the MLS was significantly increased during and after osmotic stimulation of the SON (Fig. 1B, samples 3 and 4). The authors noted that previous observations (Engelmann, Ludwig, and Landgraf, unpublished results) ruled out the probability that diffusion of AVP from the SON was responsible for the increase in AVP content in the MLS. In the second series of experiments the effect of osmotic stimulation of the SON on SRM was investigated. For these experiments, separate groups of rats were implanted with a U-shaped microdialysis probe either (1) into the right SON alone, (2) in combination with a concentric microdialysis probe into the MLS, or (3) in combination with a guide cannula for microinjection into the medial nucleus of the ipsilateral amygdala. After a 2-day period of recovery from surgery, the microdialysis probes were connected with a microperfusion pump via polyethylene tubing suspended over the center of the home cage and dialysates were collected in vials during concomitant behavioral testing. The subjects were tested in the conventional SRT with juvenile (20- to 25-day-old) rats of both sexes as social test stimuli, and with a 120- or 30min IEI. SRM was assessed by the reduction of SIT with the familiar juvenile, expressed as the ratio of SITs during the second and first presentation trials [RID (ratio of investigation duration) scores]. Pilot studies had shown that untreated male rats recognize a familiar juvenile after a 30-min IEI (RID range, from 0.5 to 0.6), but not after a 120-min IEI (RID, approximately 1.0). In the first experimental test (described below), those rats that showed enhanced SRM were retested the next day with a different juvenile during the second presentation to ensure that the significant reduction in investigative time with the familiar juvenile was due to factors specific to the individual juvenile (i.e., expected RID, approximately 1.0). Two types of experimental tests were performed with the SRT. In the first test SRM was assessed in rats that were also monitored for release of AVP within the SON in response to osmotic stimulation of this brain site. SON microdialysis probes were perfused for two consecutive periods, each lasting 30 min. The first perfusion was started 35 min before the first presentation with the juvenile, and the second perfusion followed immediately thereafter during the first presentation. The rats were assigned to one of four groups (n ¼ 9 rats/group) depending on whether the first presentation trial occurred in the absence of SON microdialysis (untreated group), during microdialysis of the SON with isotonic aCSF (isotonic group) or with aCSF containing 1 M NaCl (hypertonic group), or after microdialysis of the SON with hypertonic aCSF (after-hypertonic group).

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The results of this experimental test are presented in Fig. 2. The individual bars in Fig. 2A depict the AVP content per 30-min sample of dialysate recovered from the SON during isotonic, during hypertonic, and after hypertonic stimulation of this nucleus. As noted, osmotic stimulation (during hypertonic) tended to increase AVP release from the SON, and the amount of AVP released during the typical rebound AVP release effect that occurs during the poststimulation interval (after-hypertonic group) was significantly greater than that observed during SON microdialysis with isotonic and hypertonic aCSF. The bar graphs (columns) in Fig. 2B represent the RID scores obtained under the different osmotic treatment conditions used in this test. Analysis of the data presented in Fig. 2B indicated that SRM was improved only during the rebound release of AVP within the SON in the 30 min after osmotic stimulation. Moreover, reexposure to a different juvenile (toned column in Fig. 2B) showed that the memory-enhancing effect was specific to the familiar juvenile. Figure 2C depicts the relationship between RID scores and AVP levels in SON dialysates collected simultaneously from the same rats. The weak but significant correlation between the two variables indicated that the better the SRM (the lower the RID scores) the greater the osmotically induced release of AVP from SON. For the second test additional groups of rats were used to investigate the effects of a V1 receptor antagonist, d(CH2)5[Tyr(Me)]AVP, on the SRM effects induced by osmotic stimulation of the SON. The SRT was given over a 2-day period, the first day without the V1 antagonist (aCSF alone), the second day with the V1 antagonist (aCSF containing the V1 antagonist, 40 ng) delivered into the right SON, mediolateral septum (MLS), or the ipsilateral central amygdala during the two 30-min dialysis periods before and during osmotic stimulation of the SON. The osmotic stimulus (hypertonic aCSF) was delivered via the SON microdialysis probe 35 min before the first presentation of the juvenile. A control group implanted with the SON dialysis probe was tested under the same conditions but was not osmotically stimulated (i.e., SON dialyzed with isotonic aCSF). Effects of the V1 antagonist delivered to the SON and the MLS are presented in Fig. 3. On day 1, SRM was enhanced during the hypertonically induced intranuclear rebound release of AVP that followed osmotic stimulation of the SON in rats that had received aCSF alone in the SON and MLS [RID scores were significantly reduced in both osmotically stimulated groups (after-hypertonic SON and after-hypertonic MLS) relative to nonstimulated controls (isotonic)]. On day 2, the osmotically induced facilitation of SRM observed on day 1 was partially abolished by delivery of the V1 antagonist to the SON and MLS [no significant difference in RID scores between nonstimulated untreated controls (isotonic plus aCSF) and the two osmotically stimulated V1 treatment groups (the after-hypertonic plus V1 antagonist-treated SON and the after-hypertonic plus V1 antagonist-treated

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FIGURE 2 Effects of microdialysis with isotonic or hypertonic aCSF on the release of AVP within the right SON and on performance in the social recognition paradigm. The rats (n ¼ 9 per group) were behaviorally tested in the social recognition paradigm with first exposure to the juvenile without SON microdialysis (untreated), during SON microdialysis with isotonic or hypertonic aCSF or after SON microdialysis with hypertonic aCSF (i.e., during ‘rebound’ release of AVP). (A) Each bar represents the mean pg AVP þ/SEM recovered during 30-min microdialysis with the treatment indicated. Osmotic stimulation (hatched bar) tended to increase AVP release within the SON (during hypertonic). Note the typical ‘rebound’ release of the neuropeptide during the poststimulation interval (after hypertonic; þþp < 0.01 vs isotonic and during hypertonic, ANOVA). (B) The mean ratio of investigation duration (RID) þ/SEM is shown for treatment via microdialysis probe during the first exposure. RID was not altered during SON microdialysis with isotonic or hypertonic aCSF. However, first exposure to the juvenile after microdialysis with hypertonic aCSF followed by isotonic aCSF (i.e., during the ‘rebound’ release of AVP) caused significantly improved social recognition (**p < 0.01 vs all other columns, ANOVA), whereas exposure to a different juvenile during the same period showed this to be juvenile-related memory (toned column; p < 0.01 vs same juvenile after hypertonic. (C) The correlation between RID and AVP levels in SON microdialysis samples collected simultaneously from the same animals was significant (r ¼ 0.425. p < 0.05). Source: Engelmann et al., 1994 (Fig. 2, p. 393). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

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FIGURE 3 Ratio of investigation duration (RID; means þ SEM) of adult male Wistar rats implanted with a microdialysis probe in the right SON alone (n ¼ 11, toned columns), or additionally in the mediolateral septum (n ¼ 8, hatched columns); the animals received the osmotic stimulus (hypertonic aCSF) via the SON microdialysis probe 35 min before first exposure to a juvenile. For comparison, a control group implanted with a microdialysis probe in the SON was tested under the same conditions except that no osmotic stimulus was applied (n ¼ 9, open columns). Again, first exposure during the intranuclear ‘rebound’ release of AVP (after hypertonic) on the first day decreased RID (indicating improved social recognition) in both experimental groups (*p < 0.05 vs isotonic control, ANOVA). However, microdialysis administration of the V1 antagonist d(CH2)5[Tyr(Me)]AVP either into the SON (toned column, after hypertonic þ V1 antagonist) or into the septum (hatched column, after hypertonic þ V1 antagonist; 40 ng of the antagonist was delivered during 2 consecutive 30-min dialysis periods in either brain area) on the second day partially abolished this memory-facilitating effect. Source: Engelmann et al., 1994 (Fig. 3, p. 394). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

MLS groups)] (see Fig. 3). Although not presented in Fig. 3, the results also showed that direct injection of the V1 receptor antagonist into the ipsilateral medial nucleus of the amygdala significantly interfered with the improved recognition memory induced by osmotic stimulation of the SON (i.e., IEI, 120 min; RID after control injection of aCSF, 0.5; RID after injection of the V1 antagonist, 1.8; p < 0.01). The findings that osmotic stimulation of the SON increased AVP release within both the SON and MLS, and also enhanced SRM, suggested that the endogenous AVP released at this time was responsible for the enhanced SRM. The observation that blockade of VP-ergic transmission in both

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brain sites interfered with this osmotically induced memory enhancement supported this interpretation. It was suggested that direct osmotic stimulation via microdialysis activated a ‘‘functional SON–septum axis’’ (pathway from the SON to the septum) causally responsible for the observed SRM improvement. The present findings together with the observation that electrical stimulation of the SON increased septal release of AVP (DemotesMainard et al., 1986) support this suggestion. Moreover, the finding that microinjection of the V1 antagonist into the medial amygdala interfered with the SRM improvement induced by osmotic stimulation of the SON led to the proposal that this brain site is part of the ‘‘functional SON–septum axis’’ involved in VP-ergic mediation of olfactorybased SRM. The direct anatomical connections between the olfactory system and the medial amygdala (Brennan et al., 1990) are in accord with this proposal. c. Landgraf et al. (1995) Landgraf et al. (1995) designed a study to test the ability of antisense oligodeoxynucleotide (AS oligo) treatment, an AVP receptor knockdown strategy to influence SRM and anxiety in male Wistar rats when chronically infused into the septal area. Only the testing pertinent to SRM is discussed here. Application of AS oligo to a brain area interferes with synthesis of the AVP V1 receptor and is an alternative to antagonists or immunotoxins as a strategy for disrupting AVP neurotransmission in that area. The authors noted several potential advantages of this technique compared with AVP antagonists in studying the behavioral effects of endogenous AVP (see Landgraf et al., 1995, for further discussion). In this study, the AS oligo was chronically infused via an osmotic minipump into the septum of adult male rats, and Ringer’s solution (vehicle), scrambled sequence oligodeoxynucleotide (SS oligo), and sense oligodeoxynucleotide (S oligo) served as controls. The social discrimination test (SDT) was used to assess SRM, with a 4-min exposure period during the first and second presentations and a 30- or 120-min IEI. Behavioral testing was carried out on the evenings of days 3 and 4 after implantation of the minipump–tubing– cannula device used for delivery of the treatments to the septal area. After behavioral testing the animals were killed and the brains were removed and either examined for histological verification of the infusion site (all rats were treated with intracerebroventricular AVP) or the septal brain areas were dissected and prepared for receptor autoradiography. The first experiment was designed as an attempt to verify that the experimental conditions per se did not interfere with social discrimination abilities. Subjects received 3 days of vehicle infused into the mediolateral septum (MLS) via the osmotic minipumps. Immediately after the first presentation on day 3, they additionally received intracerebroventricularly administered vehicle solution (5
l infused over a 1-min period) and similarly, on day 4, either intracerebroventricularly administered V1 AVP

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receptor antagonist d(CH2)5[Tyr(Me)]AVP (100 ng/5
l) or synthetic AVP (1 ng/5
l) (IEI, 30 and 120 min, respectively). The results of the first experiment indicated that (1) on day 3 after intraseptal implantation, intracerebroventricularly administered vehicle immediately after the first presentation trial did not affect normal SRM [i.e., the simultaneously present novel juvenile was investigated for a significantly longer time than the preencountered (same) juvenile during the second presentation trial after a 30-min IEI]; and (2) on day 4, the control rats intracerebroventricularly injected with the V1 receptor antagonist immediately after the first presentation were impaired in SRM (no significant difference in investigative time directed to the same and different juveniles during the second presentation trial 30 min after the first one), whereas those similarly injected with AVP were facilitated in this type of memory (significantly shorter time investigating the same compared with the different juvenile in the second presentation trial 120 min after the first one). In a second experiment they examined the effects of oligo treatment on SRM. Independent groups of septally implanted rats received vehicle, SS oligo, S oligo, or AS oligo, and were tested in the evening of day 3 (30-min IEI). After the first presentation period on day 4, some of the rats from the vehicle, S oligo, and AS oligo treatment groups were infused intracerebroventricularly with synthetic AVP (1 ng/5
l) over a 1-min period, and tested with a 120-min IEI. The results of the second experiment indicated that (1) intraseptally infused AS oligo over a 3-day period significantly interfered with normal SRM (both the same and different juveniles were equally investigated after a 30-min IEI); (2) chronic intraseptal infusion with either SS oligo or the vehicle solution did not affect normal SRM (the shorter time investigating the same relative to the different juvenile after the 30-min IEI was highly significant for both groups); (3) although the animals intraseptally infused with S oligo were still able to recognize the same juvenile after the 30-min IEI, the difference in investigative behavior directed toward the same and different juveniles was less significant than that for the vehicle- and SS oligotreated animals; and (4) on day 4, intracerebroventricularly injected AVP given to the AS oligo and S oligo treatment groups immediately after the first presentation trial did not preserve SRM after the 120-min IEI, although it did for the vehicle control group (i.e., both types of intraseptally infused oligos similarly interfered with the SRM-enhancing effects of the exogenous peptide). Results of the postmortem analyses performed on whole brains or septal areas were as follows: (1) histological study indicated that the infusion cannulas of the rats treated with intracerebroventricular AVP were precisely located in the MLS; (2) receptor autoradiography, which assessed [3H]AVP binding to V1 receptors in the septum, indicated that in contrast to vehicle or SS oligo, receptor density was markedly reduced by AS oligo treatment, and

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slightly reduced by S oligo treatment. It also showed that these treatment effects did not spread to the central amygdala or to the bed nucleus of the stria terminalis; and (3) RNA analysis demonstrated that after infusion of AS oligo into the septal region,V1 receptor mRNA levels were markedly increased compared with those of vehicle- and SS oligo-infused rats. The S oligo-treated rats showed a reduction in V1 receptor mRNA. Taken together, the results of this study suggested that AS oligo treatment selectively reduced synthesis of the V1 receptor in the infused limbic site (septal area) and that this receptor subtype is critically involved in the mediation of SRM. The findings also suggested that some degree of interference with the AVP–receptor interaction occurred in the septal area after local administration of S oligo. The authors noted that the antisense-targeting technique has been used to manipulate synthesis/release of AVP (Skutella et al., 1994) and OT (Neumann et al., 1994). In addition to the absence of the drawbacks associated with VP antagonist treatment (e.g., crossreacting with OT receptors, thereby also preventing behavioral effects of OT; Di Scala-Guenot et al., 1990) and the Brattleboro rat model (see Chapter 4), the present findings demonstrate that this antisense-targeting technique is a highly useful tool for revealing ‘‘relationships between local gene expression, neuropeptide–receptor interaction in distinct brain areas, and behavioral performance’’ (Landgraf et al., 1995, p. 4250). d. Everts and Koolhaas (1997) Everts and Koolhaas (1997) investigated whether the involvement of the septal VP-ergic system in SRM (Dantzer et al., 1988; Engelmann and Landgraf, 1994; Landgraf et al., 1995) also extends to the inanimate environment. To this end, they tested adult male Wistar rat subjects in their home cages for the effect of a VP receptor antagonist in the lateral septum (LS) in two comparable paradigms, one designed to test social, the other object, recognition. The object recognition task was comparable to the social memory test in both time course and test settings. The object equivalent of the same juvenile was a gray plastic food cup, that of the different juvenile, a transparent Erlenmeyer flask equal in size to the food cup. Social investigative behavior consisted of anogenital sniffing, close following, and pawing directed toward the juvenile; object investigative behavior included object dragging, pushing, gnawing, and sniffing. Recognition memory was defined by a significant reduction in investigative time in the second relative to the first encounter with the same juvenile (or object). Rats were initially tested in the two tasks under nontreatment conditions, with a 30- or 120-min IEI in tests with the same juvenile (or object), and a 30-min IEI for sessions with a different juvenile (or object). The results were as follows: (1) SRM occurred when the animals were presented with the same juvenile after the 30-min IEI. Time spent in investigative behavior (mainly anogenital sniffing) directed toward the same, but not the different,

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juvenile was significantly decreased during the second versus the first encounter; (2) as expected, SRM did not occur for the same juvenile after a 120-min IEI (no significant reduction in social investigative time during the second encounter with the same juvenile); (3) the time spent investigating the object (mainly sniffing and manipulating the object) during the first presentation was the same as that spent with the juvenile (about 180 s); (4) investigative time during the second trial was significantly reduced from the first trial whether the subject was shown the same or the different object (i.e., investigative time reduced by 40 s for both objects); and (5) an IEI of 120 min resulted in a slight (nonsignificant) reduction in investigative behavior toward the preencountered object during the second trial. After the completion of initial baseline testing, osmotic minipumps and brain cannula guides were implanted for bilateral infusions of the vasopressin V1 receptor antagonist [dPTyr(Et)]AVP (1 ng/0.5
l per hour) or physiological saline into the LS. Behavioral testing began 7 days after recovery from surgery. All rats were first tested for object recognition [a first investigative trial (5 min) followed 30 min later by a second trial with the same object]. After 1 day of rest all rats were tested for social recognition using the same paradigm (two exposures with a 30-min IEI). The data for the object recognition task indicated that (1) treatment with the VP antagonist resulted in a slight but not significant reduction in initial object investigative behavior (saline controls tended to spend more time investigating the object than did the VP antagonist-treated rats during the first encounter); and (2) the VP antagonist did not influence object recognition after the 30-min ITI (during the second encounter, both controls and antagonist treatment groups significantly decreased the duration of their investigation of the reencountered object by approximately 40 s). The results of the SRT indicated that (1) the VP antagonist did not interfere with social investigative behavior during the first encounter [both the saline- and AVP antagonist-treated rats spent equal amounts of time investigating the juvenile (about 180 s)]; and (2) the VP antagonist impaired normal SRM (when tested 30 min later with the same juvenile, the saline controls significantly reduced their investigative time by 40 s, whereas those given the VP antagonist increased their investigative time to above 200 s). The authors made the following points in the course of discussing these results: (1) this study upheld previous findings indicating the important involvement of LS V1 receptor-mediated neurotransmission in SRM (Dantzer et al., 1987; Popik et al., 1992; Van Wimersma Greidanus and Maigret, 1996), and also showed that object recognition appears to be independent of this system; (2) however, the present findings should not be interpreted to suggest that the LS itself is not important for object recognition, because there is evidence to the contrary. Lesioning the LS reduced the animal’s preference to investigate a novel object, probably because of insufficient processing of sensory information (Myhrer, 1989), and disruption of

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large parts of the septum after septal or medial frontal cortical damage impairs exploratory activity and habituation during object displacement (Poucet, 1989); and (3) relevant to the present findings is the importance of processing in the olfactory vomeronasal system (includes projections to the septal area via the medial amygdala and BNST) for social recognition memory (Bluthe and Dantzer, 1993; Popik et al., 1991; Simerly, 1990). This pathway processes species-specific olfactory cues used for identifying conspecifics, and appears not to be involved in object recognition, which instead may be mediated through the main olfactory system. e. Everts and Koolhaas (1999) Everts and Koolhaas (1999) tested the effect of infusing a V1//V2 receptor antagonist into the lateral septum (LS) on SRM (tested with the SRT), spatial memory (tested with the Morris water maze, MWM), and anxiety-related behavior (tested with elevated plus maze) in male Wister rats. Only the testing for SRM is reported here. The V1/V2 receptor antagonist [1-(-mercapto-,-pentamethylenepropionic acid)-2-(O-ethyl)-d-tyrosine, 4-valine]arginine vasopressin [d(CH2)5[dTyr(Et)]VAVP] used in this study was shown to be as potent as the most commonly used V1 antagonist d(CH2)5[Tyr(Me)]AVP (Engelmann et al., 1992a,b). 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 be present in the septum as well (Engelmann et al., 1992a; Landgraf et al., 1991a; Ramirez et al., 1990). Saline or the V2/V1 antagonist (2 ng/
l, sufficient 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 rats were tested in the SRT with juvenile male conspecifics as social test stimuli and with a 30-min IEI. The time spent investigating the same juvenile (anogenital sniffing, close following, and pawing) in the second relative to the first trial was the measure of SRM. The results of this testing were as follows: (1) both treatment groups were comparable in the time spent investigating the juvenile in the first presentation trial (about 180 s); and (2) unlike the saline-treated controls, which decreased investigative activity by 40 s on reencountering the juvenile 30 min later, the rats treated with the VP antagonist increased it by 20 s (significant treatment  exposure interaction on ANOVA, and significant difference between the two groups confirmed by post hoc t testing). These findings offer additional support for a role for septal AVP in SRM in the male rat, and also indicate that a VP antagonist relevant to V2 as well as V1 receptors blocks this form of memory. Moreover, additional behavioral testing with the MWM (see Chapter 10) suggested that the LS VP system is fairly specific for SRM, because it appears to be involved neither in spatial learning and memory, nor in object recognition memory (Everts and Koolhaas, 1997).

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A. General Comments Dluzen and coworkers (1998a,b, 2000) carried out several studies with the social discrimination test (SDT) to examine the role of olfactory bulb (OB) VP and OT in mediating SRM in the male rat (described below). Several lines of evidence stimulated their interest in this research question. First, these peptides act in various brain sites to modulate SRM (Popik and Van Ree, 1991; Popik et al., 1992; Van Wimersma Greidanus and Maigret, 1996). Second, this memory appears to be heavily reliant on the olfactory system (Sawyer et al., 1984). Third, the peptides (Dogterom and Buijs, 1980; Halasz and Shepherd, 1983), as well as VP-binding sites (Levy et al., 1992), are present in the OB. Fourth, these peptides are released within the OB of ewes during social interactions involving recognition processing (Levy et al., 1995). 1. Selected Studies a. Dluzen et al. (1998a) Dluzen et al. (1998a) examined the effects of infusions of VP, OT, and their antagonists into the olfactory bulb (OB) on SRM in male rats. The subjects, adult male Wistar rats, were tested in the SDT with male or female juvenile (21–30 days of age) social test stimuli. Depending on the agent tested, the IEI was either 30 or 120 min, because SRM is typically present with the former and absent with the latter (e.g., Dantzer et al., 1987; Thor and Holloway, 1982). On the day of testing, the vehicle or peptide was infused into the OB over a 10-s interval via two infusion cannulas inserted through previously implanted guide cannulas. Behavioral testing began within 1 min of infusion. At the conclusion of behavioral testing, the animals were killed and their brains were visually inspected to verify cannula placement. The rats in a given treatment group received a 1-
l solution of one of the following agents: sterile Ringer’s solution (vehicle), AVP (0.5 ng/
l), OT (0.5 ng/
l), the V1 antagonist d(CH2)5[Tyr(Me)]AVP (AVP-Ant, 5.0 and 50.0 ng/
l), or the OT receptor antagonist desGly-NH2d(CH2)5[Tyr(Me)2, Thr4]OVT (OT-Ant, 5.0 and 50 ng/
l). The duration of the IEI for each treatment was as follows: vehicle, 30 and 120 min; AVP and OT, 120 min; AVP-Ant and OT-Ant, 30 min. The 120-min IEI in the AVP and OT treatment conditions assessed the recognition-preserving effects of the peptides, and the 30-min IEI used in the AVP-Ant and OT-Ant treatment conditions tested the putative recognition-interference effects of these antagonists. A t test determined whether the tested males exhibited SRM under each treatment condition (i.e., directed significantly more investigation time to the novel juvenile, compared with the preencountered juvenile, during the second trial). A separate sample of urethane-anesthetized subjects received

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an infusion of 1
l of radiolabeled AVP (125I-labeled AVP) into the OB to determine the approximate area of peptide diffusion through this structure and into the frontal cortex, septal area, and cerebrospinal fluid (CSF). The results of the statistical analyses indicated that (1) the vehicle control subjects recognized the preencountered juvenile after the 30-min, but not the 120-min, IEI (i.e., a significantly greater amount of time was directed to the novel versus the same juvenile after an IEI of 30, but not 120, min); (2) infusion of a 0.5-ng dose of either AVP or OT into the OB facilitated SRM after an IEI of 120 min (i.e., these subjects spent significantly more time investigating the novel juvenile, compared with same juvenile, after an interval when control animals spent similar times investigating both types of juveniles); (3) infusion of AVP-Ant at both the 5- and 50-ng dose levels did not interfere with normal SRM tested at the 30-min IEI (i.e., at both dose levels the subjects spent significantly more time investigating the novel juvenile, compared with the same juvenile, in the second trial after a 30-min IEI, as did the controls); and (4) as with AVP-Ant, OB-infused OT-Ant at both the 5- and 50-ng dose levels failed to interfere with normal social recognition tested with a 30-min IEI. The results of the spread of diffusion of the 125I-labeled AVP bilaterally infused into the OB, expressed as the mean percentage of total activity relative to that in the OB, were as follows: frontal cortex, 23.1%; septal area, 0%; and CSF, 0%. This indicates that the peptide infused into the OB remained primarily localized within that structure. Most important for interpreting the present results is that although the septal area is particularly responsive to the modulatory effects of these peptides [e.g., Dantzer et al., 1987, 1988 (see Chapter 12); Engelmann and Landgraf, 1994; Engelmann et al., 1994; Everts and Koolhaas, 1997], they were unlikely to have activated this area and thus its complicating effects can be ruled out. The following points were made during discussion of these results: 1. These findings revealed that the OB, like the septal area, is a target structure that mediates memory-modulating effects of these peptides on SRM. Although these data may represent only pharmacological effects of the peptides, it was noted that in ewes the release of OB AVP and OT occurs under physiological conditions, and evidence suggests that OT release in this structure may be involved with promoting maternal recognition of her lamb offspring (Levy et al., 1995). 2. The lack of an effect on normal SRM after AVP-Ant and OT-Ant infusion into the OB was compared with the reported results of infusions of these antagonists into the septal area. This comparison indicated similar findings for intraseptal infusions of OT-Ant (Van Wimersma Greidanus and Maigret, 1996), but opposite findings for AVP-Ant, which interferes with normal SRM [Dantzer et al., 1988 (see Chapter 12); Engelmann and Landgraf, 1994; Everts and Koolhaas, 1997].

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3. The failure of these OB-infused antagonists to interfere with normal SRM (a 30-min IEI) was contrary to what would be expected if their presence in this brain site played a physiological role in this type of memory processing. Two possibilities may have accounted for this failure: (a) the dose levels of the antagonists were insufficient to produce a behavioral effect. However, two facts argue against this explanation: (i) the two dose levels used were separated by a 10-fold difference in range, and (ii) the 5-ng/
l dose of AVP-Ant, which did not block normal SRM (tested with a 30-min IEI) when infused into the OB, did so when injected into the lateral septum (Dantzer et al., 1988); and (b) at the time of treatment, there was insufficient ongoing activity in the VP and OT receptor neurons in the OB to be influenced by the blocking action of the antagonists. This condition was considered analogous to results obtained when AVP-Ant was infused into the SON. Under basal conditions (analogous to the present study), the antagonist was ineffective in influencing SRM, but blocked this memory under circumstances (i.e., osmotic stimulation) that enhanced AVP activity in the SON (Engelmann and Landgraf, 1994). Whatever the reasons for the asymmetric agonist–antagonist effects observed in this study, these researchers noted that, in general, SRM is ‘‘preserved’’ (extended in its normal duration from 30 to 120 min) after infusion of VP and/or OT agonists into relevant brain sites (e.g., Dantzer et al., 1988; Engelmann and Landgraf, 1994; Van Wimersma Greidanus and Maigret, 1996), whereas their antagonists do not in all cases block the ‘‘display’’ of normal SRM tested with the 30-min IEI (Engelmann et al., 1998; Popik et al., 1992) despite what might be expected. Taken together, these findings led to the suggestion that ‘‘the underlying mechanisms by which peptides function within the olfactory bulb differ as a function of whether they are involved with the display versus the preservation of recognition responses’’ (Dluzen et al., 1998a, p. 999). b. Dluzen et al. (1998b) Dluzen et al. (1998b) selectively depleted noradrenaline (NA) from the OB and assessed its effect on AVP- and OTinduced preservation of SRM in the male rat. The rationale of this study was based on several findings, which together suggested that when AVP and OT are infused into the OB, activation of the NA system in this site may be one mechanism by which they act to preserve SRM (i.e., extend its duration from 30 to 120 min). These findings include (1) the demonstration that release of NA into the OB was critical for memory/recognition responses associated with reproduction (Brennan et al., 1990; Kaba and Nakanishi, 1995); (2) the presence of VP and OT in the OB (Dogterom and Buijs, 1980; Halasz and Shepherd, 1983; Levy et al., 1995); and (3) the observation that these peptides activated NA release in the OB of sheep (Levy et al., 1995).

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The procedure and coordinates for cannula implantation in the OB were identical to those used by Dluzen et al. (1998a). OB depletion of NA was achieved by a bilateral infusion of 1.0
l of 6-hydroxydopamine (6-OHDA; dissolved in Ringer’s solution to a concentration of 20
g/
l) via guide cannulas into the OB. The OB in control rats was bilaterally infused with Ringer’s solution (vehicle). During behavioral testing, the subjects received a 1.0-
l infusion of vehicle, AVP (0.5 ng/
l), or OT (0.5 ng/
l) over a 10-s interval into the OB via infusion cannulas inserted through guide cannulas. The animals were tested in the SDT with a 120-min IEI to assess the recognition preservation effects of AVP and OT, and 1–2 days later under nontreatment conditions, with a 30-min IEI to determine the ability of each animal to demonstrate a normal recognition response. Paired t tests were used to determine whether recognition responses were present or absent under the different test conditions (i.e., differences in SIT spent with the same versus the novel juvenile during the second exposure period). At the completion of testing, the animals were killed and the brains were visually inspected to confirm cannula location within the OB. The OB was then removed and prepared to determine NA concentrations by highpressure liquid chromatography (HPLC) with electrochemical detection (see Feldman et al., 1997, for a description of HPLC). The mean OB NA concentrations were calculated for each of three groups defined on the basis of the status of the 6-OHDA-induced chemical lesion (i.e., NA depletion) and the peptide treatment received during behavioral testing: (1) lesioned rats given AVP treatment, (2) lesioned rats given OT treatment, and (3) nonlesioned rats given either AVP or OT treatment. The data for these three groups were statistically evaluated in a one-way ANOVA. The behavioral results were as follows: (1) when tested with the 120min IEI, AVP infusion into the OB preserved SRM in the nonlesioned rats (a longer time was spent investigating the novel versus the same juvenile), but not in the 6-OHDA-lesioned rats (no significant difference in SIT spent with the novel versus the same juvenile); (2) when retested 1–2 days later under nontreatment conditions with a 30-min IEI, the 6-OHDA-lesioned rats displayed normal SRM (a significantly greater amount of SIT was spent with the novel compared with the same juvenile); (3) when tested with the 120-min IEI, OT infusion into the OB preserved SRM in the nonlesioned rats (significantly longer time spent investigating the novel versus the same juvenile), but not in the 6-OHDA-lesioned rats (no significant difference between the times spent investigating the same and the different juvenile); and (4) retesting 1–2 days later under nontreatment conditions with a 30-min IEI indicated that the lesion itself did not impair normal SRM (a statistically significant greater amount of time was spent investigating the novel juvenile). The results of the postmortem analysis were as follows: (1) the OB NA concentrations [picograms of NA per milligram wet tissue weight

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(mean  standard error of the mean) for the lesioned rats treated with AVP, 39.2  8.7; for lesioned rats treated with OT, 47.4  8.7; and for nonlesioned rats receiving AVP or OT treatment, 205  18.3]; (2) the ANOVA and post hoc comparisons indicated that the 6-OHDA lesion significantly reduced NA levels in the OB; and (3) specifically, there was an overall significant difference among these three groups, and the post hoc pairwise comparisons indicated that the OB NA concentration in the nonlesioned AVP/OT group was significantly greater than those in the two lesioned groups, which did not differ from each other. The following points were made in the discussion of these results: 1. Previous research supports the argument that the 6-OHDA lesion effects observed in this study were primarily due to depletion of NA in the OB rather than to secondary effects of lesion-induced depletion of DA or serotonin (5HT) in the OB, or NA depletion in other brain sites (Doty et al., 1988; Guan et al., 1993; Royet et al., 1983). 2. The findings that the 6-OHDA lesion per se did not impair the display of normal social recognition with a 30-min IEI under nonpeptide treatment, and that AVP and OT infusion into the OB in nonlesioned rats preserved SRM with a 120-min IEI in non-6-OHDA-treated rats, strongly suggest a specific interaction between the peptides and OB NA in social recognition preservation. 3. Moreover, the failure of the 6-OHDA lesion to interfere with the display of normal SRM (tested with a 30-min IEI) under nontreatment conditions, whereas it obliterated the ability of AVP and OT to preserve SRM (tested with the 120-min IEI), suggests that markedly different mechanisms apply to these two testing conditions. Specifically, these findings suggest that the display of normal SRM involves an OB NA-independent process, whereas the peptide-induced preservation of SRM involves an OB NA-dependent process. The findings that AVP and OT can modulate the release of NA within the OB of sheep (Levy et al., 1995) is consistent with the proposal that infusion of these peptides into the OB preserves social recognition by activating the NA system in that brain structure. 4. The possibility that the VP/OT interaction with the NA system in the OB may preserve SRM indirectly by a selective attention effect was raised in light of the popular view that the locus coeruleus NA system, from which the OB receives substantial input (Shipley et al., 1985), promotes selective attention by releasing NA within numerous sensory target systems in the brain (Robbins, 1997; Sara et al., 1994; Vankov et al.,1995). c. Dluzen et al. (2000) Dluzen et al. (2000) carried out four experiments designed to evaluate the relationship between the OB NA system and intra-OB infusion of OT in the preservation of SRM memory in the male Wistar rat. This study was instigated by previous work, done by these investigators, suggesting that an OB NA-dependent mechanism is involved in

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the ability of OT to preserve recognition responses (Dluzen et al., 1998b; discussed above). The animals in experiments 1–3 were tested in the SDT after recovery from surgical implantation of a guide cannula as done in previous investigations of the effects on SRM of bilateral infusion of OT into the OB (Dluzen et al., 1998a,b). General testing conditions, and assessment of social recognition, were also the same as those of the earlier studies. The doses used in all these experiments were selected because of their effectiveness in other related paradigms (Kaba and Keverne, 1988; Liebsch et al., 1996; Roozendaal et al., 1992), and represent attempts to approximate physiological concentrations when taking into consideration such variables as localized administration, diffusion, and permeability through membranes (experiment 4). The purpose of experiment 1 was to determine whether blocking presumptive OT receptors within the OB would abolish the ability of OT to preserve social recognition responses. Three perfusion groups received a 1-
l intra-OB bilateral infusion of OT diluted in Ringer’s solution (0.5 ng/
l) on its own (group 1) or coinfused with a highly selective OT receptor antagonist, desGly-NH2, d(CH2)5[Tyr(Me)2, Thr4]OVT (group 2); or with a highly selective V1 receptor antagonist, d(CH2)5[Tyr(Me)]AVP (0.5 ng/
l) (group 3). These rats were then tested for OT-induced preservation of SRM in the SDT with a 120-min IEI. The rats that received the OT antagonist were retested 2 days later with no infusions, and with a 30-min IEI, to determine whether they were capable of displaying normal SRM. The results indicated that the OT-induced preservation of SRM was mediated by an OT receptor in the OB, because this facilitated memory effect was blocked by a coinfusion of OT with an OT receptor antagonist (desGlyNH2, d(CH2)5[Tyr(Me)2, Thr4]OVT), but not with a VP V1 receptor antagonist (d(CH2)5[Tyr(Me)]AVP). Experiment 2 examined whether infusion of an 2-noradrenergic agonist, clonidine, could preserve SRM, and thus ‘‘provide some potential indication for the actions of OT’’ (Dluzen et al., 2000, p. 761). To examine the specificity of its effect, a separate group of rats was similarly tested with the -noradrenergic agonist isoproterenol. The results indicated that adrenoceptors, but not -adrenoceptors, in the OB influence SRM in male rats, because infusion of an -adrenoceptor, but not a -adrenoceptor, agonist preserved SRM (rats in the former but not the latter treatment group spent significantly less time investigating the familiar juvenile, compared with the novel juvenile, after the 120-min IEI). The findings suggested that the increased output of OB NA that results from OT appears to activate -adrenoceptors to produce this preservation in recognition because infusions of clonidine into the OB preserve recognition responses in a manner similar to that observed with OT.

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The purpose of experiment 3 was to determine whether the OT-dependent preservation of recognition was mediated by postsynaptic activation of - or -adrenoceptors. The animals received either a coinfusion of OT (0.5 ng/
l) and an -adrenoceptor antagonist (phentolamine; 40 nM), or of OT (0.5 ng/
l) and a -adrenoceptor antagonist (timolol; 40 nM). The animals were all tested with the 120-min IEI. A separate group of rats receiving an infusion of the -adrenoceptor antagonist (40 nM) and tested after a 30-min IEI was included. Because this antagonist was here found to block OT-dependent recognition it was important to learn whether this -adrenoceptor antagonist itself would block normal SRM. When the -adrenoceptor antagonist (phentolamine) was combined with OT and infused into the OB, it blocked the OT-induced preservation of SRM (the rats in this treatment group showed no significant difference in investigative time spent with the same versus the different juvenile after the 120-min IEI). On the other hand, when the -adrenoceptor antagonist (timolol) was coinfused with OT into the OB, it had no effect on the OTinduced preservation of SRM (rats in this treatment group spent significantly less time investigating the same juvenile, compared with the different juvenile, after the 120-min IEI). The control test with the adrenoceptor antagonist indicated that, when infused into the OB on its own, there was no impairment of normal SRM (these rats spent significantly more time investigating the novel juvenile, compared with the same juvenile, after the 30-min IEI). Experiment 4 was designed to determine whether OT exerts any direct effect on the output of NA within the OB. Each animal was infused with either OT, OT-Ant, or normal Ringer’s solution through preimplanted microdialysis probes (retrodialyzed) to determine whether these agents directly alter OB NA output. The results indicated that OB OT is directly responsible for the release of NA in the OB by activating -adrenoceptors in this structure. NA output (measured by a microdialysis probe) was significantly greater during the 15-min collection interval in which OT was infused in the OB (2–3 ng) than during control intervals; moreover, this increase was not found during treatments with either the OT antagonist or vehicle. In their discussion the authors related these findings to studies of maternal behavior in sheep by Keverne, Levy, and associates (Kendrick et al., 1988a,b; Levy et al., 1995), as well as to their own research on rats (Dluzen et al., 1998a–c). The sheep research indicated that OT released within the OB at parturition, and in response to vaginocervical stimulation (Kendrick et al., 1988a,b), appears to be a primary agent for the onset of maternal behavior and the associated recognition of offspring required for selective bonding. An OT interaction with NA released from brainstem NA-ergic fibers projecting to the OB is an important mechanism underlying

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the OT influence. That is, in sheep, the OT-induced release of NA activates -adrenoceptors that results in offspring recognition (Levy et al., 1995). These researchers proposed that, in the male rat, a similar cascade of events might be necessary for the social recognition response to occur. Earlier studies in their laboratory support this proposal: (1) OT infusion into the OB preserves SRM in the male rat (Dluzen et al., 1998a); (2) this process is abolished by depletion of OB NA (Dluzen et al., 1998b) but not after depletion of OB serotonin (Dluzen et al., 1998c), indicating that the OT-dependent preservation of SRM involves an OB NA-mediated process; (3) additional support is provided by the present findings, and includes the demonstration that (a) OT must activate OT receptors within the OB (experiment 1), and (b) this OT-induced increase in OB NA activates the -adrenoceptor system (experiments 2 and 3), resulting in preservation of the social recognition responses; and (4) finally, the authors noted that although the present findings are based on pharmacological manipulations, they might nevertheless indicate the operation of physiologically significant processes. For example, during copulation endogenous OT is released within the male rat brain (Hughes et al., 1987) and such an increase in the release of endogenous OT within the OB may contribute to an enhanced ability of male rats to discriminate between conspecifics.

VII. VP and OT in the Medial Preoptic Area and SRM

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A. General Comments The medial preoptic area (MPA) is an anterior extension of the hypothalamus that has been implicated in the regulation of gonadal hormone secretion (Hart and Leedy, 1985; Macrides, 1976), and in male (Edwards and Einhorn, 1986; Kondo et al., 1990) and female (Caldwell et al., 1986, 1989) sexual behavior. Both OT-ergic and VP-ergic mechanisms are present in this brain site (Caldwell et al., 1989). Moreover, the MPA is regarded as one of the brain centers involved in processing olfactory information (Macrides, 1976; Pfaff and Pfaffmann, 1969). Given the crucial importance of olfactory processing in rodent SRM, Popik and Van Ree (1991; see below) designed a study to investigate whether VP and/or OT injected into this brain site may influence this processing and therefore SRM. 1. Selected Study: Popik and Van Ree (1991) Popik and Van Ree (1991) used the SRT, with a 120-min IEI, to examine the effect of local injections of VP and OT into the MPA on SRM in the male Wistar rat. A significant decrease in the duration of investigative behavior (anogenital sniffing) in the second relative to the first encounter operationally

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defined SRM. The anogenital investigative time (AIT) during the second encounter was divided by that during the first encounter, and the resulting value was multiplied by 100 to obtain the ratio of investigation duration (RID). RIDs were used in a one-way ANOVA, followed by least significant difference (LSD) tests to compare effects of different doses of the peptides on SRM. In a given treatment session, physiological saline (placebo) or one of the following peptides was bilaterally injected into the MPA: OT (0.03, 0.3, 3, 30, 300, or 1000 pg), AVP (0.03, 0.3, or 3 pg), AVP(4–8) (200 pg), AVP(4–9) (200 pg), or OT (300 pg) plus the OT antagonist desGly-(NH2)9, d(CH2)5[Tyr(Me)2Thr4]OVT (3000 pg). Each subject was tested under all treatment conditions, and successive treatments were separated by intervals of at least 48 h. Two additional experiments included: (1) a control test (presentation of a different juvenile during the second encounter) to rule out nonspecific influences that might be mistaken for peptide-induced memory effects, and (2) determining whether a peptide treatment that facilitated SRM when locally injected into the MPA did so when injected into the septal area. All treatments were given immediately after removal of the juvenile during the first presentation. The results were as follows: (1) placebo-treated subjects were unable to recognize the same juvenile after a 120-min IEI (investigative time during the second encounter was similar to that of the first encounter); (2) all but the lowest dose of MPA-injected OT dose dependently enhanced SRM of the same juvenile after a 120-min IEI (all dose levels of OT, except the 0.03-pg dose, significantly reduced RID ratios relative to those obtained by the placebo-treated rats); (3) control experiments, which presented a different juvenile in the second trial, ensured that this OT dose-dependent memory effect was not attributable to nonspecific drug factors [RID scores for OT (3.0 and 1000 pg)-treated subjects were not significantly different from those of placebo controls]; (4) OT dose levels (100 and 1000 pg) that enhanced SRM of the same juvenile after the 120-min IEI, when injected into the MPA, did not do so when injected into the septal area (RID scores for these OT-treated subjects did not significantly differ from those of placebo controls); (5) pretreatment with an OT antagonist did not block the SRM effect induced by a local injection of OT in the MPA (RID scores were significantly reduced from control values in both the placebo plus OTtreated group, and the OT-Ant plus OT-treated group, tested with the same juvenile after a 120-min IEI), (6) in contrast to OT, local injection of AVP into the MPA did not enhance SRM of the same juvenile after the 120-min IEI (no significant difference in RID scores between placebo- and AVPtreated subjects); and (7) when injected into the MPA, neither AVP(4–8), nor AVP(4–9), at the dose level used here (200 pg), enhanced SRM of the same juvenile after the 120-min IEI [RID scores for AVP(4–8) and AVP(4–9) groups did not significantly differ from control values].

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The following points were made during discussion of these results: 1. The finding that OT, microinjected into the MPA at a low dose range, dose dependently facilitated SRM resembles that reported for peripherally administered OT (Popik et al., 1991). 2. The facilitation of SRM induced by the injection of OT into the MPA appears to be specific, for three reasons: (a) the control experiments with a different juvenile ruled out nonspecific drug effects; (b) although VP is present in the MPA, and is involved in mediating certain behavioral effects [e.g., expression of sex behavior in female rats (Caldwell et al., 1989) and social dominance in hamsters (Ferris et al., 1984)], neither it nor its behaviorally active metabolites [AVP(4–8) and AVP(4–9)] influenced SRM when injected into the MPA; and (c) identical doses of OT that facilitated SRM, when injected into the MPA, were ineffective when injected into the septal area. 3. The failure of the OT receptor antagonist used in this study to block OT enhancement of SRM suggests that the MPA receptors mediating this effect are site and/or task specific because (a) this antagonist attenuated the effects of OT on passive avoidance retention after intracerebroventricular administration (De Wied et al., 1991), and (b) peripheral injection of other OT antagonists blocked OT-induced attenuation of SRM, and facilitated this memory when injected alone (Popik and Vetulani, 1991). 4. It was suggested that the SRM effects of MPA-injected OT might have some relation to the purported role of the peptide in sexual behavior. The following two sets of observations provide supportive evidence of a role for central OT in male and female sexual behavior: (a) OT microinjected into the MPA increases sexual receptivity in female rats, and OT immunoreactive levels in the MPA are higher in receptive females that were mounted by males than in control animals (Caldwell et al., 1989); and (b) OT facilitates male sexual behavior (Arletti et al., 1985), and central OT-ergic systems in males respond to mating (Hughes et al., 1987). 5. It was concluded that the mechanism underlying the MPA/OT-induced memory effect observed in this study may involve ‘‘enhancement of the olfactory signal and/or modification of the processing of olfactory information’’ (Popik and Van Ree, 1991, p. 559). This suggestion is consistent with evidence implicating MPA involvement in processing of olfactory information (Pfaff and Pfaffmann, 1969).

VIII. VP and OT Genetic Knockout Models and SRM

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A. General Comments Two types of genetic neuropeptide ‘‘knockout’’ models (genetic mutations that deplete the organism of brain VP or OT) have been used in studies with SRM. The first, the Brattleboro homozygous diabetes insipidus (HODI)

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rat, is a natural knockout model, characterized by a VP deficiency that results from a natural genetic mutation. Its use in VP/memory research began in the 1970s with studies by De Wied and colleagues (see Bohus and De Wied, 1998, for discussion of this work). The second, the OT knockout mouse, is unable to synthesize OT and has been ‘‘created’’ by genetic techniques used in laboratory-based stem cell research. It is important to recognize the difficulties inherent in interpreting the results of studies with these models (see Bohus and De Wied, 1998; and Chapter 3). In general, an observed deficit could be due to any number of secondarily arising conditions that act to obscure the specific contribution of the neuropeptide under study. Further, the absence of a clear-cut behavioral outcome may reflect the fact that CNS functions such as learning and memory are under the concurrent influence of many peptidergic, aminergic, and cholinergic chemical messengers and not just the one studied. Nevertheless, the results obtained from experiments with knockout models, together with those on intact rats and mice, have provided useful information in neuropeptide/ behavioral study. The research discussed below illustrates the use of VP and OT knockout animals in the study of SRM. 1. Selected Studies a. Engelmann and Landgraf (1994) Engelmann and Landgraf (1994) investigated the role of septal AVP in SRM, using homozygous Brattleboro (HODI) and normal Long-Evans (LE) rats. These authors compared the performance of HODI and LE rats in the SRT before and after microdialysis administration of AVP or a V1 receptor antagonist, d(CH2)5[Tyr (Me)]AVP, into the mediolateral septum (MLS). Juveniles (20–25 days old) of both sexes were used as social stimuli. The two 5-min presentations were separated by either a 30- or 120-min IEI, and the same or a different juvenile was presented during the second presentation trial. Untreated behavioral performance was tested in the first week [two sessions: 30-min IEI, same juvenile; and 30-min IEI, different juvenile; for the LE rats another session (120-min IEI, same juvenile) was added]. Subsequent to this test, the microdialysis probe was implanted with its tip end directed to the MLS. Thirty hours later treatment sessions began, during which the perfusion fluid [artificial CSF (aCSF), or aCSF containing either AVP (0.2 or 2.0 ng/rat) or the V1 receptor antagonist (5.0 ng)] was infused into the septum at a flow rate of 3
l/min. Infusion began 5 min before the first presentation and lasted 30 min. The first 5-min presentation trial occurred during this perfusion interval. Investigative behavior (anogenital sniffing, licking, pawing, and close pursuing of the juvenile) was timed during the first and second presentation trials. The microdialysis site was histologically confirmed on completion of behavioral testing. The results for the untreated sessions were as follows: (1) investigative duration during the first presentation with the same or a different juvenile

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was significantly longer for the HODI rats than for the LE rats; (2) the untreated HODI rats did not exhibit normal SRM after the 30-min IEI (SIT with the same juvenile was of comparable duration as that with the different juvenile during the second exposure); (3) in contrast to the HODI rats, the untreated normal LE rats recognized the same juvenile after the 30min IEI (SIT with the same juvenile, but not the different juvenile, was significantly reduced during the second relative to the first presentation trial); and (4) the LE rats did not recognize the same juvenile after the 120-min IEI (no significant difference between the investigation durations of both presentation trials). The results for the treated sessions indicated: (1) implantation of the microdialysis probe into the MLS and its perfusion with aCSF did not by itself influence the behavioral performance of either rat strain, compared with that in untreated sessions; (2) infusion of either dose of AVP into the HODI rats resulted in normal SRM when tested after a 30-min IEI (SIT was significantly less during the second relative to the first presentation trial with the same juvenile, indicating a juvenile-specific recognition, whereas presentation of a different juvenile resulted in investigative behavior comparable to that of untreated LE rats or aCSF-perfused rats); (3) infusion of AVP into the MLS of LE rats resulted in SRM after a 120-min IEI, and this effect was juvenile specific; (4) infusion of the V1 antagonist into the MLS of LE rats interfered with normal SRM tested after a 30-min IEI (in contrast to aCSFperfused LE rats, there was no significant difference in SIT between the first and second presentation with the same juvenile); and (5) a comparable failure to recognize juveniles after a 30-min IEI was also observed in untreated or aCSF-perfused HODI rats. In the discussion, the authors noted that the importance of intraseptally released AVP for normal SRM was supported by the findings of this study: (1) unlike the normal LE rats, untreated HODI rats were unable to recognize a preencountered juvenile after a 30-min IEI; (2) an intraseptal infusion of AVP via the virtually stress-free microdialysis probe mimicked AVP release patterns in the septum better than intraperitoneal or intracerebroventricular injections of the neuropeptide, improved SRM in the HODI rats to the level of untreated or aCSF-treated normal LE rats, whereas the converse occurred after MLS infusion of the V1 receptor antagonist into the brain of normal LE rats; and (3) an increase in the MLS level of AVP in normal rats, induced by microdialysis of synthetic AVP, significantly improved their performance and indicated that SRM may be manipulated over a relatively wide range. It was concluded that strain differences per se were not accountable for the results obtained in this study because the untreated Long-Evans rats did not behave differently from males of the Wistar strain (Bluthe et al., 1990; Dantzer et al., 1987) or Sprague-Dawley strain (Bluthe and Dantzer, 1990). Moreover, although the Brattleboro and LE rats were obtained from two different breeding farms, which could result in secondary causal effects for

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the observed behavioral differences (Ambrogi Lorenzini et al., 1991; and see Chapter 3), pilot studies using HODI rats from other breeders confirmed the impaired recognition regardless of the inbred rat strain used (Engelmann, unpublished results, cited in Engelmann and Landgraf, 1994). Therefore, it seems clear that the lack of central AVP in the HODI rats was responsible for the impaired acquisition, storage, and/or recall of olfactory cues. In addition to the present findings, observations from other behavioral pharmacological experiments, autoradiographic observations, and electrophysiological studies provide direct or indirect supportive evidence for the following: (1) excess intraseptal levels of AVP, induced by locally administered synthetic AVP, improves SRM memory (Dantzer et al., 1988; Popik et al., 1992); (2) V1 receptor blockade of septal AVP receptors interferes with normal SRM in adult male Wistar rats (Dantzer et al., 1988); (3) highly specific AVP-binding sites are present in the septal brain area of HODI rats (Shewey and Dorsa, 1986) and stimulation of these binding sites by intraseptally administered AVP (Shewey et al., 1989) probably mediated the improved SRM demonstrated in this study; and (4) observations with electrophysiological recording techniques indicate that AVP treatment increases the firing rate of neurons in hippocampal slices from HODI rats (Dreifuss and Muhlethaler, 1982). Altogether, the findings of this study and the above-cited observations support the importance of intraseptal AVP for SRM. b. Ferguson et al. (2000) Ferguson et al. (2000) compared male mice mutant for the OT-encoding gene (OT /) with those normal for this genotype (OT þ/þ or wild-type mice) in a number of tests of SRM, and in follow-up tests to analyze nonmnemonic factors that may contribute to the genotype-dependent differences observed in SRM. SRM was tested in two paradigms [a multitrial social recognition task (MSRT) and the SRT] with the subjects remaining in their home cages. The social stimuli used in this study were wild-type ovariectomized (OVX) female mice (used in each paradigm) or wild-type intact male or female mice (used only in one test with the MSRT). SRM was indicated by a reduction in olfactory investigation on prolonged exposure or repeated encounters with the same conspecific (Kendrick et al., 1997; Keverne and Brennan, 1996; Thor and Holloway, 1981). Several experimental tests were run with the MSRT. In the first test, the investigators measured the duration of social investigation time (SIT) directed toward the same OVX female reencountered during four successive 1-min trials, and that directed toward a different OVX female presented in the fifth 1-min trial. At the end of each 1-min trial the stimulus mouse was removed from the resident’s home cage and returned to an individual holding cage for the 10-min intertrial interval (ITI). SIT significantly declined over prolonged exposure to the same OVX female for the OT þ/þ subjects,

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but not for the OT / subjects. There were no genotype-dependent differences in SIT in trial 5, when a new female stimulus was presented. The investigative behavior shown by the OT þ/þ males can be interpreted as a renewal of interest in the novel social stimulus. The second test was designed to examine the possibility that changes in the female’s behavior associated with repeated encounters with males per se may have contributed to the decline in SIT scores observed in the OT þ/þ males. To this end, the females were rotated so that new males in each of the four trials investigated each female, and a different female was presented to a given subject in each 1-min trial. There was no decline in SIT for either OT þ/þ or OT / mice, supporting a social recognition explanation for the decline in SIT scores observed in the first test. In a third test, the resident mouse was presented with the same social stimulus (a reproductively intact male or female mouse) in each of four 1-min trials. The results showed that the SRM deficit of the OT / mice was not limited to OVX female stimuli (i.e., there was a significant decline in SIT over the repeated presentations for both types of social stimuli in the OT þ/þ mice, whereas the OT / mice showed persistent interest over the course of this test). The SRT with a 30-min IEI was used as a second measure of SRM. The OT þ/þ mice, but not the OT / mice, recognized the reencountered OVX female (SIT significantly declined during the second relative to the first presentation trial for the former but not the latter genotype). Subgroups of OT / and OT þ/þ mice tested for SRM were subsequently used in experimental tests designed to determine whether the observed deficits in SRM might have been due to impairments in olfactory function (olfactory foraging task) or to behavioral inhibition (habituation in olfactory and acoustic startle tests). For the olfactory foraging task, the mice were initially familiarized with the taste of chocolate chip rewards that were eaten in the home cage. The test proper consisted of four trials with a 10-min ITI. For each trial the resident mouse was removed to a holding cage while a chocolate chip was placed in its home cage, either on the surface of the bedding (trial 1) or hidden in different positions beneath leveled bedding (trials 2, 3, and 4). The latency to locate the food reward on its return to the home cage was recorded. Both the OT þ/þ and OT / mice learned to locate the buried food as rapidly as they located food placed on the surface of the cage bedding. The olfactory habituation/dishabituation task consisted of five 1-min trials (10-min ITI) in which a perforated tube containing lemon-scented cotton (trials 1– 4) or lemon plus vanilla-scented cotton (trial 5) was placed in the home cage of each mouse. The amount of time spent investigating the scent (nasal contact with the tube) during each trial was recorded. The results indicated that whereas OT / mice spent more time investigating the lemon-scented object than did the OT þ/þ mice, both genotypes rapidly

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habituated to the scent (investigative activity significantly declined over the four successive trials) and dishabituated when the scent was changed (investigative behavior increased in trial 5). The test for habituation to an acoustic startle stimulus consisted of 200 presentations of the acoustic stimulus (40 ms, 118 dB) regularly presented at intervals of 10 s. The investigators computed mean startle response amplitude data for successive blocks of 20 stimulus presentations for each subject. The results indicated that whereas the OT þ/þ mice exhibited significantly higher startle responses during habituation to the acoustic stimulus than did OT / mice, both genotypes habituated to the stimulus. Moreover, when the data were analyzed and expressed as a percentage of the average response measured in the first block of 20 trials, the rate of habituation was identical for both genotypes. Pharmacological experiments, carried out with a separate group of OT þ/þ and OT / mice implanted with intraventricular cannulas, were designed to learn the effect of OT, AVP, and an OT antagonist on SRM. The MSRT described earlier (the same female social stimulus presented for four 1-min trials, a new female presented in trial 5; 10-min ITI) was used in these experimental tests. Testing began 3 or 4 days after recovery from surgery. On successive treatment days (intertreatment interval, 48–72 h) the subject received an intracerebroventricular injection of artificial cerebrospinal fluid (aCSF, the vehicle), or a 1-ng dose of OT, AVP, or an OT antagonist (OT-Ant) and was tested 2 min later. The vehicle control trial preceded the first and third, and followed the last, peptide administration. Each subject received OT and AVP as its first and second peptide dosing. OT and OT-Ant comprised the third and fourth peptide dosing and were administered according to a counterbalanced design within subjects. Only data from the mice showing correct intracerebroventricular cannula placement on histological verification were included for analyses. Acute treatment with intracerebroventricularly injected OT completely restored social memory in the OT / mice, as indicated by the significant decline in SIT with the same female on presentation trials 2, 3, and 4 relative to trial 1, and by the recovery of interest when a new female was presented. OT-Ant treatment did not influence SRM in the OT / mice, but impaired it in the OT þ/þ mice (relative to the vehicle control condition, intracerebroventricularly injected OT-Ant had no measurable effect on olfactory investigation in the former, but significantly delayed its decline in the latter). At the dose tested, AVP had no significant effect on SRM of either genotype group (no significant difference in behavior between AVP and CSF vehicle treatment conditions for OT þ/þ or OT / mice). In their discussion, these investigators reported that similar testing for SRM ability in OT þ/þ and OT / females resulted in a pattern of deficits resembling that shown by the males. However, it had been much more difficult to find a robust deficit for the females because their initial level of

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investigation was less vigorous than that of the males in this paradigm. A comparable sex difference in social investigative behavior has been observed in the conventional social recognition paradigm (Bluthe and Dantzer, 1990; see Chapter 12). Taken together, the results of this study have shown that male mice genetically deficient in OT (OT /) do not have the SRM abilities shown by OT-intact wild-mice (OT þ/þ). The importance of OT to this olfactory-based memory processing was further substantiated by the findings that OT, but not AVP, stimuli tested in habituation/dishabituation and food-foraging paradigms, or in tests of spatial memory treatment, repaired the SRM deficit observed in the OT / mice, and the OT receptor antagonist produced a social amnesia-like effect in OT þ/þ mice. Moreover, the neural processing underlying SRM appears to be independent of that required for olfactory foraging and olfactory habituation involving nonsocial stimuli, and habituation to an acoustic startle stimulus, because these abilities were intact in OT / mice.

IX. Chapter Summary and Commentary

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A. General Comments The contributions of the investigators whose works are discussed in this chapter have been of both methodological and substantive value. Two techniques applied to this field of study, the antisense oligonucleotide treatment used by Landgraf et al. (1995) and the OT genetic knockout model used by Ferguson et al. (2000), permitted precise strategies for removing (1) targeted VP receptors in selected brain sites in the rat, and (2) OT in the OT / mutant mouse. Yet another methodological advance has been the microdialysis technique, which provides a means of applying exogenous VP/OT and of collecting endogenous VP/OT released from activated brain structures with negligible interference with normal activity in the conscious behaving animal [Engelmann and Landgraf, 1994; Engelmann et al., 1994; and see Landgraf et al. (1998) for further discussion of this technique as applied to VP/OT memory research]. The remainder of this section summarizes and discusses the contents of this chapter in terms of relevance to the views and findings of Dantzer, Bluthe, and colleagues, who pioneered the study of VP/OT and SRM, and of De Wied and colleagues, who pioneered the general field of VP/OT and memory processing.

B. Peripherally Administered AVP and SRM Dantzer, Bluthe, and colleagues carried out several studies (see Chapter 13) showing that peripherally administered VP facilitated SRM in male rats (Dantzer et al., 1987) and female rats (Bluthe and Dantzer, 1990) and in

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male mice (Bluthe et al., 1993). In contrast, peripheral administration of an AVP V1 receptor antagonist on its own impaired SRM in sexually intact male rats (Bluthe and Dantzer, 1990) and mice (Bluthe et al., 1993), but not in female rats (Bluthe and Dantzer, 1990), castrated male rats (Bluthe et al., 1990), or castrated male mice (Bluthe et al., 1993). Noting that a peripherally administered dose of behaviorally effective AVP is unlikely to cross the blood–brain barrier, whereas that of the AVP V1 receptor antagonist can do so, they concluded that two different VP systems account for these findings. The facilitating effect of peripherally injected AVP on SRM was thought to be due to an interaction of an androgen-independent VP system with a central arousal system, as proposed for memory tested in other learning paradigms (see Chapters 6 and 7). On the other hand, the SRM impairment induced by the V1 antagonist was attributed to an androgen-dependent VPergic system in sexually intact males, exclusively involved in the olfactory processing of conspecific social signals necessary for SRM (Dantzer, 1998; and see Chapter 12). The findings of Popik and colleagues are relevant to the view outlined above. First, while replicating the Dantzer et al. finding that peripherally injected AVP extended the duration of SRM, these researchers also showed that the endocrine (pressor) activity of AVP was not necessary for its ability to facilitate SRM. Thus, AVP derivatives lacking the endocrine effects of the parent peptide nevertheless facilitated SRM (Popik et al., 1991; Sekiguchi et al., 1991a), as they do memory tested in appetitive [Vawter and Van Ree, 1995; Vawter et al., 1997 (see Chapter 2)] and avoidance learning paradigms (De Wied et al., 1987; see Chapter 5). Second, Popik and colleagues obtained evidence that the AVP facilitation of SRM consists of both long-term and short-term memory components, which are differentially sensitive to various AVP-related peptides. That is, AVP peptides containing the covalent ring structure [e.g., AVP(1–8), AVP(1–7), and AVP(1–6)] exerted a long-term memory effect on SRM, extending its duration to at least 24 h, whereas AVP derivatives lacking this structure [e.g., AVP(4–9) and AVP(4–8)] exerted only a short-term memory effect that extended SRM for 2 h, but not for 24 h (Popik and Van Ree, 1992). Although the study of Popik and Van Ree (1992) is consistent with the possibility that two different VP mechanisms are responsible for the effects of exogenous AVP on SRM, they are not comparable to the two VP-ergic mechanisms postulated by Dantzer and colleagues. In accordance with the ‘‘VP Dual Action Theory,’’ Dantzer and colleagues postulate that the arousal-dependent VP system is peripherally associated with a pressor effect and influences SRM as it does long-term memory tested in a number of appetitive and avoidance learning paradigms. However, the findings of Popik and Van Ree (1992) suggested that the VP mechanism responsible for the long-term component of SRM is not an arousaldependent mechanism, because it was activated by peripherally injected DG-AVP, which lacks the pressor arousal effects of AVP.

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C. OT and SRM The role of OT in SRM, demonstrated in the studies reviewed in this chapter, extends the research carried out by De Wied and colleagues on the role of OT in long-term memory tested in avoidance conditioning paradigms (see Chapters 2–5). Dantzer and colleagues (see Chapter 12) studied the role of OT in SRM mainly to compare it with that of VP. In their one study with OT and SRM, Dantzer et al. (1987) showed that peripheral administration of OT and VP resulted in the same reciprocal (opponent) actions of these neuropeptides previously shown for long-term memory tested with avoidance paradigms (Bohus et al., 1978b; see Chapter 2). That is, at the dose levels used, OT impaired normal SRM, and VP facilitated it, that is, extended its duration (Dantzer et al., 1987). Subsequent research has suggested that the OT influence on olfactorybased SRM is more complicated than originally suspected. The studies reviewed in this chapter have found that exogenous OT may impair or enhance SRM depending on the dose range used in the study. That is, administered in a dose range typically used in learning/memory paradigms, OT has been found to impair memory in avoidance paradigms (Bohus et al., 1978b; see Chapter 2) and in the social recognition test (Dantzer et al., 1987; Popik and Vetulani, 1991). However, when administered at a low dose range, exogenous OT has been shown to facilitate SRM whether the peptide is administered peripherally (Arletti et al., 1995; Popik et al., 1992, 1996), intraventricularly (Benelli et al., 1995), or locally into specific brain structures (Popik and Van Ree, 1991). There is evidence indicating that endogenous OT modulates SRM as it does memory involved in avoidance behavior. Van Wimersma Greidanus and Maigret (1996) used intracerebroventricularly and locally injected anti-OT serum to reduce endogenous OT in the brain. Their findings indicated that the intracerebroventricularly injected antiserum delivered in a 2-
l volume at a sufficiently high concentration (1:10, but not 1:30 dilution) extended the duration of SRM from the normal 30 min to at least a 120-min interval. When the antiserum was locally applied at the same volume and concentration (2
l, 1:10 dilution) used with intracerebroventricular treatment to facilitate SRM, it was similarly effective when injected into the ventral hippocampus, but was ineffective when injected into the dorsal hippocampus, septal region, or olfactory nucleus. Their findings suggested that endogenous OT in the ventral hippocampus and the brain structures reached by intracerebroventricularly injected antiserum is involved in the modulation of SRM, and opposes the facilitative SRM effect inferred for endogenous VP, as it does in avoidance learning paradigms (see Chapters 2–5). Other studies reviewed in this chapter, in accord with the abovedescribed findings, suggest a physiological role for brain OT in modulating SRM, but the findings point to a facilitative, as opposed to a retarding, effect

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[Engelmann et al., 1998; Ferguson et al., 2000 (see below)]. Engelmann et al. (1998) found that an intracerebroventricularly injected OT antagonist blocked normal SRM in female rats whereas a similarly injected AVP antagonist was without effect. Taken together, these data point to the need for more detailed study to more clearly understand the role(s) that OT plays in memory modulation. It is possible that the memory-facilitative effect of both exogenously administered and endogenous OT is specific to SRM, and is mediated by olfactory pathways and structures activated by conspecific social stimuli directly or indirectly associated with prosocial reproduction-related activities such as mate bonding and offspring nurturance. On the other hand, the memoryimpairment function of OT may be involved in reduction of the conditioned anxiety and fear that occurs in painful or stressful situations that must be reencountered for reproductive success (e.g., parturition in females, or agonistic encounters associated with defense of mates, territories, and offspring).

D. Brain Structures and Pathways Mediating the VP and OT Influence on SRM The results of the studies reviewed in this chapter have reinforced the importance of olfactory pathways containing VP-ergic and OT-ergic circuitry in mediating SRM. Bluthe and Dantzer (1993; see Chapter 12) demonstrated the importance of the vomeronasal system [accessory olfactory system, engaged in the analysis of surface-deposited rather than airborne odorous molecules (pheromones)] in mediating the effect of androgendependent AVP on SRM. The studies in this chapter detailed their beginning insight into the SRM–olfactory connection. The research findings of Dluzen et al. (1998a,b, 2000) demonstrated that (1) the olfactory bulb (OB) is a target structure that mediates the SRM enhancement effects of exogenous AVP and OT (Dluzen et al., 1998a); (2) noradrenaline released in the OB is critical for the SRM preservation effects of these peptides (Dluzen et al., 1998b); and (3) in the case of OT, 2-adrenoceptors mediated the OT–NA interaction necessary to the influence of the neuropeptide on SRM. Anatomical study has indicated that both the main and accessory components of the OB project to the amygdala and the bed nucleus of the stria terminalis (BNST), which in turn connect with the septal area. Dantzer et al. (1988; see Chapter 12) demonstrated the importance to SRM of the septally released AVP, originating from the sexually dimorphic circuitry localized in the amygdala and BNST. The importance of the septal AVP system to this type of memory processing has been repeatedly reaffirmed in the studies reviewed in this chapter. The VP/OT antiserum study of Van Wimersma Greidanus and Maigret (1996) provided evidence of a physiological role of

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septal AVP, but not OT, in modulating SRM. Landgraf et al. (1995) further validated the AS oligo treatment technique by showing its ability to confirm the well-demonstrated septal AVP recognition memory effect. The OB is also indirectly connected with the medial preoptic area (MPA), and the MPA has been shown to be involved in processing olfactory information (Macrides, 1976; Pfaff and Pfaffmann, 1969) and in mediating the SRM-facilitative action of exogenously applied OT (Popik and Van Ree, 1991). Engelmann et al. (1994) using microdialysis and push–pull perfusion, provided evidence of a connection between the SON and the septal area in mediating a VP influence on SRM. It is noteworthy that SON AVP, which is not sexually dimorphic (see Chapter 1), is nevertheless a contributory factor to the enhancement of SRM when activated by osmotic stimulation. This may be interpreted as indirect support for the proposal by Koob, Dantzer, and colleagues that arousal-modulating, androgen-independent VP modulates SRM as it does other types of learning and memory. The VP (and presumably OT) connections with the arousal system were considered responsible by them for the opponent effects on SRT performance observed after peripherally injected AVP and OT (Dantzer et al., 1987; see Chapter 12), and for the SRM enhancement resulting from osmotic stimulation (Bluthe et al., 1991), which increases peripheral and central levels of both neuropeptides (see Koob et al., 1985a). The ability of peripherally administered V1 receptor antagonist, which decreases the pressor effect associated with high levels of peripherally circulating AVP, to block the osmotically induced SRM enhancement was interpreted as support for their proposal of a VP arousal effect on SRM (Bluthe et al., 1991). Finally, the research of Everts and Koolhaas (1997, 1999) was relevant to septal AVP involvement in SRM. The results of both studies suggested the specificity of this septal AVP memory effect, because VP antagonist treatment applied to the lateral septum produced the expected impairment in SRM but had no effect on a parallel object recognition test (Everts and Koolhaas, 1997).

E. VP/OT Knockout Models and SRM The role of endogenous brain AVP, and particularly septal VP, in SRM processing received further confirmation in a study by Engelmann and Landgraf (1994). They showed that SRM was impaired in untreated Brattleboro HODI rats, but was normal in Long-Evans (LE) rats with genetically intact AVP. Moreover, microdialysis-infused AVP into the lateral septum restored SRM to its normal level in the HODI rats, whereas similar infusion of the V1 antagonist into this structure impaired SRM in AVP-intact LE rats. Ferguson et al. (2000) confirmed a role for endogenous OT in rodent SRM in their study with OT-mutant male and female mice. In contrast to

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mice with OT-intact genotypes, these mice showed no evidence of SRM in a multitrial retention test or in the social recognition test, with genetically normal adult females (ovariectomized or sexually intact) and sexually intact males as social test stimuli. This suggests that the VP/OT neuropeptide effect on SRM is not limited to juveniles but extends to conspecifics of all ages. Their results also suggested that the influence of endogenous OT on SRM appears to be specifically linked to the role of the peptide in olfactory-based social memory processing because it was not essential for normal performance in tests reliant on olfactory-based processing of nonsocial stimuli tested in habituation/dishabituation and food-foraging paradigms, or on tests of spatial memory (see Chapter 10).