De Wied and Colleagues III: Brain Sites and Transmitter Systems Involved in the Vasopressin and Oxytocin Influence on Memory Processing

Barbara B. McEwen

De Wied and Colleagues III: Brain Sites and Transmitter Systems Involved in the Vasopressin and Oxytocin Influence on Memory Processing

I. Introductory Remarks

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This chapter discusses evidence relevant to the brain structures that may be target sites for the effects of vasopressin (VP) and oxytocin (OT) on memory processing, as well as the monoamine classic transmitter systems with which these neuropeptides may interact to produce these effects.

II. Localizing Central Sites for the Memory-Modulating Effects of VP and OT by Means of Lesioning and Microinjection Techniques

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Lesion and microinjection techniques have been used to localize the central sites at which VP and OT exert their effects on memory storage and retrieval. It is rationalized that lesioning (injuring or destroying) a Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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structure (e.g., nucleus or pathway) important to the influence of the peptide on storage or retrieval will attenuate or prohibit the relevant retention behavior on subsequent peptide treatment of the lesioned subjects. The lesion technique is not without problems of interpretation because failures to produce impairment do not necessarily indicate that the structure is irrelevant to the effect of the peptide on retention. The failure may simply be due to insufficient tissue damage from the lesion, or other participating sites that can substitute for the damaged component. Further, demonstration of lesion-induced impairment could also be misinterpreted because ‘‘fibers of passage’’ (i.e., fibers in the damaged area deriving from cell bodies originating elsewhere in the brain), rather than the cell bodies in the damaged brain nucleus, may be the basis of the observed behavioral deficit. Microinjection of a peptide or its antiserum into a specific brain site is justified on the basis that increasing or reducing its level in a brain site that normally mediates the influence of the peptide on retention should enhance and impair retention behavior, respectively. Studies utilizing this technique are also not without interpretive problems. For example, microinjection of the peptide or its antiserum could fail to modulate memory because of an insufficient dose level. This may account for several early failures in studies that used unilateral injections in bilaterally distributed brain structures (De Wied et al., 1976). Despite these caveats, lesion and microinjection studies have provided evidence of the localization of a number of brain sites involved in mediating the effects of the neurohypophysial peptides on memory consolidation and/or retrieval. These include forebrain limbic structures implicated in memory processing, and brainstem sites that give rise to catecholamine and serotonin projections to these forebrain structures.

A. Thalamic–Limbic Lesions: Influence on the Memory-Modulating Effects of Adrenocorticotropic Hormone-Like Peptides and of VP and/or OT De Wied and colleagues had earlier reported that adrenocorticotropic hormone (ACTH)-like peptides, as well as vasopressin, prolonged extinction of conditioned avoidance behavior, although the former produced short-term, the latter, long-term effects on this behavior (De Wied, 1971; De Wied and Bohus, 1966; see Chapter 2). ‘‘ACTH-like peptides’’ refer to ACTH(1–39) derivatives, such as ACTH(1–10), as well as hormones such as -melanocyte-stimulating hormone (-MSH), which are derived from the same prohormone as ACTH. A series of studies conducted by Van Wimersma Greidanus and associates, described below, examined the possibility that the different behavioral effects of the two neuropeptides involve different brain structures, or the same structures functioning in different operative modes.

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1. Selected Studies a. Van Wimersma Greidanus et al. (1974) Van Wimersma Greidanus et al. (1974) tested the ability of lesions in the thalamic parafascicular nucleus (pfc nucleus) of male Wistar rats to interfere with the effects on extinction behavior usually induced by lysine vasopressin (LVP) (experiment 1) and ACTH(4–10) (experiment 2). The pfc nucleus is part of the diffuse thalamic projection system, and it has been shown that microinjection of LVP (Van Wimersma Greidanus et al., 1973) and implantation of ACTH(1–10) (Van Wimersma Greidanus and De Wied, 1971) into this nucleus help maintain a conditioned avoidance response. After recovery from surgery, the lesioned animals and the sham operates in both experiments were trained in a pole-jump avoidance task (days 1–4, 10 trials/day), tested for extinction (days 5 and 8, 10 trials/day), given reacquisition training (10 trials on day 9), and tested for extinction on days 10, 11, 12, and 15 (10 trials/day). In experiment 1, all subjects received a subcutaneous injection of placebo (physiological saline) or LVP (1.8 or 5.4 g/rat) immediately after the extinction session on day 10. In experiment 2, the pfc-lesioned rats received placebo or ACTH(4–10) (1.0 or 9.0 g/rat) and the sham operates were given placebo or ACTH(4–10) (1.0 or 3.0 g/rat), 1 h before each extinction session on days 10, 11, 12, and 15. After completion of behavioral testing, histological examination was carried out to determine the exact location and size of the lesion. The results were as follows: (1) the lesion in experiment 1 destroyed the pfc nucleus and extended into the mediodorsal thalamus; (2) this lesion impaired acquisition and hastened extinction of the avoidance response in placebo-treated rats; (3) the single injection of LVP (1.8 or 5.4 g/rat, subcutaneous) dose dependently reversed the effects of the lesion on extinction. The low dose of LVP preserved the learned response for the first 2 days of the 4-day extinction period whereas the high dose preserved it for the duration of extinction testing; (4) the thalamic pfc lesion in experiment 2 was more restricted than in experiment 1 and did not affect acquisition, but did reduce response during extinction relative to the sham operates; and (5) the chronically injected ACTH(4–10), which lacks the corticotrophic effects of ACTH(1–39), dose dependently inhibited extinction in the sham operates; however, neither the low dose nor the extremely high dose of the peptide was able to reverse the effect of the lesion on extinction. The results were interpreted as follows: (1) although the thalamic pfc nucleus is normally involved, along with other brain sites, in mediating the effect of vasopressin on extinction, it is not essential for that effect to occur. Thus, whereas low amounts of the peptide preserved the pole-jumping response in sham operates throughout extinction, it did not do so for pfclesioned rats. However, higher doses were able to overcome the effects of the lesion; (2) on the other hand, the pfc nucleus must be intact for

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ACTH(4–10) to exert its retrieval effect on avoidance retention, because neither the low dose nor the high dose of this peptide was able to overcome the lesion-induced reduction of avoidance response during extinction. b. Van Wimersma Greidanus et al. (1975b) Van Wimersma Greidanus et al. (1975b; and cited in Van Wimersma Greidanus et al., 1983) reported the effects of extensive bilateral lesions in the rostral septal area on the ability of ACTH-like peptides and LVP to influence extinction of a polejump avoidance response. The lesion almost completely destroyed the medial septal nucleus and partially destroyed the lateral septal nucleus and the nucleus accumbens. This region proved to be another brain site essential for the short-term behavioral action of ACTH-like peptides, because ACTH(1–10) treatment, given during extinction of the pole-jump task, was unable to maintain the learned response in these septally lesioned animals. This limbic site also appears to be essential for mediating the ability of vasopressin to preserve a learned avoidance response because a single subcutaneous injection of a 9-g dose of LVP, given before extinction, failed to maintain avoidance responding in lesioned subjects even when the much smaller, 3-g dose did so for the sham operates. c. Van Wimersma Greidanus and De Wied (1976b) Van Wimersma Greidanus and De Wied (1976b) studied the effect of an anterodorsal hippocampal lesion on extinction of a pole-jump shock avoidance response in male inbred Wistar rats after treatment with either LVP (experiment 1) or ACTH(4–10) (experiment 2). In experiment 1, the subjects received either sham operations, small, or large bilateral lesions. After recovery from surgery, 10 training trials were given on each day of acquisition: days 1–4 for sham operates and the small-lesioned operates, and days 1–5 and day 8 for the large-lesioned operates. After reaching the learning criterion (eight or more correct avoidances), they received a single subcutaneous injection of either placebo or LVP (1.0 or 3.0 g/rat for sham operates, 1.0, 3.0, or 9.0 g/rat for the small-lesion groups, and 1.0 or 9.0 g/rat for the largelesion groups). Extinction testing occurred on days 5, 8–12 and 17 for sham operates; on days 6, 8, 9, and 11 for small-lesioned groups; and on days 9–12 and 17 for the large-lesioned groups. The results of experiment 1 were as follows: (1) for the sham operates, LVP produced a long-term, dose-dependent inhibition of extinction. Thus, LVP-treated rats continued to exhibit a high rate of avoidance responding on the last day of extinction, when placebo controls no longer responded; (2) the small lesion did not interfere with acquisition, but tended to accelerate the rate of extinction among the placebo controls. This lesion prevented the LVP-induced inhibition of extinction in subjects given the 1.0- or 3.0-g dose, but not in those given the 9.0-g dose; and (3) the large lesion retarded acquisition of the avoidance response and prevented LVP-induced inhibition

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of avoidance extinction whether the subjects received the 1.0-g or the 9.0-g dose level. This finding suggests that an intact anterodorsal hippocampus is essential for mediating the LVP-induced prolonged extinction effect in this task. In experiment 2, the subjects received either sham operations or large bilateral lesions in the anterodorsal hippocampus. After recovery from surgery, the sham operates were trained on days 1–3 (10 trials/day) and, having attained criterion performance, were tested for extinction on days 4, 5, and 8 (10 trials/day). These sham operates received a subcutaneous injection of placebo or ACTH(4–10) (3.0 g/rat, subcutaneous) 1 h before each day of extinction. The lesioned subjects were given 6 days of training (days 1–5 and day 8; 10 trials/day) and 3 days of extinction (days 9, 10, and 11; 10 trials/day), provided they reached criterion performance. These subjects received a subcutaneous injection of placebo or ACTH(4–10) (9 g/rat, subcutaneous) 1 h before each day of extinction. The results of experiment 2 were as follows: (1) relative to the placebo-treated subjects, a 3.0-g dose of ACTH(4–10) inhibited extinction of the avoidance response in the sham operates; and (2) the large lesion retarded acquisition of the avoidance response as was observed in experiment 1, and prevented ACTH(4–10) from producing its inhibitory effect on response extinction, even though the large (9.0 g) dose of the peptide was used. Taken together, the results of this study indicated that an intact anterolateral hippocampus is as essential for the short-term behavioral effects of ACTH-like peptides as it is for the long-term behavioral effects of vasopressin treatment. d. Van Wimersma Greidanus et al. (1979b) Van Wimersma Greidanus et al. (1979b) observed the effect of bilateral lesions of the amygdala complex on the ability of peripherally administered ACTH(4–10) and desglycinamide-lysine vasopressin (DG-LVP) to prolong active avoidance extinction in male inbred Wistar rats. Experiment 1 tested the effect of DG-LVP treatment on pole-jump shock avoidance behavior in sham operates and amygdala-lesioned subjects. After recovery from surgery, the subjects were given 4 days of acquisition training (10 trials/day) followed by 4 extinction sessions (10 trials/session): 2 on day 5, and 1 each on days 6 and 8. Immediately after the extinction session on day 5, the subjects received a subcutaneous injection of placebo (physiological saline) or DG-LVP (3 g/rat for the sham operates, and up to 5 g/rat for the lesioned rats). The effect of this single injection was observed 4 h later in a second 10-trial extinction session on day 5 and thereafter in the extinction sessions on days 6 and 8. Experiment 2 employed a similar training/extinction procedure to test the effect of ACTH(4–10) on response extinction in amygdala-lesioned and sham-operate subjects. However, in this experiment the rats were tested for

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extinction on days 5 and 6 (10 trials/day), and received an injection of placebo or ACTH(4–10) (3.0 g for sham operates and 9.0 g for lesioned rats) 1 h before each 10-trial extinction session. The results were as follows: (1) the lesion damaged the central and basolateral parts of the amygdaloid complex; (2) the lesion did not influence acquisition or extinction of the conditioned avoidance response in the placebo-treated rats; (3) DG-LVP treatment induced a long-term inhibitory effect on extinction in the sham operates but failed to do so in the amygdala-lesioned subjects (Fig. 1); and (4) ACTH(4–10) produced its short-term inhibitory effect on extinction in the sham operates on both days of extinction testing but failed to influence extinction, at either dose level, in the amygdala-lesioned subjects (Fig. 2). e. Van Wimersma Greidanus et al. (1979c) Van Wimersma Greidanus et al. (1979c) investigated whether the various limbic system structures act individually, or as an integrated system in mediating the inhibitory effect on avoidance extinction exerted by ACTH(4–10) and DG-LVP. Fibers of the fornix (connects hippocampus with other limbic sites and with the hypothalamus) and the stria terminalis (connects amygdala with other limbic sites and with the hypothalamus) were transected. The effect of this on extinction of a pole-jump avoidance task, after treatment with placebo or each of

FIGURE 1 Effect of DG-LVP on extinction of a pole-jumping avoidance response in rats with lesions in the amygdaloid complex and in sham-operated animals. AM, amygdaloid; CAR, conditioned avoidance response; s.c., subcutaneous. Source: Van Wimersma Greidanus et al., 1979b (Fig. 2, p.294). Copyright ß 1979 by Pergamon Press and Brain Research Publications Inc.

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FIGURE 2 Effect of ACTH (4–10) on extinction of a pole-jumping avoidance response in rats with lesions in the amygdaloid complex and in sham-operated rats. Source: Van Wimersma Greidanus et al., 1979b (Fig. 3, p.294). Copyright ß 1979 by Pergamon Press and Brain Research Publications Inc.

the peptides, was tested in male Wistar rats. The surgery left intact the individual limbic system sites but prevented operation of the limbic system as an integrated unit. The subjects received 10 acquisition trials per day until they reached a learning criterion of 75% correct avoidance responses in a single 10-trial session. Extinction testing (10 trials/day) began the day after the final day of training. The long-term effect of DG-LVP treatment on response maintenance was tested by administering a single subcutaneous injection of placebo (physiological saline) or DG-LVP (3 g/rat for sham operates and 3 or 9 g/rat for operates) immediately after the last acquisition trial, and was followed by response extinction carried out for three consecutive days. The short-term effect of ACTH(4–10) on response maintenance was evaluated

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by chronic subcutaneous injections of placebo or ACTH(4–10) (3 g/rat for sham operates; 3.0 or 9.0 g/rat for operates) administered 1 h before each of the two consecutive days of extinction testing. The fornix–stria terminalis transection hastened acquisition of the avoidance response, a result previously obtained in avoidance paradigms after damage of the fornix pathway (Alvarez-Palaez, 1973; De Castro and Hall, 1975). This enhanced acquisition may be related to the decrease in freezing behavior and increase in spontaneous motor activity previously noted in similarly lesioned rats (Alvarez-Palaez, 1973; Liss, 1968). The transection did not interfere with maintenance by vasopressin of the avoidance response, because DG-LVP dose dependently inhibited extinction in both the lesioned and sham-operate rats. In contrast, the inhibitory action of ACTH(4–10) on extinction, observed in the sham operates, was prevented by the lesion even when a high dose of the peptide was employed. The overall results were interpreted to suggest that the limbic system acts as an integrated functional unit when mediating the proposed attention/arousal/retrieval effects of ACTH, but as separate and independent substrates for the proposed consolidation/retrieval effects of vasopressin on avoidance extinction. 2. Summary: Lesion Studies Taken together, the results of these studies indicated that (1) the thalamic pfc nucleus, part of the diffuse thalamic arousal system, is involved in mediating the effects on avoidance retention produced by both sets of peptides. However, although it is essential for the retrieval effect induced by the ACTH-like peptides, the pfc nucleus is not as important for the effect of vasopressin on memory consolidation (Van Wimersma Greidanus et al., 1974); (2) other limbic system structures such as the septal region (Van Wimersma Greidanus et al., 1975b), hippocampus (Van Wimersma Greidanus and De Wied, 1976b), and amygdala (Van Wimersma Greidanus et al., 1979b) do appear to play a crucial role in mediating the effects of ACTH-like peptides as well as vasopressin on retention behavior; and (3) transections of pathways interlinking components of the limbic system are more detrimental to ACTH-like peptide effects on avoidance retention than effects induced by vasopressin (Van Wimersma Greidanus et al., 1979c). This latter finding is in accord with the hypothesis that the septal region, hippocampal area, and amygdala complex act as separate substrates in mediating the effects of vasopressin on extinction and therefore when engaged in the process of memory consolidation. On the other hand, these limbic system components function as an integrated unit mediating the effects of ACTH on behavior, and therefore when engaged in the neuronal activity that contributes to attention, motivation, and retrieval (Van Wimersma Greidanus et al., 1979c).

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B. Microinjection of VP, OT, or Their Antisera into Discrete Brain Sites 1. Microinjections of VP or OT into Selected Brain Sites a. Van Wimersma Greidanus et al. (1973) Van Wimersma Greidanus et al. (1973) demonstrated prolonged maintenance of a conditioned pole-jump shock avoidance response after posttraining unilateral microinjections of arginine vasopressin (AVP) into the parafascicular nucleus of the thalamus, but not in numerous other structures that may be involved in memory processing (e.g., the reticular formation, midbrain gray, substantia nigra, putamen, dentate gyrus of the hippocampus, and cortex). The authors suggested that an insufficient amount of peptide administered by the unilateral injection procedure may have been responsible for some of these failures. This was supported by bilateral microinjection studies that employed the passive avoidance paradigm, as described below. b. Kovacs et al. (1979a) Kovacs et al. (1979a), noting the evidence suggesting that vasopressin and oxytocin produce opposing effects on CNS mechanisms involved in memory processing (Bohus et al., 1978a,b; see Chapter 2), designed several experiments to study the brain sites activated by these peptides in producing their effects on avoidance behavior. Using male inbred Wistar rats and a passive avoidance (PA) memory consolidation design, these peptides were microinjected into midbrain/limbic sites that former lesion studies suggested were involved in mediating the effect of vasopressin on avoidance extinction (see previous section of this chapter). The dose levels for both peptides were 0 (saline controls) or 20–25 pg for bilateral injections into the hippocampus, dorsal septal area, and amygdaloid nuclei, and 0 (saline controls) or 50 pg for midline injection into the dorsal raphe nucleus; these dose levels were behaviorally ineffective for both peptides when injected into the lateral ventricle (Bohus et al., 1978b). PA behavior was tested 24 and 48 h after the single footshock (FS) trial. The results were as follows: (1) when microinjected into the hippocampal dentate gyri, AVP significantly increased and OT significantly decreased median reentry latency relative to saline controls; (2) when microinjected into the dorsal septal nuclei, both peptides significantly increased median reentry latency relative to the saline controls in both retention tests; (3) relative to the saline controls, neither AVP nor OT influenced median reentry latency on either retention test when injected into the central amygdaloid nuclei; and (4) when microinjected into the dorsal raphe nucleus, AVP significantly increased, and OT significantly decreased, median reentry latencies relative to saline controls in the first, but not the second, retention test. Taken together, these results suggest that all the brain sites tested, except the central nucleus of the amygdala, are sites at which VP and/or OT influence memory consolidation. The failure of either AVP or OT, microinjected

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into this area, to influence memory consolidation suggests that this amygdala nucleus is not involved in the influence of these peptides on memory consolidation. The findings that, when microinjected into the hippocampal dentate gyri or the dorsal raphe nucleus, AVP facilitated and OT inhibited PA memory consolidation, agree with the effects produced by much larger quantities of these peptides injected into a lateral ventricle (Bohus et al., 1978a,b). The finding that OT microinjected into the septal nuclei mimicked the effect of VP when microinjected into this region, although unexpected, has been observed in biochemical studies (Van Heuven-Nolsen et al., 1984a; this chapter), and in electrophysiological studies (Joels and Urban, 1982; Urban, 1981; see Chapter 5) conducted by De Wied’s colleagues. The authors suggested two reasons why OT facilitated PA memory consolidation when injected into the septal nuclei. Noting that peripherally injected OT, especially at high doses, sometimes produces VP-like effects on active and passive avoidance retention (Bohus et al., 1978b), it was suggested that the septal area may be the target site for these effects of OT. Alternatively, the observation that the C-terminal fragment of OT [Pro-Leu-Gly (PLG)] attenuates puromycin-induced amnesia (Walter et al., 1975) and prolongs extinction in a pole-jump shock avoidance task (Walter et al., 1978) led to the suggestion that brain region differences in degradation of this peptide (Burbach et al., 1980a) may have produced this effect. c. Kovacs et al. (1979b) Kovacs et al. (1979b) tested the effect of AVP microinjected into either the dorsal raphe nucleus or the locus coeruleus on memory consolidation in a step-through passive avoidance paradigm. The subjects, male inbred Wistar rats, were assigned to various groups: an AVP/ locus coeruleus group (25 pg bilaterally injected into the locus coeruleus on each side of the brain); a saline/locus coeruleus control group (bilateral injection of an equal volume of physiological saline); an AVP/dorsal raphe group (50 pg unilaterally injected into the dorsal raphe nucleus at midline); and a saline/dorsal raphe control group (a unilateral injection of an equal volume of physiological saline). The single treatment was given immediately after the FS learning trial, and median reentry latency was tested 24 and 48 h later. The results demonstrated that (1) AVP microinjected into the locus coeruleus had no effect on memory consolidation (no significant difference between VP-treated and saline-treated controls on reentry latency scores) and (2) AVP microinjected into the dorsal raphe nucleus improved memory consolidation at the 24-h retention test (reentry latency was significantly greater in the VP-treated subjects than in the saline-treated subjects). d. Bohus et al. (1982) Bohus et al. (1982) reported the outcomes of several experimental tests carried out to determine brain sites mediating the effects of VP on memory retrieval, in a passive avoidance (PA) paradigm. Three experiments tested the ability of peripheral, intracerebroventricular,

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and localized injections of AVP into specific brain structures to reverse retrograde amnesia (RA) in a step-through passive avoidance task. RA was induced by an intraperitoneal injection of pentylenetetrazole (PT, 45 mg/kg) given immediately after the single learning trial. The rationale for this paradigm is that amnesia can be viewed as a deficit in memory retrieval (Spear, 1973). In each experiment the subjects received a single treatment of placebo (physiological saline) or AVP 1 h before the 24-h retention test. In experiment 1, PT significantly reduced reentry latency relative to the nonamnestic controls, and peripherally injected AVP (2 g/rat, subcutaneous) substantially reversed this amnestic effect (i.e., median reentry latency was significantly increased in the AVP-treated subjects relative to the placebo controls). In experiment 2, intracerebroventricularly injected AVP (10 ng/ rat) fully reversed the severe amnestic effect induced by PT treatment. In experiment 3, AVP was microinjected into specific brain structures, either bilaterally (100 pg on each side of midline) into the hippocampal dentate gyrus, dorsal septum, or central nucleus of the amygdala, or unilaterally (200 pg) along the midline into the dorsal raphe nucleus. In this experiment, pretreatment with PT produced a severe amnestic effect, and AVP induced a relatively small but significant reversal of this amnesia when injected into either the central nucleus of the amygdala or the hippocampal dentate gyrus, but not the dorsal septum or dorsal raphe nucleus. The results of the experiments reported above, together with those of Kovacs et al. (1979a,b), suggest that individual brain sites may or may not mediate both consolidation and retrieval effects of these peptides. Thus, according to these findings the hippocampal dentate gyrus mediates the effects of these peptides in both memory consolidation and retrieval; the central nucleus of the amygdala is involved in the effect of AVP on retrieval, but not in the influence of either peptide on consolidation; and the dorsal septal nucleus and the dorsal raphe nucleus are involved in the effects of the peptides on consolidation, but not in the influence of AVP on retrieval. 2. Microinjections of VP or OT Antiserum into Selected Brain Sites a. Kovacs et al. (1980a) Kovacs et al. (1980a) demonstrated that VP antiserum microinjected into the dorsal raphe nucleus immediately after the learning trial attenuates passive avoidance behavior in a 24-h retention test trial. This finding suggests that endogenous VP localized in this structure is involved in memory consolidation. This study is discussed later in this chapter because of its relevance to interactional effects between VP and brain catecholamines. b. Kovacs et al. (1982a) Kovacs et al. (1982a), noting evidence that the dorsal hippocampal complex plays a role in mediating the retention effects of peripherally (Van Wimersma Greidanus and De Wied, 1976b, described above) and centrally (Kovacs et al., 1979a, described above) injected

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vasopressin, tested for a role of endogenous hippocampal VP in memory processing. Using male inbred Wistar rats in a PA consolidation design, hippocampal VP was neutralized by a bilateral microinjection of VP antiserum (1 l of a 1:50 dilution) into the dentate gyrus in the dorsal part of the hippocampal complex. For comparison, VP antiserum (2 l of a 1:50 or 1:10 dilution) was injected into a lateral ventricle (intracerebroventricular injection) in two other groups of subjects. Normal rabbit serum was injected into the control groups. All solutions were delivered via previously implanted cannulas in freely moving subjects immediately after the PA learning trial, and retention was tested 24 h later. Histological study at the conclusion of behavioral testing permitted localization of the tip of the cannulas as well as immunocytochemical detection of spread of the injected antiserum in the brain. Results indicated that the intracerebroventricularly injected antiserum, at the stronger (1:10) but not the weaker (1:50) dilution, significantly inhibited PA retention in the 24-h retention test. However, microinjection of the weaker solution into the hippocampal dentate gyrus did severely impair PA behavior. Because the 1:50 dilution of the antiserum had no effect when injected directly into the cerebrospinal fluid, it was concluded that the microinjected antiserum had its effect in the brain and did not result from leakage into the ventricular fluid. Histological findings indicated that the antiserum microinjected into the dorsal hippocampus, although maximally concentrated in a 1-mm surround of the cannula tract, also spread rostrally toward the dorsolateral septum and ventrocaudally into a small region of the ventral hippocampus. Because of the uptake of the antiserum in the septal area and ventral hippocampus, it is possible that a combination of these sites mediated the effect observed in this study. c. Veldhuis et al. (1987) Veldhuis et al. (1987) used a memory consolidation and retrieval design with a step-through PA task to investigate the effects of VP antiserum injected intracerebroventricularly or locally into each of several brain sites (dorsal hippocampus, ventral hippocampus, dorsolateral septum, or caudate nucleus). Injections of antiserum (1:50 dilution) or normal rabbit serum (control serum) were administered via preimplanted cannulas into freely moving male Wistar rats. The single injection was given either immediately after the PA learning trial (consolidation design) or 1 h before the 24-h retention test (retrieval design). Retention (reentry latency) was measured 24 and 48 h after the learning trial. For the antiserum dilution used, the results of the consolidation design indicated the following: (1) there was no significant difference between subjects receiving an intracerebroventricular injection of antiserum or control serum in median reentry latency on either retention test; (2) subjects injected via either the ventral or dorsal hippocampus with anti-VP serum

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had a sharply and significantly reduced median reentry latency in both retention tests relative to those injected with control serum; and (3) there was no significant difference in median reentry latency for either retention test between those subjects receiving VP antiserum in the dorsolateral septum or the caudate nucleus and those receiving the control serum. The results for the retrieval design indicated that (1) the subjects receiving an intracerebroventricular injection of either anti-VP serum or control serum exhibited maximum PA behavior (median latency, 300 s in duration) in both retention tests; (2) anti-VP serum, injected into the dorsal or ventral hippocampus or into the dorsolateral septum, significantly reduced reentry latencies in both retention tests relative to serum controls; and (3) median reentry latency in subjects microinjected with anti-VP serum in the caudate nucleus did not differ significantly from that of their serum controls. These results were interpreted as indicating that endogenous vasopressin in the dorsal and ventral hippocampus is important for both memory consolidation and retrieval, whereas that in the dorsolateral septum appears to be functionally involved only in memory retrieval. The failure to obtain evidence of a role of caudate nuclear VP in memory consolidation or retrieval is not surprising given the sparse and diffuse input into this region from extrahypothalamic VP-ergic projections (Buijs, 1983) and failure to detect VP-receptor sites in this region (Ostrowski et al., 1994). d. Van Wimersma Greidanus and Baars (1988) Van Wimersma Greidanus and Baars (1988) tested the effects of locally injected OT antiserum on PA behavior in male inbred Wistar rats. Either normal rabbit serum (control group) or OT antiserum was microinjected into the dorsolateral septum or the ventral hippocampus immediately after the PA learning trial or 1 h before the 24-h retention test. Reentry latencies were measured (to a maximum of 300 s) both 24 and 48 h after the PA learning (FS) trial. Results indicated that, relative to the control groups, reentry latencies were significantly increased (1) in both retention tests after a posttraining injection of anti-OT serum (1:100 dilution) into the dorsolateral septum or the ventral hippocampus, and (2) in the 24-h but not the 48-h retention test after a preretention injection of OT antiserum (1:50 dilution) into the dorsolateral septum or the ventral hippocampus. These results suggest that endogenous OT within the ventral hippocampus and dorsolateral septum is normally involved in attenuating memory consolidation and retrieval in this passive avoidance paradigm. 3. Summary: Microinjection Studies These microinjection studies have extended and been generally consistent with the previously discussed lesion research. One potential source of inconsistency pertains to the role of the amygdala in memory consolidation assessed in the microinjection study of Kovacs et al. (1979a), and the lesion

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study of Van Wimersma Greidanus et al. (1979b). The microinjection study found no support, and the lesion study positive evidence, of a role for the amygdala in mediating the influence of VP in memory consolidation. However, the two studies are not directly comparable because a fairly restricted area (central nucleus of the amygdala) was affected in the microinjection study whereas extensive damage (central and basolateral nuclei of the amygdala) was inflicted in the lesion study. The microinjection studies also support the hypothesis of an amnesic effect of OT on PA retention because the effects of locally injected OT typically oppose those of VP, whereas neutralizing endogenous levels of OT in selective brain sites has effects mimicking those of VP treatment. The studies that used the active avoidance (AA) or PA experimental paradigm pointed to roles for the parafascicular thalamus (AA) and various limbic system structures (PA) in mediating the effects of locally microinjected VP and/or OT on memory consolidation or retrieval. Specifically, these studies suggested the following: (1) VP improved memory consolidation when injected into the thalamic pfc nucleus, dorsal septal area, hippocampal dentate gyri, or dorsal raphe nucleus, but had no effect when injected into the central nucleus of the amygdala or the locus coeruleus; (2) VP improved memory retrieval when injected into the hippocampal dentate gyri or the central nucleus of the amygdala, but had no effect on retrieval when injected into the dorsal septal area, or the dorsal raphe nucleus; and (3) OT attenuated memory consolidation when injected into the hippocampal dentate gyrus or the dorsal raphe nucleus, improved it when injected into the dorsal septal nucleus, and had no effect when injected into the central nucleus of the amygdala. The unexpected finding that OT mimicked, rather than opposed, the behavioral effect of VP when microinjected into the septal area may be related to the ability of OT or the C-terminal tripeptide derivative of OT (PLG) to stimulate VP receptors in this brain site and thereby produce a VP-like effect. The observation that VP receptors are abundant, whereas OT receptors are sparse, in the lateral septal area (Barberis and Tribollet, 1996) is consistent with this possibility. The VP antiserum studies demonstrated that neutralizing endogenous AVP in some limbic system structures implicated in memory processing produced effects opposite to those observed after microinjection of the peptide itself. Specifically, these studies suggested that endogenous VP released in the dorsal or ventral hippocampus enhances both memory storage and retrieval, that in the dorsal raphe nucleus enhances consolidation, and that in the dorsolateral septum enhances retrieval. The caudate nucleus, however, appears not to be involved in endogenous vasopressin effects on either memory consolidation or retrieval. Neutralization of endogenous OT has suggested that both dorsal septal and ventral hippocampal OT has a physiological role in modulating (attenuating) PA memory consolidation and retrieval (Van Wimersma Greidanus and Baars, 1988).

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III. Interaction between VP/OT Peptides and Brain Catecholamines in Memory Processing

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There has been much theoretical speculation and research on the possibility that the three brainstem–telencephalic monoaminergic projection systems [noradrenergic (NA-ergic), dopaminergic (DA-ergic), and serotonergic (5HTergic)] modulate memory processing. These monoaminergic projection systems originate in cell populations localized in the locus coeruleus of the pons (NA-ergic projection), substantia nigra and ventral tegmental areas (DA-ergic projections), and dorsal raphe nucleus (5HT-ergic projection) of the midbrain, and they project to telencephalic structures implicated in memory processing (i.e., the hippocampus, septum, amygdala, and striatum). These same structures also receive terminals from extrahypothalmic VP- or OT-containing fiber projections or contain receptors for these peptides (see Chapter 1). The presence of this anatomical substrate is in accord with a putative neurohypophysial peptide–monoaminergic interaction in memory processing. Although the animal literature is not entirely consistent, there is some degree of support for the propositions that NA-ergic (Ellis, 1985; Lee and Ma, 1995; Sara, 1985) and DA-ergic (Beninger, 1983; Packard and White, 1991) projections facilitate, and that the 5HT-ergic projection inhibits (Altman and Normile, 1988; McEntee and Crook, 1991; Ogren, 1985), memory processing, at least in certain types of learning and memory tasks. De Wied and colleagues, and related groups (e.g., Kovacs and colleagues), have investigated the possibility that the central neurohypophysial peptidergic fiber systems modulate memory processing by influencing neurotransmission in these brainstem–telencephalic monoaminergic projections. This research has particularly focused on the fiber projections containing the catecholamine neurotransmitters [i.e., noradrenaline (NA) or dopamine (DA)], and has employed three research protocols: (1) the behavioral/biochemical protocol includes experiments that investigate the ability of neurohypophysial peptides or their antisera to influence PA retention as well as catecholaminergic (CA-ergic) neurotransmission; (2) the behavioral protocol examines VP or OT modulation of avoidance retention after experimental lesions in a selected catecholamine-containing projection system; and (3) the biochemical protocol examines the ability of neurohypophysial peptides to modulate neurotransmission in CA-ergic terminals innervating selective brain sites implicated in memory processing. CA-ergic neurotransmission is assessed by determining the rate at which the neurotransmitter disappears (rate of utilization) once further synthesis of the catecholamine is prevented by pretreatment with -methylparatyrosine (-MPT), an inhibitor of the enzyme necessary for synthesis of either NA or DA. Thus, if VP activates the neurotransmitter-containing fiber, the rate of neurotransmitter disappearance (utilization) should be increased; if the peptide inhibits it, the disappearance rate will be decreased.

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A. The Behavioral/Biochemical Protocol: Influence of VP, OT, or Their Antisera on PA Behavior and Catecholaminergic Neurotransmission 1. Selected Studies a. Kovacs et al. (1977) Kovacs et al. (1977) carried out behavioral and biochemical experiments to investigate an AVP/CA-ergic interaction in memory processing. The behavioral experiment determined the effect of CA synthesis inhibition on the ability of peripherally administered LVP to influence PA learning and retention in male CFY rats. The biochemical experiment investigated the effect of peripherally administered LVP on -MPT-induced utilization of NA and DA in selective brain structures. In the behavioral experiment, the CA-synthesizing enzyme inhibitor -MPT was injected alone or in combination with peripherally administered LVP. The effect of these treatments on learning and retention of a benchjump passive avoidance (PA) task was assessed. The rats were trained to jump onto a bench and to remain there for 180 s (PA component), and total time required for them to remain on the bench for 180 s (PA learning criterion) was scored (step-on latency). Retention was tested 24 h later when the rats were returned to the apparatus and placed on the bench; the latency to step down onto the grid floor was used as the measure of PA retention. On both training and retention test days, the subjects received either vehicle control, LVP (300 mU/kg, intraperitoneal), -MPT (80 mg/ kg, intraperitoneal), or LVP and -MPT. The -MPT injection was given 3 h before, and the vehicle or LVP injection 10 min before, the behavioral session. Neither LVP nor -MPT treatment alone or in combination influenced learning (i.e., no significant difference in step-on latency between the vehicle controls and the three treatment groups). On its own, -MPT did not influence PA retention, whereas LVP alone facilitated PA response (significantly increased step-down latency relative to the vehicle control). When combined with LVP treatment, the enzyme inhibitor prevented the significant retention effect induced by LVP, indicating the importance of an intact CA-ergic projection system for mediating the influence of vasopressin on PA retention. The biochemical experiments tested the ability of LVP to influence NA and DA: (1) levels in the hypothalamus, septum, and striatum and (2) turnover rate (utilization) in these structures. Naive rats were used in these experiments to avoid an interaction of LVP and behavioral training effects on NA and DA metabolism. The subjects received either no treatment, LVP (300 mU/kg, intraperitoneal), or vehicle control solution 10 min before sacrifice, and the effects of these treatments on NA and DA levels in the hypothalamus, septum, and striatum were assessed. Results indicated that, relative to intact and vehicle controls, peripherally administered LVP

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decreased DA content in the hypothalamus, septum, and striatum but had no significant effect on NA content in any of these brain structures. In biochemical testing of the effect of LVP on NA and DA turnover in these structures, the subjects were either nontreated, or received -MPT injected together with either LVP (300 mU/kg, intraperitoneal) or vehicle control solution 4 h before sacrifice. The results demonstrated that LVP increased the -MPT-induced disappearance of DA in the septum and the striatum, and of NA in the hypothalamus. The results of this study confirm earlier findings that vasopressin, although not importantly involved in avoidance learning, does play a role in its retention (see Chapter 2), and also suggested that brain catecholamines (CAs) appear to be involved in mediating the VP retention effect because inhibiting their synthesis prevented the effect. Moreover, the observation that LVP influenced CA utilization in some areas of the brain is consistent with this suggestion. b. Kovacs et al. (1979a) Kovacs et al. (1979a) further investigated the effects of AVP and OT on memory consolidation in a PA task, and on CA neurotransmission. The behavioral experiments, discussed earlier in this chapter, demonstrated that VP and OT significantly modulated memory consolidation when microinjected into the hippocampal dentate gyrus, the dorsal septal nucleus, and the dorsal raphe nucleus, but not the central nucleus of the amygdala. The biochemical studies were done 1 week later, and used those subjects for which treatment resulted in significant PA retention. As in the behavioral experiment, AVP (20–25 pg for bilateral injections or 50 pg for a midline injection) or placebo was microinjected into either the hippocampal dentate gyrus, dorsal septal nucleus, or dorsal raphe nucleus. The microinjection was given 30 min after intraperitoneal injection of -MPT, and 3 h later the rats were killed and the brains dissected. The following nuclei were removed and examined for NA and DA content: dorsal septal nucleus, medial septal nucleus, hippocampal dentate gyrus, thalamic parafascicular nucleus, caudate nucleus, red nucleus, dorsal raphe nucleus, and locus coeruleus. AVP injection into the hippocampal dentate gyrus increased utilization of NA in that structure and also in the red nucleus. AVP injection into the dorsal septal nucleus decreased NA utilization in the septal nucleus itself and increased it in the red nucleus. AVP injected into the dorsal raphe nucleus did not influence NA utilization in any of the brain nuclei studied but did increase DA disappearance in both the locus coeruleus and the red nucleus. Whether the increased utilization of NA and DA in the red nucleus after VP injection into the septal–hippocampal system and locus coeruleus, respectively, is related to mnemonic and/or motor processing remains to be clarified. The behavioral and biochemical data were interpreted as indicating that (1) VP and OT modulate memory consolidation in limbic system structures

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that also receive input from extrinsic catecholaminergic projection systems, and (2) the ability of AVP to modulate catecholaminergic neurotransmission is related to this behavioral effect. c. Veldhuis et al. (1987) Veldhuis et al. (1987) carried out behavioral and biochemical experiments to investigate the effects of endogenous AVP on PA retention and CA utilization in selected limbic and striatal brain sites of male Wistar rats. The behavioral experiments, described earlier in this chapter, showed that anti-VP serum attenuated PA memory consolidation and retrieval when microinjected into the dorsal and ventral hippocampus, and retrieval when injected into the dorsal septal nucleus. The biochemical experiments were carried out 1 week after completion of the behavioral experiments. Thirty minutes after intraperitoneal injection of -MPT, vehicle or anti-VP serum was microinjected into the same brain structures treated during the behavioral experiments. Three hours later the animals were killed and the injection regions were dissected for study. Anti-VP serum influenced NA utilization when microinjected into the dorsal or ventral hippocampus (decreased utilization) or into the dorsolateral septum (increased utilization). Influence on DA utilization was not applicable because, after -MPT administration, the concentration of this catecholamine was below the limit of detection in all three of these brain structures. Microinjection of the antiserum into the caudate nucleus had no effect on DA utilization, and NA concentration was below the limits of detection after -MPT treatment. The biochemical data suggest that endogenous VP modulates NA neurotransmission in each of the three limbic system sites influenced by the antiserum in the behavioral experiments (i.e., dorsal and ventral hippocampus, and dorsal septal nucleus). The authors noted the discrepancy between their failure to demonstrate an interaction between VP and the nigrostriatal system and the results of several behavioral and biochemical studies that have obtained positive findings (e.g., Van Heuven-Nolsen and Versteeg, 1985; see Section III.C.1.a). They suggested that the present findings could be due to the fact that the influence of VP on DA neurotransmission in the caudate nucleus is indirect (i.e., mediated by VP-modulated neurons that impinge on DA terminals but are located outside the area treated in this study). 2. Summary Each of the studies described above (including both behavioral and biochemical experiments) provided support for a VP/CA-ergic interaction in memory processing. Kovacs et al. (1977) confirmed the importance of CA-ergic neurotransmission for mediating the influence of VP on PA retention, and also indicated that VP modulates CA-ergic neurotransmission in a number of areas of the brain (e.g., hypothalamus, striatum, and septum). Kovacs et al. (1979a) observed that AVP microinjected into selected limbic

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system structures (hippocampus, septal area, and dorsal raphe nucleus) facilitates PA memory consolidation, whereas OT microinjected into some of these structures (hippocampus and dorsal raphe nucleus) attenuates it. Moreover, AVP modulates NA neurotransmission in both the hippocampus and dorsal septal nuclei. Veldhuis et al. (1987) demonstrated that reducing endogenous VP in the hippocampus and septal area attenuates memory consolidation and/or retrieval and that VP modulates NA neurotransmission in these sites.

B. The Behavioral Protocol: Effect of Selective Lesions in a Catecholaminergic Projection System on VP-Induced Avoidance Retention 1. Selected Studies The two studies described in this section focused on a potential interaction between VP and the NA-ergic system originating in the locus coeruleus, in the upper pontine level of the brainstem, and projecting to the telencephalon (ceruleo–telencephalic NA pathway). This pathway was interrupted by chemical lesions placed in the dorsal noradrenergic bundle, and the effect of this lesion on the ability of the peptide to facilitate PA retention was studied. The rationale for this protocol is that a failure of VP to influence retention in the absence of the fully functioning transmitter system indicates the necessity for that system in mediating the retention effect of VP. The first study employed exogenous vasopressin, and the second manipulated central levels of endogenous vasopressin. Both studies provided support for the proposed VP/CA-ergic interaction in memory processing, and also for a secondary role for the 5HT-ergic projection in mediating the VP influence on memory processing. a. Kovacs et al. (1979b) Kovacs et al. (1979b) lesioned the dorsal noradrenergic bundle (DNAB, a major part of the ceruleo–telencephalic pathway) and studied the effect on VP-induced PA retention. This pathway innervates structures that have been implicated, by lesion (Van Wimersma Greidanus and De Wied, 1976b) and microinjection studies (Kovacs et al., 1979a), in the effects of vasopressin on avoidance behavior. Because VP has been reported to influence DA (Kovacs et al., 1977, 1979a) and serotoninergic neurotransmission (Ramaekers et al., 1977), and the DNAB interacts with these neurotransmitter systems (e.g., Anderson et al., 1977; Antelman and Caggiula, 1977), the lesion technique was also applied to a brain structure containing cells producing DA (nucleus accumbens), and serotonin [5HT; dorsal raphe nucleus (DRN)]. The subjects received either sham operations (microinjected physiological saline) or chemical lesions in the DNAB [microinjected 6-hydroxydopamine (6-OHDA) toxin], nucleus accumbens (microinjected 6-OHDA toxin),

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or the DRN [microinjected 5,6-dihydroxytryptamine (5,6-DHT) toxin]. The chemical lesions were made by microinjection of the designated neurotoxic compound via a metal guide cannula acutely implanted in the target structure until completion of the lesion. The chronically implanted cannula(s) permitted a subsequent injection of AVP [50 pg, unilaterally into the DRN; 25 pg, bilaterally into the locus coeruleus (LC)] into freely moving conscious subjects during behavioral testing. Behavioral testing began 10 days after lesioning and cannula implantation. Behavioral testing was carried out in a single-trial step-through passive avoidance task (2-s FS at 0.25-mA intensity). A single postlearning injection of physiological saline or AVP was given either subcutaneously (AVP dose of 5 g/rat, subcutaneous) or intracerebrally via the implanted cannula (AVP at a bilateral dose of 25 pg or 50 pg for midline injection). Retention test trials (up to maximum of 300 s) were given 24 and 48 h after the PA learning trial. The behavioral results were as follows: (1) for the sham operates, the 5-g dose of AVP significantly increased reentry latency relative to the saline controls in both retention tests; (2) comparisons between lesioned and shamoperate saline controls indicated that the lesion interfered with PA retention in the 48-h, but not the 24-h, retention test. However, the lesion did prevent the AVP-facilitated retention effect in both tests; (3) neither the nucleus accumbens lesion nor the DRN lesion prevented the facilitation of PA behavior produced by postlearning peripherally administered AVP; (4) the postlearning microinjection of AVP into the LC did not influence PA behavior in either retention test; (5) a posttraining microinjection of AVP into the dorsal raphe nucleus significantly facilitated PA retention in the 24-h retention test. Although chemically lesioning the DRN did not influence PA behavior per se, it did prevent the facilitated PA retention induced by the AVP microinjection into this region; and (6) injection of the NA toxin into the DRN (destructive of NA terminals synapsing on 5HT cell bodies in the nucleus) did not influence PA behavior per se but prevented the facilitated PA retention effect induced by postlearning microinjection of AVP into the nucleus. After completion of the behavioral studies, neurochemical techniques applied to dissected brains determined catecholamine (NA and DA) content in the brain as well as serotonin uptake in the midbrain and dorsal hippocampi. Results of the neurochemical analysis indicated that (1) the 6OHDA-induced lesion of the DNAB significantly decreased NA content in the LC (site of A6 cell population, the source of the NA fibers of the DNAB) and in the hippocampal dentate gyrus (a target structure of NA fibers in the DNAB). However, this lesion did not affect DA content assessed in the nucleus accumbens or caudate nucleus (sites that receive synaptic input from the DNAB). This finding is consistent with studies that have shown that 6-OHDA damage to the DNAB depletes NA levels mainly in the forebrain (Mason and Iversen, 1977), but produces no changes in brain

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DA levels (Roberts et al., 1976); and (2) the 5,6-DHT lesion of the dorsal raphe nucleus significantly decreased serotonin uptake in the midbrain and in the dorsal hippocampus (target areas receiving serotonin input from the dorsal raphe nucleus). The results of this study led the authors to three major conclusions. First, the DNAB–NA fiber pathway is important for expression of the AVPinduced facilitation of PA memory consolidation. This conclusion rests on the observations that the 6-OHDA lesion of the DNAB prevented AVP facilitation of PA memory consolidation, and that this lesion depleted NA content in a brain site implicated in the AVP effect on retention while failing to influence DA content in the brain. Second, the interaction between AVP and the DNAB–NA fiber system appears to occur in the region of the fiber terminals of this pathway. This conclusion rests on the findings that (1) locally applied AVP facilitated PA memory consolidation when microinjected into the DRN, which contains NA terminals synapsing on 5HT cell bodies, but not when microinjected into the LC, which contains the cell bodies of the DNAB–NA fibers, and (2) lesioning the DRN prevented the PA response normally maintained by posttraining microinjection of AVP into this structure. Third, this study indicated that the serotonin (5HT) projection to the telencephalon, which originates in the DRN, is of secondary importance to the VP-induced facilitation of memory, because activity in the 5HT pathway is modulated by NA input from the LC. Figure 3 provides a schematic illustration of the neurotransmitter pathways involved in the influence of AVP on PA behavior.

FIGURE 3 Neurotransmitter pathways involved in the action of vasopressin. Key: Norepinephrine (NE) pathways, solid arrows; serotonin (5HT) pathways, dashed arrows; HPC, hippocampus; SEPT, septum; LC, locus coeruleus; DNB, dorsal noradrenergic bundle. Source: Kovacs et al., 1979b (Fig. 5, p. 83). Copyright ß 1979 by Elsevier/North-Holland Biomedical Press.

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b. Kovacs et al. (1980a) Kovacs et al. (1980a) obtained further support for an NA–VP interaction mediating the retention effects of the peptide. The DRN contains serotonin cell bodies and also terminals of both NA and DA projection systems. Reducing endogenous AVP in this area by microinjection of anti-VP serum impaired PA behavior in a 24-h retention test. The antiserum interacted with the CA terminals rather than the 5HT cell bodies in the region, as indicated by the failure of the antiserum to influence PA retention once the CA terminals in the region were destroyed.

C. The Biochemical Protocol: The Effect of VP, OT, or Their Antisera on Catecholaminergic Transmission in Selected Brain Sites Studies examining the effect of the neurohypophysial peptides on catecholaminergic utilization fall into two categories depending on whether vasopressin or oxytocin levels in selected brain sites were elevated by microinjected VP or OT, or reduced by microinjection of their antisera. 1. Effect of Centrally Injected AVP or OT on Catecholamine Neurotransmission a. Selected Studies i. Tanaka et al. (1977a) Tanaka et al. (1977a) studied the effect of intracerebroventricularly administered AVP (30 ng/rat) on catecholaminergic (CA-ergic) utilization in 37 individual brain sites dissected out of the following brain regions: hindbrain, midbrain, hypothalamus, thalamus, preoptic area, amygdala, and hippocampus. Vasopressin influence on CA-ergic utilization was assessed by evaluating the rate of disappearance of NA and DA after treatment with the CA synthesis enzyme inhibitor, -MPT. An AVP-induced increase in this rate would signify activation by the peptide, whereas a decrease would indicate an inhibitory effect of the peptide. Intracerebroventricularly injected vasopressin significantly increased the rate of NA disappearance (increased utilization) in several regions of the hindbrain (locus coeruleus, A1 region, and nucleus of the solitary tract), midbrain (dorsal raphe nucleus), thalamus (parafascicular nucleus), and hypothalamus (anterior hypothalamic nucleus), and in the medial forebrain bundle and the dorsal septal nucleus. It significantly decreased the rate of NA disappearance in the red nucleus of the midbrain and in the supraoptic nucleus of the preoptic area. The vasopressin-induced increase in NA utilization in the subiculum and dentate gyrus of the hippocampus was close to statistical significance. A vasopressin-induced increase in the rate of DA disappearance (increased utilization) was observed in the basal ganglia (caudate nucleus), hypothalamus (median eminence), and midbrain (dorsal raphe nucleus and in the A8 region, the origin of the DA nigrostriatal projection system).

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Several of the regions in which CA-ergic utilization was increased by AVP have been implicated, by lesion or microinjection studies described earlier, in a vasopressin facilitation of retention in avoidance behavior. These regions include the parafascicular thalamus, dorsal septal nucleus, locus coeruleus, and dorsal raphe nucleus. The nearly significant VP increase in NA utilization in the hippocampus is also noteworthy. ii. Van Heuven-Nolsen et al. (1984a) Van Heuven-Nolsen et al. (1984a) studied the effect of intracerebroventricularly administered OT (0, 1, 10, 100, or 1000 ng/rat) on the rate of disappearance of NA and DA after -MPT treatment. OT was administered 30 min after -MPT treatment and the animals were killed 3 h later. Twenty-eight nuclei were dissected from various brain regions for study. At one or more dose levels, intracerebroventricularly injected OT significantly modulated NA utilization relative to saline controls in a few restricted brain sites: it increased NA utilization in the supraoptic nucleus, and decreased it in the lateral septal (LS) nucleus, medial septal (MS) nucleus, and the anterior hypothalamic area. OT did not significantly influence DA utilization in any of the brain nuclei studied, although it did exhibit a nonsignificant tendency to increase it in the caudate nucleus, globus pallidus, and medial septal nucleus. None of the midbrain sites (i.e., red nucleus, central gray, and A8 region) showed an OT effect on DA utilization. The authors concluded that, in comparison with the effects of VP on NA utilization (Tanaka et al., 1977a,b), these results suggest that OT interactions with the catecholamines are less widespread and, where effective, it generally tends to attenuate NA utilization whereas VP tends to increase it. iii. Van Heuven-Nolsen et al. (1984b) Van Heuven-Nolsen et al. (1984b) carried out two experiments to study the effects of intraamygdalainjected AVP and two related peptides (the AVP derivative cyclo[Lys-Gly], and the C-terminal tripeptide of oxytocin, Pro-Leu-Gly-NH2 [PLG]) on CA utilization in various brain structures. In the first experiment, vehicle, AVP (0.01, 0.1, or 1 pmol/rat), cyclo[Lys-Gly] (2.7 or 27 pmol/rat), or PLG (1.8 or 18 pmol) was microinjected into the amygdala 30 min after intraperitoneally injected -MPT. The rats were killed 3 h later and the amygdala was dissected and examined for NA and DA utilization. The results indicated that DA utilization in the amygdala was increased by the highest dose of AVP (1 pmol), the lower dose of cyclo[Lys-Gly] (2.7 pmol), and the higher dose of PLG (18 pmol). None of the peptides influenced NA utilization in the amygdala. In the second experiment, vehicle, cyclo[Lys-Gly] (0.27, 2.7, or 27 pmol), or PLG (0.18, 1.8, or 18 pmol/rat) was microinjected into the amygdala 30 min after intraperitoneal injection of -MPT. The animals were killed 3 h later, the brain was dissected, and NA and DA utilization was assessed in the nucleus

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accumbens, each of five nuclei of the amygdala complex (basal, central, cortical, lateral, and medial nuclei), the thalamic parafascicular (pfc) nucleus, and the hypothalamic paraventricular nucleus (PVN). The results indicated that the OT fragment PLG, at doses of 1.8 and 18 pmol, significantly enhanced DA utilization in the cortical amygdaloid nucleus, and a similar effect in the central amygdaloid nucleus neared statistical significance. The AVP residual cyclo[Lys-Gly], at a dose of 2.7 pmol, facilitated DA utilization in the central amygdaloid nucleus, and a similar effect in the cortical amygdaloid nucleus neared statistical significance. DA levels in the other nuclei studied were either below the limit of detection after -MPT pretreatment or were not influenced by these peptides. In summary, the peptides had no influence on NA utilization in any of the brain nuclei studied. The increase in local DA utilization after their microinjection into various nuclei of the amygdala suggests that VP and related peptides may exert at least some of their effects on retrieval processes via DA-containing terminals in the amygdala. The observation that local microinjection of AVP, cyclo[Lys-Gly], and PLG into the amygdala reverses experimentally induced amnesia (Bohus et al., 1982) is in accord with this suggestion, because retrograde amnesia may involve a deficit in memory retrieval (Dunn, 1980). iv. Van Heuven-Nolsen and Versteeg (1985) Van Heuven-Nolsen and Versteeg (1985) carried out microinjection, push–pull perfusion, and in vitro experiments to test the hypothesis that AVP interacts with the nigrostriatal DA system, and to learn more about the site and nature of this interaction. Two microinjection experiments were conducted. In one experiment, saline or AVP (10, 100, or 1000 pg/rat) was bilaterally injected into the A9 region of the substantia nigra, the cell body region of this pathway, and its effect on DA utilization in the caudate nucleus (CN) and the nucleus accumbens (NA), two terminal regions of the pathway, was assessed. In the other experiment, saline or AVP (10, 100, 1000, or 10,000 pg/rat) was bilaterally microinjected into the CN and evaluated for its effect on DA utilization in that structure. In both experiments the rats received an intraperitoneal injection of -MPT 30 min before the microinjection experiments. The rats were killed 3 h after the microinjections, their brains were removed and sectioned, and cannula placements were histologically verified. The tissue sections from selected parts of the CN and/or NA were homogenized and DA concentration levels in the homogenates were measured. The results indicated that AVP, at a dose of 100 pg/rat or greater, significantly increased DA utilization in the CN when microinjected into that brain site, but had no influence on DA utilization in either the CN or the NA when microinjected into the A9 region of the substantia nigra.

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In the push–pull experiment, a push–pull cannula was implanted in a site in the CN that previous experiments had indicated increased DA utilization from microinjected AVP. AVP was either added to or omitted from (control procedure) the medium used to perfuse the implanted brain area, permitting an in vivo test of the effect of AVP on the activity of the cells that release the neurotransmitter (DA) into the perfusate. The percentage of DA release during the 30 min of VP perfusion relative to that calculated from the first four fractions obtained before VP treatment was determined for each rat. Addition of AVP to the medium at a final concentration of 10 6 M strongly enhanced DA efflux from the CN, an effect that was immediate and lasted only during the time that the peptide was present in the perfusion medium. The effect of VP on DA synthesis in the CN was deduced by two experiments that used in vitro assessment of tritiated DA conversion from tritiated tyrosine, its precursor molecule. In the first experiment, AVP (concentration range, 10 8 to 10 4 M) was added to CN slices in vitro from the beginning of the incubation period. In the second experiment, VP (0.1, 1.0, or 5.0 nmol in 1 l of saline) or 1 l of saline was initially injected in vivo, that is, into a lateral ventricle of rats via a preimplanted cannula. Each rat was decapitated 1 h after the injection, its brain was removed, and the CN was dissected and assessed in vitro for tritiated DA content. The results of the first experiment indicated that AVP exerted a biphasic effect, inhibiting DA accumulation at a low concentration (10 7 M) and progressively increasing it up to a concentration of 10 5 M. The results of the second experiment indicated no significant effect on DA accumulation in CN slices for any of the doses administered in vivo. Taken together, the results of these various experiments suggest that although VP may have a modest effect on DA synthesis, its primary influence is the enhancement of DA release in the nigrostriatal pathway by an action exerted at the terminus (CN) rather than at the origin (A9 region) of this pathway. This VP effect may involve a direct VP presynaptic action on DA terminals or may influence other neurons, which in turn modulate this DA activity. An indirect effect on DA release could explain the repeated failures of these researchers to observe an AVP influence on DA release from the CN, using in vitro preparations that presumably removed circuitry essential for mediating the influence of VP on this release (unpublished observations cited in Van Heuven-Nolsen and Versteeg, 1985). In addition to the present study, a number of lines of behavioral and biochemical evidence, cited by these authors, also support a VP influence on DA neurotransmitter activity in the nigrostriatal system: (1) intracerebroventricularly injected LVP produced ipsilateral turning in rats with 6-OHDA-induced lesions in the substantia nigra (Schultz et al., 1979); (2) systemically injected DG-AVP modulated self-stimulation of the substantia nigra and ventral tegmental area in rats preimplanted with electrodes in these brain sites (Dorsa and Van Ree, 1979);

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and (3) DA utilization was decreased in the striatum–caudate nucleus region after intracerebroventricular administration of VP antiserum (Versteeg et al., 1978) and in the VP-deficient Brattleboro HODI (homozygous diabetes insipidus) rat (Kovacs et al., 1980b). b. Summary Centrally (intracerebroventricularly) administered AVP (Tanaka et al., 1977a), and AVP and the OT fragment PLG (Van HeuvenNolsen et al., 1984a), have been shown to influence NA and/or DA neurotransmission in a number of brain sites, several of which have been implicated in memory processing. Van Heuven-Nolsen et al. (1984a) found that VP and PLG (which has a VP-like effect on memory processing) (Walter et al., 1975; see Chapter 2) produced similar effects on catecholamine neurotransmission (i.e., enhanced DA, but not NA, neurotransmission in the amygdala). This finding suggested that DA terminals in the amygdala may be modulated by VP input to mediate the influence of the peptide on memory retrieval. Van Heuven-Nolsen and Versteeg (1985) demonstrated that the influence of AVP on DA neurotransmission in the nigrostriatal pathway was mediated at its terminal region, and not its cell body region, thereby enhancing the release of DA into the caudate nucleus and nucleus accumbens, two major target regions of this pathway. 2. Intraventricularly Injected VP or OT Antiserum: Effect on Catecholamine Neurotransmission in Selected Brain Sites a. Selected Studies i. Versteeg et al. (1979) Versteeg et al. (1979) studied the effect of intracerebroventricularly injected anti-VP serum on the -MPT-induced disappearance of catecholamines (NA and DA) in selective brain sites of male Wistar rats. Antiserum or control rabbit serum was injected into a lateral ventricle 30 min after intraperitoneal injection of -MPT, and 3 h later the rats were killed and the brain regions dissected for radioenzymatic assay. NA and DA concentrations were measured in various sites in the telencephalon (caudate nucleus, dorsal septal nucleus, and dorsal hippocampus), diencephalon (thalamic parafascicular nucleus and the following hypothalamic areas: paraventricular nucleus, arcuate nucleus, median eminence, and supraoptic nucleus), midbrain (dorsal raphe nucleus), and hindbrain (locus coeruleus, A1 and A2 regions, and nucleus of solitary tract). The results indicated that anti-VP serum significantly decreased NA utilization in the dorsal septal nucleus, the thalamic parafascicular nucleus, and the nucleus of the solitary tract and DA utilization in the caudate nucleus and the A2 region of the medulla. In comparison with intracerebroventricularly injected AVP (Tanaka et al., 1977a; described earlier), anti-VP serum effects on catecholamine utilization, although involving fewer brain sites, were in the expected opposite direction. The difference in the number of brain sites

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influenced in the two studies was attributed to the dose level used in the AVP study, which resulted in a relatively high VP concentration at its sites of action. It was also noted that although the anti-VP serum undoubtedly decreased the bioavailable AVP at these sites, the extent of this decrease is not known. The data were interpreted as supporting the postulate that endogenous AVP exerts a tonic influence on CA-ergic neurons terminating in various brain regions, several of which have been implicated in memory processing. ii. Kovacs and Telegdy (1983) Kovacs and Telegdy (1983) studied the effect of peripherally and intracerebroventricularly injected OT, and of intracerebroventricularly injected desglycinamide oxytocin (DG-OT) and anti-OT serum, on CA utilization in the brains of an inbred strain of Sprague-Dawley rat. The role of these peptides in CA-ergic neurotransmission was of interest because previous work had indicated that OT may function as a natural amnestic (e.g., Bohus et al., 1978a; Kovacs et al., 1978; see Chapter 2) and that DG-OT, although practically devoid of the endocrine activities of OT, is as potent as the parent peptide in its ability to attenuate avoidance retention (Kovacs et al., 1982b). OT was administered peripherally (0, 0.81, 8.1, or 81.0 g/kg, intraperitoneal) or centrally (1 g/rat, intracerebroventricular). DG-OT (1 g/rat, intracerebroventricular) and anti-OT serum [diluted 1:10 with cerebrospinal fluid (CSF)] were injected into a lateral ventricle in separate groups of rats. Vehicle solution, or normal rabbit serum diluted 1:10 with CSF, served as appropriate controls for the various peptide treatments, and intraperitoneal saline served as the control for -MPT treatment. Peptides and antiserum were injected 30 min after -MPT treatment and the rats were killed 3 h later. The following brain regions were dissected for examination of NA and DA levels: midbrain, hypothalamus, septum, and striatum. On the basis of comparisons with control data, the results indicated that (1) depending on the dose, peripherally administered OT accelerated NA and decreased DA neurotransmission in the midbrain, failed to affect utilization of either neurotransmitter in the hypothalamus or the septum, and had opposing effects on striatal DA utilization depending on whether OT was simultaneously administered, or given 30 min after inhibition of CA synthesis (i.e., inhibited DA disappearance in the former case, and facilitated it in the latter); (2) intracerebroventricularly administered OT failed to influence NA and decreased DA neurotransmission in the midbrain, increased NA and decreased DA neurotransmission in the hypothalamus, failed to influence utilization of either transmitter in the septal area, and facilitated DA disappearance in the striatum; (3) intracerebroventricularly administered DG-OT had no influence on NA and decreased DA utilization in the midbrain, had no influence on the utilization of either neurotransmitter in the hypothalamus, increased NA and decreased DA

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utilization in the septal area, and had no influence on DA utilization in the striatum; (4) intracerebroventricularly administered anti-OT serum had no influence on NA but increased DA utilization in the midbrain, decreased NA but was without influence on DA utilization in the hypothalamus, and had no influence on NA but decreased DA utilization in the septum; and (5) in contrast to the facilitation of striatal DA utilization by intracerebroventricularly administered OT (see result 2 above), intracerebroventricularly injected OT antiserum normalized the accelerated -MPT-induced striatal DA utilization that resulted from intracerebroventricularly administered normal rabbit serum. Although the peptides and antiserum influenced either NA or DA utilization in several of the regions studied, the most interesting finding relevant to memory processing was the pattern of effects on DA utilization in the midbrain. Central (intracerebroventricular) treatment with OT or DG-OT had opposite effects, compared with anti-OT serum, on DA neurotransmission. This parallels the attenuation of PA retention induced by intracerebroventricular injection of OT (Bohus et al., 1978b) or DG-OT (Kovacs et al., 1982b) and its improvement by intracerebroventricularly injected anti-OT serum (Bohus et al., 1978b). The authors suggested that the pattern of effects on DA utilization in the midbrain might be related to the influence of OT on DA utilization in memory processing, a suggestion awaiting experimental evidence. They pointed out that monoaminergic-containing cell bodies in this region [e.g., the dorsal raphe nucleus and the DA-containing cells (A9 and A10 groups) that project to limbic and striatal structures, respectively] are known to receive OT terminals of neurons originating in the hypothalamic PVN. It is also noted that OT-induced inhibition of steady state DA levels in the midbrain was previously observed by Schwarzberg et al. (1981). Further study is needed to clarify the role of the OT interaction with DA projections to learn whether this interaction is involved in modulation by the peptide of conditioned avoidance retention behavior. b. Summary The two studies described above conducted experiments that reduced the bioavailability of brain levels of VP or OT and studied the effect on CA-ergic neurotransmission in various brain sites implicated in memory processing. Relevant to the putative role of VP in memory modulation were the findings by Versteeg et al. (1979) that endogenous VP interacts with NA-ergic neurotransmission in the dorsal septal nucleus and the thalamic parafascicular nucleus, and with DA-ergic neurotransmission in the caudate nucleus. Relevant to the putative role of OT in memory modulation was the pattern of findings by Kovacs and Telegdy (1983) indicating OT/DA-ergic interactions in the midbrain that paralleled earlier described behavioral findings of OT effects on retention in avoidance conditioning tasks.

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IV. Interaction between VP and Catecholamines of Peripheral Origin during Memory Processing

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Whereas studies described in the preceding section of this chapter support an interaction between central catecholaminergic systems and the neurohypophysial peptides in memory processing, experiments described below indicate the importance of the adrenal medullary hormone adrenaline (epinephrine) for the contribution of vasopressin to memory processing. The role of peripheral catecholamines in memory processing is documented by a number of lines of evidence: (1) PA retention was enhanced after posttraining peripherally injected adrenaline or noradrenaline (Gold et al., 1977), whereas peripherally administered inhibitors of catecholamine synthesis impaired retention (Palfai and Walsh, 1979); (2) PA retention was significantly impaired after surgical removal of the adrenal medulla (the endocrine source of peripherally circulating epinephrine), and epinephrine replacement therapy dose dependently normalized this retention deficit (Borrell et al., 1983c); and (3) more recent evidence indicates that central effects of peripherally administered epinephrine entail the release of noradrenaline in the basolateral medulla, mediated by a pathway that involves activation of vagus nerve endings by peripherally circulating catecholamine (Cahill and McGaugh, 1996; Williams et al., 1998).

A. Selected Studies: Borrell et al. (1983a,b) Experiments by Borrell and colleagues, cited below, investigating a potential interaction between vasopressin and peripherally circulating epinephrine in memory processing were reported in two sources: Borrell et al. (1983a,b). These experiments demonstrate the importance of peripherally circulating epinephrine for expression of (1) the facilitating effects of exogenous AVP on memory consolidation and retrieval (experiments 1–4) and (2) the normal role played by endogenous vasopressin in memory processing (experiments 5 and 6). A single-trial step-through PA task was used for testing retention in both normal Wistar rats and Brattleboro HODI and HEDI (heterozygous diabetes insipidus) rats. Manipulation of hormonal epinephrine was accomplished by surgical removal of either the entire adrenal gland (adrenalectomy, ADX), which depleted the subjects of both adrenocortical hormones and the catecholaminergic hormone, or the adrenal medulla (adrenomedullectomy, ADMX), which deprived the animals only of the hormonal catecholamine, and by the use of hormonal replacement therapy (various doses of epinephrine for ADMX experiments, and of epinephrine and corticosterone for ADX experiments). Experiments testing the effects of exogenous vasopressin on PA retention or retrieval injected vehicle or AVP either peripherally (1 g/rat, subcutaneous) or centrally

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(5 ng/rat, intracerebroventricular), using a memory consolidation or retrieval test design. In experiments with Brattleboro rats, exogenous AVP was used for replacement therapy. In all experiments PA retention was tested 24 h after the PA learning trial. In experiment 1, rats that had received sham operations, adrenalectomies, or adrenomedullectomies were injected either peripherally with physiological saline or AVP (1 g/rat, subcutaneous), or centrally with saline or AVP (5 ng/rat, intracerebroventricular). To test AVP effects on memory consolidation, the injections were given immediately after the learning trial (2-s FS of 0.50-mA intensity); to test the effects of VP on memory retrieval, the injections were given 1 h before the 24-h retention test (reentry latency measured up to a maximum of 300 s). The results demonstrated that (1) ADX and ADXM significantly impaired PA retention (reentry latencies in the two operate groups were significantly below the 300-s median reentry latency of the sham operates); (2) neither a posttraining nor preretention injection of subcutaneously or intracerebroventricularly administered AVP normalized the retention deficit observed in the ADX and ADXM rats; and (3) the lack of an AVP effect on memory consolidation and retrieval, even when the ADX rats were given corticosterone replacement therapy, indicated that the failure of VP to facilitate PA behavior was not due to the absence of this adrenal cortical hormone. Experiment 2 was designed to determine the degree to which epinephrine replacement therapy in ADX rats could correct the PA retention deficit in both saline- and AVP-treated rats. The ADX rats received a subcutaneous injection of either vehicle control solution or epinephrine (0.0025, 0.05, or 50 g/kg, subcutaneous) immediately after the learning trial. The three doses of epinephrine selected for study were based on the inverted U-shaped dose– response curve relating PA retention to posttraining epinephrine treatment in intact rats (i.e., the facilitatory effect disappears with further increases in dose level; Gold and Van Buskirk, 1975). Accordingly, the three dose levels of epinephrine were judged as least effective (0.0025 g/kg, subcutaneous), submaximally effective (0.05 g/kg, subcutaneous), and as a high, normally amnesia-inducing dose (50.0 g/kg, subcutaneous). Physiological saline or AVP (1 g/rat, subcutaneous) was injected either immediately after the PA learning trial or 1 h before the 24-h retention test. Testing for VP influence on memory retrieval occurred only in ADX rats given the low (0.0025 g/kg) or high (50 g/kg) dose of epinephrine. Results indicated that ADX rats given posttraining injections of AVP showed no improvement of PA retention after the least effective dose of epinephrine, improved PA retention after the submaximal dose of epinephrine (i.e., significantly more rats showed maximal avoidance behavior in the AVP-treated group), and significantly improved PA retention after the normally amnesia-inducing high dose of epinephrine. On the other hand, the ADX rats given preretention injections of AVP showed no improvement in

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memory retrieval whether treated with the ineffective low or the amnesiainducing high doses of epinephrine. Taken together, these findings (1) indicate that the VP facilitation of memory consolidation occurs only in the presence of an adequate amount of peripherally circulating epinephrine and (2) suggest that the failure of AVP to influence retrieval among the epinephrine-treated ADX rats might have been because postlearning-administered epinephrine was no longer present in the circulation at the time of vasopressin treatment. It was also noted that the memory consolidation effect induced by AVP in ADX rats that received the normally amnesia-inducing dose of epinephrine is consistent with the antiamnesic property of vasopressin (e.g., Rigter et al., 1974; Chapter 2), and indicates the importance of peripherally circulating epinephrine for the expression of this VP function. In experiment 3, the authors tested the effect of AVP on memory retrieval in ADX or sham operates given preretention injections of epinephrine. That is, epinephrine (0.0025, 0.50, or 125 g/kg, subcutaneous) and AVP (1 g/rat, subcutaneous) were both injected 1 h before the 24-h retention test. The vehicle for AVP and epinephrine was used for control injections. The results indicated that (1) given alone, AVP failed to normalize memory retrieval in ADX rats; (2) on its own, only the middle dose of epinephrine (50 g/kg) markedly facilitated memory retrieval in ADX rats; and (3) AVP markedly improved PA memory retrieval in all ADX rats that received preretention epinephrine injections. These data were interpreted as indicating that AVP-induced facilitation of memory consolidation, as well as retrieval, requires an adequate level of peripherally circulating epinephrine at the time each phase of memory processing is active. The finding that the peptide was active in ADX rats that received the high dose of epinephrine also reinforces the earlier interpretation that AVP has an antiamnesic property as well as a facilitative influence on memory consolidation and retrieval. Experiment 4 tested the effect of posttraining centrally administered AVP (5 ng/rat, intracerebroventricular) on PA behavior in ADX and shamoperated rats, given an injection of vehicle control or a high dose of epinephrine (50 g/kg, subcutaneous) immediately after the training trial. On its own, this high dose of epinephrine has amnestic properties when injected into intact rats (Gold and Van Buskirk, 1975). It was observed that centrally administered AVP treatment was ineffective in the vehicle-treated ADX rats but significantly improved PA retention in those ADX rats given the posttraining high dose of epinephrine. Because intracerebroventricularly injected AVP likely acted at central receptors, the effects of the peptide were probably not mediated by an initial influence on receptors in the adrenal medulla; instead, the peripherally circulating epinephrine was in some way necessary for mediating the central effects of AVP on PA retention. Experiment 5 investigated PA retention in HODI and HEDI Brattleboro rats given sham operations or ADX surgery 48 h before the PA learning trial.

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These subjects were peripherally injected with vehicle control or AVP (2 g/rat, subcutaneous) immediately after the learning trial. The results were as follows: (1) in accordance with previous observations (De Wied et al., 1975; see Chapter 3), sham-operated, vehicle-treated HODI rats were significantly impaired in PA retention relative to vehicle-treated sham-operated HEDI rats. AVP treatment significantly improved PA behavior in both groups of Brattleboro rats, relative to the vehicle-treated, sham-operated HEDI comparison group; (2) adrenalectomy significantly impaired PA behavior of the HEDI rats, but did not further impair the already severely deficient PA performance of the HODI rats; and (3) the AVP dose that improved PA performance in the sham-operated HODI and HEDI rats had no effect on the deteriorated PA performance of these subjects after adrenalectomy. These results again demonstrated the necessity of peripherally circulating epinephrine for expression of the facilitative influence of AVP on PA retention. Experiment 6 tested the effect of a low (0.05 g/kg, subcutaneous) or a high (50 g/kg, subcutaneous) dose of epinephrine on PA retention in ADX or sham-operated HODI or HEDI rats. Results indicated that the ADXproduced retention deficit was significantly improved in HEDI rats given the low but not the high dose of epinephrine. On the other hand, the same high dose of epinephrine improved PA retention in HEDI rats with intact adrenal glands. Different results emerged for HODI rats given epinephrine replacement therapy. Posttraining injections of either the low (0.05 g/kg) or high (50.0 g/kg) dose of epinephrine failed to influence the behavioral deficit of HODI rats, whether they were given ADX or not. This suggests that in the absence of brain AVP, peripherally circulating epinephrine loses its behavioral effects, indicating multiple interactions between peripheral epinephrine and AVP in memory processing. In summary, these experimental findings indicate that whether injected intracerebroventricularly or subcutaneously, the influence of VP on PA memory consolidation and retrieval is not expressed in rats deprived of the peripherally circulating catecholamine, epinephrine. Furthermore, noting the importance of the ceruleo–telencephalic NA projection system for mediating the facilitative influence of the peptide on PA retention (Kovacs et al., 1979a,b; discussed earlier), it was concluded that the findings suggest a close interaction between vasopressin and central catecholaminergic systems in modulating memory processes, and a dependence on an intact hormonal epinephrine system for expression of this VP/CA-ergic interaction. The authors noted that the exact nature of the involvement of VP with peripherally circulating epinephrine needs to be clarified. As noted in earlier discussion, McGaugh and colleagues (e.g., Williams et al., 1998) have shown that one aspect of the central effect of epinephrine on memory processing involves NA release in the basolateral amygdala initiated by activation by the hormone of the vagus nerve. It is possible

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that a similar hormonal action results in activation of the locus coeruleus via a branch of the NA projection to the amygdala that originates in the nucleus of the solitary tract. If so, this could account for the finding that the expression of VP influence on memory processing depends on an intact peripheral epinephrine system. This is so, because interaction of VP with the cerulean/NA-ergic projection pathway is an important mechanism by which the peptide influences memory processing (Kovacs et al., 1979a). However, support for this speculation relies, in part, on demonstration of the required anatomical substrate.

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

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The research studies described in this chapter provided evidence relevant to two additional propositions of the ‘‘VP/OT Central Memory Theory.’’ These propositions and their supportive evidence are discussed below.

A. Proposition 7: The Central Anatomical Substrate for the Memory-Modulating Effects of VP and OT Includes Brainstem and Forebrain Limbic System Structures That Are Implicated in Memory Processing Microinjection and lesion studies have provided evidence in support of proposition 7. Lesion studies have indicated that VP-induced prolonged maintenance of a conditioned avoidance response is partially or totally prevented by a bilateral lesion of the thalamic parafascicular nucleus (Van Wimersma Greidanus et al., 1974), the septal/nucleus accumbens region (Van Wimersma Greidanus et al., 1975b), the anterolateral hippocampus (Van Wimersma Greidanus and De Wied, 1976b), and the central and basolateral amygdala (Van Wimersma Greidanus et al., 1979b). Lesion studies have also suggested that the various forebrain limbic system structures act more or less independently of one another in mediating the longterm effect of vasopressin on retention, but act in concert in mediating the short-term, presumably arousal-inducing effects of ACTH-like peptides on memory processing (Van Wimersma Greidanus et al., 1979c). Microinjection studies have indicated that exogenous AVP facilitates memory consolidation when locally injected into the thalamic parafascicular nucleus (Van Wimersma Greidanus et al., 1973); dorsal septal nuclei, hippocampal dentate gyrus, and dorsal raphe nuclei (Kovacs et al., 1979a); and retrieval when injected into the central nucleus of the amygdala (Bohus et al., 1982). Microinjection studies with anti-VP serum have indicated that endogenous VP facilitates memory consolidation when released into the dorsal raphe nucleus (Kovacs et al., 1980a), dorsal hippocampal

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dentate gyrus (Kovacs et al., 1982b), dorsal and ventral hippocampi (Veldhuis et al., 1987), and memory retrieval when released into the dorsal and ventral hippocampi and the dorsolateral septal area (Veldhuis et al., 1987). Local microinjections of OT into midbrain–limbic brain sites have demonstrated that exogenous OT opposes the influence of VP and attenuates PA memory consolidation when microinjected into the dorsal raphe nucleus and hippocampal dentate gyrus, but mimicks the influence of VP and facilitates memory consolidation when injected into the dorsal septal area (Bohus et al., 1982; Kovacs et al., 1979a). The similarity of VP and OT actions when injected into the septal area may be related to brain regional differences in rate of metabolism of these peptides, and the consequent generation of OT metabolites having VP-like effects on avoidance behavior (Bohus et al., 1982). Moreover, microinjection of anti-OT serum into the dorsolateral septum as well as the ventral hippocampus facilitated PA memory consolidation and retrieval, suggesting that endogenous OT released into these brain sites opposes the action of VP and attenuates these phases of memory processing (Van Wimersma Greidanus and Baars, 1988).

B. Proposition 8: Neurohypophysial Peptides Interact with Central Catecholaminergic Neurotransmitters in Mediating Their Influence on Memory Processing, and the VP/NA-ergic Interactional Effect Appears to Be Dependent on an Intact Hormonal Epinephrine System for Its Expression According to proposition 8, centrally located VP-ergic and OT-ergic neuropeptide systems interact with central NA as well as DA projection systems in exerting their influence on memory consolidation or retrieval in avoidance tasks. This interaction involves VP- or OT-induced modulation of release of NA or DA at relevant brain sites, which in turn mediates the influence of the neuropeptides on memory processing. In addition, peripherally circulating epinephrine plays an essential role in modulation by vasopressin of these phases of memory processing. Although proposition 8 emphasizes catecholamine involvement in the neurohypophysial peptide influence on memory processing, it does not deny participation of the serotoninergic and cholinergic forebrain projection systems. Thus Kovacs and colleagues suggested that serotonin-containing fibers originating from the dorsal raphe nucleus may play a secondary role in mediating VP memory effects, because vasopressin appears to interact with noradrenergic terminals that modulate neural activity in this serotonin pathway (Kovacs et al., 1979b, 1980a). Interaction between central CA-ergic projections and the neurohypophysial peptides in memory processing has been supported by both behavioral

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and biochemical paradigms. Behavioral paradigms test the ability of exogenous VP or OT to influence PA retention after manipulation of a given CA-ergic pathway. Three behavioral studies support this proposition. Kovacs et al. (1977) demonstrated that inhibition of CA synthesis in the brain prevented facilitation of PA retention induced by peripherally injected LVP. Kovacs et al. (1979b, 1980a) showed that VP interacts with NA-containing fiber systems in mediating its effect on PA retention and that this interaction occurs at the NA terminals and not in the cell body region. Thus, chemically lesioning the dorsal noradrenergic projection to the forebrain, which originates in the locus coeruleus, prevented the facilitation of PA memory consolidation induced by peripherally injected AVP (Kovacs et al., 1979b). It has been demonstrated that NA terminals innervating the dorsal raphe nucleus are responsible for the facilitated PA retention induced by endogenous VP released in this structure (Kovacs et al., 1980a) as well as by locally microinjected AVP (Kovacs et al., 1979b). The biochemical paradigm tests the ability of VP or OT to influence neurotransmission in CA-ergic pathways innervating forebrain structures implicated in memory processing. If VP facilitates memory storage and retrieval by activating these CA-ergic pathways, and OT attenuates memory processing by inhibiting them, intracerebroventricularly or locally administered VP should increase, and OT decrease, pathway activation (the rate of utilization or release of the catecholamine). Moreover, correspondingly opposite effects induced by administration of the antisera of these peptides would further support an interaction between endogenous VP and OT and these CA-ergic pathways. The following observations concerning vasopressin are consistent with these predictions: (1) locally microinjected AVP significantly increased NA release in the hippocampal complex (Kovacs et al., 1979a), and locally microinjected anti-VP serum significantly decreased it in the dorsal and ventral hippocampus (Veldhuis et al., 1987); (2) intracerebroventricular injection of AVP (Tanaka et al., 1977a) and anti-VP serum (Versteeg et al., 1979) increased and decreased NA release in the thalamic parafascicular nucleus, respectively; (3) Van Heuven-Nolsen and Versteeg (1985) observed that microinjected AVP increased DA release in the caudate nucleus, a structure innervated by the nigrostriatal DA pathway, and this finding was supported by a subsequent push–pull perfusion experiment. Moreover, intracerebroventricular injection of AVP increased (Tanaka et al., 1977a), and anti-VP serum decreased (Versteeg et al., 1979) DA utilization in the caudate nucleus; and (4) locally microinjected AVP and AVP-like fragments (cyclo[Lys-Gly] and the OT tripeptide LPG), which exert VP-like effects on memory processing (Walter et al., 1975), increased DA utilization (release) in the amygdala, an interaction believed to be involved in mediating the retrieval effects of the neurohypophysial peptides (Van Heuven-Nolsen et al., 1984b). However, a study by Winnicka (1996) obtained results

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that indicated that chemical lesioning, which prevented DA release in the central amygdala, did not influence the facilitated retrieval effect induced by intracerebroventricularly injected AVP. Discrepant findings have been obtained for the NA-ergic pathways innervating the dorsal raphe nucleus and the dorsal septal region: (1) whereas intracerebroventricularly injected AVP increased NA utilization in the dorsal raphe nucleus (Tanaka et al., 1977a), a microinjection of AVP in this structure did not influence NA utilization (Kovacs et al., 1979a); and (2) in accordance with expectations, intracerebroventricular injection of AVP (Tanaka et al., 1977a) and of anti-VP serum (Versteeg et al., 1979) significantly increased and decreased NA utilization (release) in the dorsal septal nucleus, respectively. However, when microinjected into this brain site, the opposite pattern of effects was observed: when microinjected into the dorsal septal nucleus, AVP significantly decreased (Kovacs et al., 1979a), and antiVP serum significantly increased (Veldhuis et al., 1987), NA utilization in this structure. Although fewer studies have been carried out with oxytocin, these too have been interpreted as generally consistent with the prediction that OT inhibits CA innervation of structures implicated in memory processing: (1) an intracerebroventricular injection of OT decreased the rate of NA utilization in the lateral and medial septal nuclei (Van Heuven-Nolsen et al., 1984a); and (2) an intracerebroventricular injection of OT and/or DG-OT decreased DA utilization in the midbrain and septal area but unexpectedly increased it in the striatum. These effects were reversed by an intracerebroventricular injection of anti-OT toxin, except for the septal area, where the antitoxin mimicked the effects of the peptide (Kovacs and Telegdy, 1983). Borrell and colleagues have suggested an interaction between AVP and peripherally circulating catecholamines, especially epinephrine. These researchers demonstrated the importance of circulating epinephrine for the expression of AVP facilitation of PA retention (Borrell et al., 1983a,b). Specifically, their experiments showed that whether administered centrally (intracerebroventricular) or peripherally, AVP-induced facilitation of PA retention is not observed in rats deprived of the medulla of both adrenal glands and hence of the peripherally circulating catecholamines secreted by these structures. These data are in accord with the possibility that peripherally circulating catecholamines interact with central catecholaminergic pathways in mediating the influence of AVP on memory processing (De Wied et al., 1988).