Contribution of brainstem structures in modulating ...

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Contribution of Brainstem Structures in Modulating Memory Storage Processes Cedric L. Williams Edwin C. Clayton

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consistent theme that has emerged from the laboratory of James McGaugh over the past three decades is that effective storage of memory involves both the secretion of peripheral hormones such as epinephrine (EPI) in response to arousing events and a subsequent release of norepinephrine (NE) in limbic structures that encode new experiences. Although the foundation for understanding how emotional arousal modulates the memory storage process had been firmly established by the findings from the McGaugh lab, several questions remained regarding the mechanisms that enabled peripherally secreted EPI to regulate NE release in the limbic system. An understanding of this process was hindered by the fact that EPI does not freely enter the brain and, therefore, cannot directly affect central noradrenergic activity One focus of our research as graduate students (I, Williams, studied under Dr. Robert Jensen) was to contribute to the understanding of the memory modulation process laid down by the McGaugh laboratory by identifylng the missing link that enabled these two systems to interact to modulate memory. The research described in this chapter is a direct result of the efforts directed toward understanding this relationship. This research was guided by the premise that one putative mechanism by which hormones such as EPI influence the brain during emotional arousal involves activation of neurons in an area of the brainstem known as the nucleus of the solitary tract (NTS). The NTS receives input regarding fluctuations in autonomic and hormonal states following exposure to arousing experiences by ascending fibers of the vagus nerve. NTS neurons, in turn, convey this information to brain areas that process memoryrelated information. Recent findings indicate that activation of the NTS by pepOur research supported by the National Institute of Mental Health Grant MH01450501 to the first author.

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tides, hormones, or noradrenergic agonists enhance retention in appetitive and aversive learning conditions. In contrast, the memory-enhancing effects of several compounds are attenuated by severing the connection between peripheral nerves and the NTS with vagotomy or by inactivating NTS neurons with reversible anesthetics. currently, the mechanisms by which the NTS influences memory formation are not known. However, limbic structures that process memory such as the amygdala or hippocampus may utilize neural information provided by the NTS during the initial encoding and processing of new experiences. This chapter is divided into four sections to provide an adequate illustration of the contribution of peripheral hormones in modulating memory and their interaction with specific brain systems during memory processing. The first section includes an overview of the anatomical connections among peripheral autonomic fibers, the NTS, and limbic structures involved in memory. The second part discusses the effects of manipulating neuronal activity in the NTS on retention of responses acquired in spatial and emotionally arousing learning tasks. This section is followed by the presentation of findings from in vivo microdialysis experiments demonstrating that amygdala norepinephrine activity is regulated, in part, by noradrenergic neurons originating in the NTS. The chapter concludes with a discussion of how memory processing in the hippocampus may be regulated by structures in the lower medulla that receive direct projections from the NTS.

Anatomy of the Vagal Modulating System: Bridging the Gap Between Peripheral Hormones and the Amygdala Extensive evidence indicates that the enhancement in memory produced by peripheral hormones that are released by emotionally arousing experiences or administered directly to laboratory animals are mediated by influences on the amygdala NE system. For example, the memory deficits observed following adrenalectomy, a surgical procedure that severely depletes peripheral concentrations of EPI (Borrell, de Kloet, Versteeg, & Bohus, 1983; Liang, Juler, & McGaugh, 1986; Silva, 19741, can be reversed by direct infusion of NE into the amygdala (Liang et al., 1986). A relationship between EPI and the amygdala noradrenergic system is further supported by the finding that the enhancement in memory produced by systemic posttraining injection of epinephrine is completely abolished by either lesioning the amygdala (Cahill & McGaugh, 1991), blocking noradrenergic receptors in the amygdala (Liang et al., 1986), or by depleting amygdala NE concentrations with the selective neurotoxin DSP-4 (Liang, Chen, & Huang, 1995). Despite these findings, which clearly delineate

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a functional relationship between the amygdala and EPI in processing emotionally based memories, the pathway by which EPI exerts modulatory influences on the amygdala has not been well defined because peripherally released EPI does not freely enter the central nervous system (CNS; Weil-Malharbe, Axelrod, & Tomchick, 1959; see Figure 8.1). The experiments described in the remainder of this chapter were developed to address the hypothesis that EPI’s effects on memory are mediated by vagal activation of brainstem nuclei in the NTS that project to the amygdala. Findings supporting this view are summarized in Figure 8.2. The first investigation of a possible relationship between the adrenal gland, which releases EPI and the vagus nerve, was published by Kollmann (1860), who traced vagal fibers from an area below the diaphragm to the adrenals in humans. Using more sophisticated anatomical procedures, Teitelbaum (1933) and Coupland, Parker, Kesse, and Mohamed (1989) confirmed the finding that dorsal and ventral branches of the vagus nerve innervate the adrenal glands. Other studies have shown that electrical stimulation of the adrenal nerve evokes action potentials in the vagus nerve (Niijima, 1992). This finding can be interpreted to suggest that activated vagal fibers convey input regarding changes in hormonal release to the brain since ascending fibers of the vagus contain adrenergic receptors that bind beta-adrenergic agonists such as EPI in both rats (Schreurs, Seelig, & Schulman, 1986) and humans (Lawrence, Watkins, &Jarrott, 1995). Vagal afferents terminate on a number of catecholamine and noncatecholaminergic neurons in the NTS. Following activation by vagal afferents, NTS neurons transmit information to brain areas that process memory-related information such as the amygdala (Ricardo & Koh, 1978; Zardetto-Smith & Gray, 1990). Recent findings using triple fluorescence labeling revealed that norepinephrine is the major neurotransmitter in projection neurons from the NTS to the amygdala. The cell bodies of these neurons were detected in the A2 catecholamine region of the NTS following injection of a retrograde tracer into the amygdala (Petrov, Krukoff, & Jhamandas, 1993). In addition, experiments using the expression of immediate early genes as a marker for activated neurons report that vagal nerve stimulation produces a statistically significant increase in the number of amygdala neurons that express c-fos (Naritoku, Terry, & Helfert, 1995). Other findings from electrophysiological studies show that the firing rates of amygdala neurons are increased following electrical stimulation of either the vagus nerve (Radna & MacLean, 1981) or the NTS (Rogers & Fryman, 1988). This collection of experiments provides in a limited sense a description of some putative mechanisms involved in the processing of emotionally arousing events into memory storage. According to the available evidence, physiological arousal produced by stress or emotional learning experiences elicits the release

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FIGURE 8.1

Schematic view of EPI effects on memory mediated via influences on amygdala nomdrenergic systems.

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This view is complicated by the fact that EPI does not freely cross the blood-brain barrier to produce direct actions on central nervous system structures. EPI = epinephrine; NE = norepinephrine.

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FIGURE 8 . 2

Schematic view of possible mechanisms by which €PI influences the amygdak during memory formation.

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EPI binds to beta-adrenergic receptors on afferent fibers of the vagus nerve, which terminate in the NTS. Stimulation of NE containing neurons in the NTS that have terminal projections in the amygdala would subsequently result in increased release of NE in this structure. EPI = epinephrine; NTS = nucleus of the solitary tract; NE = norepinephrine.

of peripheral hormones from the adrenal medulla, which in turn bind to betaadrenergic receptors on the vagus. Activation of these receptors influences the conduction properties of vagal afferent fibers that synapse on NE containing neurons in the NTS. Stimulation of NTS-A2 neurons that have terminal projections in the amygdala would subsequently result in increased release of NE in the amygdala during memory formation. However, until 1993, this scenario was only speculative because no experiment had examined whether or not EPI’s effects on memory were influenced by manipulation of either the vagus nerve or the NTS.

Beyond Vagal Activation: Effects of Manipulating NTS Neuronal Activity on Memory Processing One method of determining if the vagal-NTS pathway is involved in mediating EPI’s effects is to examine whether this hormone improves memory for an arous-

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ing experience in the absence of the NTS. To address this issue, Williams and McGaugh (1993) assessed whether functional neural blockade of the NTS with the reversible local anesthetic lidocaine hydrochloride would alter the memoryenhancing effects of EPI. In this experiment, animals were trained for 5 days to consume 5 food pellets placed in the left alley and 10 pellets placed in the food cup of the right alley of a Y maze (Figure 8.3). On Day 6 of training, the cardboard inserts covering the metal footshock plates in the right alley were removed. After each animal consumed all of the pellets and reentered the right alley, a brief footshock (0.35 m A for 0.5 seconds) was administered. To determine if the vagal-NTS pathway mediates EPI’s effects on memory for this type

FIGURE 8.3

Reversible lesions of the nucleus of the solifary tract (NTS) attenuate the memory-modulating effects of positraining epinephrine.

Effects of posttraining reversible inactivation of the NTS (with lidocaine) and peripheral injections of EPI (mg/kg) on the latencies to consume food pellets in the right alley where footshock was administered during training. * p < .05 as compared with buffer + saline or L EPI 0.05 mg/kg; **p c .01, Fisher‘s post hoc test. NTS = nucleus of the solitary tract; EPI = epinephrine. From Figure 3 in “Reversible Lesions of the Nucleus of the Solitary Tract Attenuate the MemoryModulatory Effects of Posttraining Epinephrine,” by C. L. Williams and J. L. McGaugh, 1993, Behavioral Neuroscience, 107, p. 959. Copyright 1993 by the American Psychological Association. Adapted with permission of the publisher.

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of arousing and perhaps unexpected experience, animals received an intra-NTS infusion of either phosphate buffered saline (PBS) or the local anesthetic lidocaine to produce a functional blockade of neural activity in the NTS. Two minutes after the brain injections, each animal received a systemic injection of saline, 0.01, or 0.05 m g k g of EPI. On a retention test given 24 hours later, the mean latency to enter the alley where footshock was received during training (data not shown) or to consume all of the food pellets in the right alley (Figure 8.3) was significantly longer in animals given a posttraining injection of 0.05 mgkg of EPI than in salinetreated controls ( p < .05). However, the enhancement in memory produced by the 0.05 mgkg dose of EPI was attenuated by inactivation of the NTS. The retention latencies of animals given the effective dose of EPI immediately after a functional blockade of the NTS were significantly lower than those of animals given 0.05 mgkg EPI and PBS, and their latencies did not differ from the saline controls. These findings confirm those of earlier studies (Gold Q van Buskirk, 1978) and extend their conclusions by suggesting that activation of the NTS via vagal afferents may be one mechanism by which peripheral EPI exerts modulating influences on brain systems that regulate memory storage. The findings from the Williams and McGaugh (1993) experiment also suggest that if inactivation of receptors in the NTS attenuates the mnemonic actions of EPI, then, by implication, activation of these receptors should produce effects on memory comparable to those observed following systemic injection of EPI. Although the results of the previous study suggest participation of the NTS in regulating hormone-induced changes in memory formation, the neurotransmitter systems involved in initiating this process in the NTS is currently not known. Electron microscopic studies have revealed that there is an abundance of noradrenergic terminals, cell bodies, and receptors in the NTS (Aoki Q Pickel, 1992; Aoki, Zemcik, Strader, Q Pickel, 1989; Smith, Egle, & Adams, 1982) and vagal afferents terminate on some noradrenergic neurons in this area (Sumal, Blessing, Joh, Reis, & Pickel, 1983). Furthermore, administration of drugs that increase sympathetic activity produce a significant elevation in NE levels in the NTS of normal rats (Dev & Philip, 1996). Given these findings, it is possible that NE may play an important role in influencing NTS activity during memory formation. To examine this hypothesis, a separate group of animals were trained in the Y-maze discrimination task using procedures identical to those described above (Williams, Men, 6s Clayton, 2000). After footshock training on Day 6, animals received an intra-NTS infusion of either PBS or the noradrenergic agonist clenbuterol (10, 50, or 100 n d 0 . 5 pl), which has been shown to facilitate the release of norepinephrine in several brain regions (Murugauah Q O’Donnell, 1994, 1995). On a retention test given 24 and 48 hours later, groups receiving 100 ngjO.5 k l of clenbuterol took significantly

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longer to enter the right alley or consume food pellets in the alley where the original footshock was administered (Figure 8.4). It is worth noting that the clenbuterol-treated animals exhibited good retention of the footshock experience even though the most prevailing cue associated with the shock (i.e., stainless steel footshock plates) was covered with the cardboard inserts on the 24-hour retention test. In addition, a similar pattern of memory enhancement was observed on the second retention test with the stainless steel plates uncovered 48 hours later. These findings were instrumental in demonstrating that memory processes can be substantially improved by posttraining activation of noradrenergic receptors in the NTS. The effects of clenbuterol may be mediated by facilitating NE output from terminals that innervate the NTS as opposed to any specific actions of this compound on the A2 catecholamine neurons in the NTS, because these cells are activated only by the binding of NE to postsynaptic a1 receptors (Feldman & Felder, 1989). This view is based on anatomical studies indicating that the p2 receptors that bind clenbuterol are localized presynaptically on noradrenergic axons (Misu Q Kubo, 1986) and function in a positive feedback loop to increase NE release from nerve terminals. Given that clenbuterol does not directly stimulate the noradrenergic A2 neurons in the NTS, Clayton and Williams (2000a) examined whether or not memory storage processes are also influenced by catecholamine agonists that bind directly to postsynaptic NE receptors in the NTS. To answer this question, laboratory animals received injections of PBS or EPI, which has a high affinity for a1 receptors immediately after training with a 0.4 mA, 0.5 second footshock in an inhibitory avoidance task. On a retention test given 48 hours later, animals administered 125 n g 0 . 5 ~1 of EPI after training had retention latencies that were significantly longer than the PBS-injected controls (Figure 8.5A). One-week after the retention test, the animals were placed on a weight maintenance schedule and then trained in an eight-arm radial arm maze task. During training, the animals learned to obtain one food pellet from each of four open arms. After a delay, the rats were returned to the maze for a retention test with all eight arms open, but food pellets were placed only in the four arms that were blocked during the initial training trial. Drug administration began on the day after each individual animal reached a criterion of 80% correct performance on 2 consecutive days. On this day, the animals were trained as before and PBS, 50, 125, or 250 n d 0 . 5 ~1 of EPI was infused into the NTS immediately after they obtained food pellets from the four open arms. On a retention test given 18 hours later, the percentage of correct responses in obtaining the pellets from the four new maze arms were recorded. As shown in Figure 8.5B,administration of a dose of EPI (125 ng/0.5 ~ 1 that ) facilitated retention following footshock training also produced a significant enhancement in memory for the radial maze task. Animals given an intra-NTS infusion of

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F I G U R E 8.4

Effects of posttraining intra-NTS infusion of clenbuterol on retention in a Y-maze discrimination task.

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