Theme : Dreams

Prolactin Microinjections into the Amygdalar Central Nucleus Lead to Decreased NREM Sleep

Larry D. Sanford1, Peter Nassar1, Richard J. Ross2, Jay Schulkin3 and Adrian R. Morrison1,2
1Laboratory for Study of the Brain in Sleep, Department of Animal Biology, School of Veterinary Medicine, 2Department of Psychiatry, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA and 3Behavioral Neuroscience Unit, Clinical Neuroendocrinology Branch, National Institute of Mental Health, Bethesda, MD 20892, USA
Abstract
Prolactin administered systemically, intracerebroventricularly or locally into the lateral hypothalamus enhances rapid eye movement sleep (REM) when given diurnally and decreases REM when given nocturnally. The amygdala is being recognized as an important modulator of behavioral state, and the central nucleus of the amygdala (CNA) has a high concentration of prolactin fibers and receptors. We microinjected prolactin (10, 100, 250 ng/0.2 µl saline) or saline alone into CNA of rats and measured the effect on behavioral state. Prolactin produced a dose-dependent decrease in non-REM (NREM), with the effect becoming significant at the high (250 ng) dose. REM was not significantly affected at any dosage. The results indicate a role for prolactin in CNA in the control of NREM. The results are discussed in terms of the amygdala having a broad role in the regulation of behavioral state.

Current Claim: Prolactin in the central nucleus of the amygdala influences waking and NREM but not REM sleep.

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Prolactin is being linked to the regulation of rapid eye movement sleep (REM) (Roky et al., 1995). Studies in pontine cats (Jouvet et al., 1986), rats (Roky et al., 1993, 1994) and rabbits (Obal et al., 1989) have demonstrated increases in REM after systemic administration of prolactin, whereas antiserum to prolactin has been found to decrease REM in rats (Obal et al., 1992). Further, injections of vasoactive intestinal peptide (VIP), which stimulates the secretion of prolactin (Abe et al., 1985), increases REM in much the same manner as prolactin itself (Obal et al., 1989). It is possible that prolactin could have its effect on REM through a circadian mechanism (Roky et al., 1995). Prolactin, administered subcutaneously (SC), intracerebroventricularly (ICV) (Roky et al., 1993) or locally into the dorsolateral hypothalamus (Roky et al., 1994), an area containing prolactin immunoreactive neurons (Paut-Pagano et al., 1993), increases REM when given diurnally and decreases REM when given nocturnally. In addition, the circadian rhythm of REM, but not non-REM (NREM) is reversed in hypoprolactinemic rats (Valatx and Jouvet, 1988).

The amygdala is rapidly being recognized as an important modulator of REM. Increased cerebral blood flow in the amygdala during REM has been demonstrated in humans (Maquet et al., 1996). In rats, microinjections of serotonin (5-HT) into the amygdala terminate sleep states, and microinjections of the broad spectrum 5-HT antagonist, methysergide, increase sleep and increase the frequency of ponto-geniculo-occipital (PGO) waves in waking (W) and NREM (Sanford et al., 1995). Likewise, in rats, microinjections of the cholinergic agonist, carbachol, into the central nucleus of the amygdala (CNA) suppress REM for up to six hours post-injection (Sanford et al., 1997), whereas in cats, similar microinjections produce a prolonged enhancement of REM and of NREM with PGO waves for up to 5 days (Calvo et al., 1996).

The role CNA plays in modulating REM and its related phenomena, coupled with its high concentration of prolactin-immunoreactive fibers and receptors (Roky et al., 1996; Siaud et al., 1989), raise the question of whether CNA is a site where prolactin influences REM. The present study examined this possibility using the microinjection technique to infuse prolactin locally into CNA and then to observe the effect on behavioral state.

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The subjects were 8 male Sprague-Dawley strain rats of approximately 90 days of age at the time of surgery. The rats were implanted with skull screws for recording the electroencephalogram (EEG) and with stainless-steel wire electrodes sutured to the dorsal neck musculature for recording the electromyogram (EMG). Leads from the recording electrodes were routed to a nine-pin miniature plug that mates to one attached to a recording cable. The recording plug, cannulae and stimulating electrodes were affixed to the skull with dental acrylic and anchor screws. Guide cannulae (26 ga.) were bilaterally implanted with their tips aimed 1.0 mm above the injection site in CNA (P: 2.3, L: 4.0, V: 7.0; Paxinos and Watson, 1986). All surgical procedures were performed stereotaxically under aseptic conditions. Ketamine (85 mg/kg) and Xylazine (15 mg/kg) were administered intraperitoneally for anesthesia. Buprenorphine (0.1 mg/kg) was administered for potential post-operative pain.

For microinjections, cannulae (30 ga.) were lowered via the guide cannulae into CNA bilaterally. Prolactin (10.0, 100.0, 250.0 ng/0.2 µl saline) or saline alone (0.2 µl) was infused in quiet waking during the lights-on period. Microinjections were administered at a rate of 0.1 ml/min. Microinjections were counterbalanced across conditions and were separated by a minimum of 7 days.

The rats were maintained on a 12:12 light-dark cycle. Six-hour polygraphic studies were conducted on each test day beginning 4 hours after light onset at 11:00 a.m. and concluding at 5:00 p.m. Waking, NREM, transition (TRANS) and REM were determined by trained observers. Transition was defined as epochs with low delta activity, high amplitude spindles and greater than 50% theta rhythm. The parameters examined were: total recording period (TRP), time spent asleep (TSA), sleep efficiency (TSA/TRP), REM percentage (REM time/TSA), number and mean duration of waking, NREM, TRANS and REM episodes, and REM latency (to the first REM episode post-injection). The data were analyzed over the first 3 hours as well as over the entire 6-hour recording period. Analyses were conducted with single factor within subjects ANOVAs with repeated measures across drug conditions. For significant F tests, differences among means were examined using planned comparisons procedures.

Upon completion of the experiment the rats were overdosed with sodium pentobarbital (50-100 mg/kg intraperitoneally) and perfused intracardially with 9% saline and 10% formalin. Evans blue dye of equal volume to the experimental microinjections was infused to assist in locating the injection site. The brains were processed to determine cannula and electrode placements. For this purpose the brains were embedded in celloidin; 40 µm slices were made through the areas of interest, and the sections were stained with cresyl violet.

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Figure 1

Figure 2

The effects of prolactin on behavioral state were observed within the first 3 hours of recording. When the entire 6-hour recording period was considered, there were no significant differences in any parameter we examined. During the first 3 hours of recording, however, prolactin produced a significant reduction in total NREM (Fig. 1) and in the number of NREM episodes (Table 1) at the 250 ng dosage. NREM episode duration was decreased at the 10 ng dosage, but not at higher dosages. The decrease in NREM was accompanied by non-significant increases in total W (Fig. 1) and average W episode duration but a significant increase in the number of W episodes (Table 1). In W, prolactin produced an apparent elevation in exploratory activity, as judged by the experimenter, but no other obvious changes in behavior were observed. There were no significant changes in any measure of REM or transitional sleep, although the latter was somewhat increased in some rats (Table 1).
Figure 2 demonstrates the location of the injection sites in the amygdala. The sites were in the vicinity of CNA, although the most ventral extent of two cannulae lay just dorsal, in the globus pallidus. There was no indication that this rat was affected differently, suggesting that the injection site was close enough for diffusion of prolactin into CNA.

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The present study follows previous work in our laboratory indicating that the amygdala plays an important role in modulating behavioral state and suggests that prolactin in CNA is a factor in that modulation. The greatest effect of prolactin in CNA on sleep was seen in the decrease in NREM that was accompanied by an increase in W during the first 3 hours post-injection. A change in NREM was not reported in studies using different modes of administration or locally into the hypothalamus or ventricles (Roky et al., 1993, 1994), suggesting the effects we observed may be specific to prolactin in CNA. The relative changes in episode counts and durations for W and NREM at the 250 ng dose of prolactin suggests a tendency for fewer, longer periods of W, whereas NREM was characterized by fewer episodes with less change in episode duration. Increased exploratory activity in W after microinjection of prolactin may have been a factor influencing alterations in the amounts of W and NREM, though this should be examined systematically.

Prolactin administered systemically, ICV or via local microinjection into the lateral hypothalamus (Roky et al., 1993, 1994) during the diurnal period produces an increase in REM episode duration. In contrast, we found that prolactin microinjected into CNA during the diurnal period did not increase REM episode duration at the dosages tested. The increase in REM found with other modes and sites of administration appears to develop slowly over time suggesting the possibility that we might have seen an increase in REM if we had recorded for longer than 6 hours. However, while not significant, we observed a tendency toward decreased REM in some of the parameters we examined (Table 1). This coupled with the fact that increases in REM were apparent within 1 hour after microinjections into the hypothalamus (Roky et al., 1994) suggests that finding increased REM at longer post-injection recording times would not be likely. Interestingly, transitional sleep between NREM and REM was considerably lengthened in some rats, although this was not significant in the group statistics, suggesting that part of the difference between our results and those of others could have been due to differences in record scoring. We distinguished between transition and REM, whereas other studies examining prolactin have not (Roky et al., 1993; 1994). However, analyses using combined REM + transition measures also did not result in a significant increase in REM. Microinjections of 10 ng prolactin into the hypothalamus during the diurnal period produced significant increases in REM (Roky et al., 1994) whereas we were unable to produce significant alterations in REM with dosages up to 250 ng. Our failure to observe effects on REM at 10 ng and even higher dosages suggests that the influence of prolactin in CNA on the control of REM is minimal.

Research has suggested roles for 5-HT (Sanford et al., 1995), noradrenaline (Sanford et al., 1996) and acetylcholine (Sanford et al., 1997; Calvo et al., 1996) in the amygdala in modulating behavioral state. Recently, Rueter and Jacobs (1996a) used microdialysis to examine 5-HT in several forebrain regions, including the amygdala. They found increased 5-HT release in the amygdala in response to behavioral manipulations that increased alert waking, and a similar increase in 5-HT was observed at the onset of the dark period when rats are most active (Rueter and Jacobs, 1996b). Alpha-1 adrenergic sites in the amygdala have been implicated in cataplexy in narcoleptic dogs (Mignot et al., 1988a; 1988b; 1989). These findings and the anatomical evidence indicating reciprocal connections between the amygdala and DRN and LC, sources of 5-HT and NA input, respectively, to the amygdala (Fallon and Ciofi, 1992) suggest a synergy between the brainstem and the amygdala in controlling behavioral state. The effects of microinjections of carbachol suggest a role for the basal forebrain, which provides the major cholinergic input to the amygdala (Heckers and Mesulam, 1994), as well as the possibility of influence from afferents from brainstem cholinergic nuclei (Woolf and Butcher, 1982). The decrease of NREM produced by prolactin microinjected into CNA also suggests that amygdalar modulation of behavioral state may be influenced by rostral regions, though the exact origin of prolactin innervation of the amygdala is not yet clear. Possibilities include prolactin cell bodies located in the lateral hypothalamic area (Siaud et al., 1989) and bed nucleus of the stria terminalis (ibid).

The present results provide further evidence that the amygdala, particularly CNA, can influence sleep and waking states. The differing effects of various pharmacological manipulations (Calvo et al., 1996; Sanford et al., 1995; 1996; 1997) suggest a broad role for the amygdala in regulating behavioral state, not just REM. This regulation may entail complex interactions with both forebrain and brainstem regions, in which different phases of behavioral state are modulated.

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Supported by USPHS grants MH42903 (Adrian R. Morrison) and Department of Veterans Affairs (Richard J. Ross).

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